REVIEW ARTICLE
High-Dose
Aprotinin
Therapy:
A Review
of the First Five Years’ Experience
David Royston, FFARCS “A Scientist must be like a child. If he sees a thing he must say that he sees it, whether he thought he was going to see it or not. Otherwise you only see what you are expecting (most scientists forget that). See first, think later, then test.” So Long, and thanks for ail the fish Douglas Adams
T
HE ROLES OF HEPARIN and protamine in manipulating anticoagulation are well known, but their actions were discovered and recognized by accident rather than by design. Heparin was shown to be a potent anticoagulant while investigators were looking for a procoagulant in ether extracts from various tissues including the liver. The activity of protamine to reverse the anticoagulant action of heparin was recognized during studies investigating whether protamine would prolong the duration of action of heparin. In the same way, serendipity, and not pure scientific reasoning, led to the use of the serine protease inhibitor aprotinin to substantially reduce operative and postoperative bleeding in a variety of operations, but specifically during cardiac and vascular surgery. Historically, a novel and relatively high-dose regimen for this agent was used in the pivotal early study conducted by Royston et al not to prevent bleeding, but in an attempt to inhibit cell activation, thereby preventing tissue injury that may occur during and after cardiac surgery.’ The original high dose used in the first study was further increased, and the dose regimen now recommended was subsequently administered to patients having reoperations.* It produced dramatic reductions in bleeding and the need for donor blood transfusions. Since the original observations published in 1987, there has been considerable interest and enthusiasm for the use of aprotinin in Europe, and now North America, to prevent bleeding associated with major operations, and particularly during cardiac surgery. The aims of this review are (1) to discuss briefly the chemistry of aprotinin and the background to the use of the dose regimen currently recommended; (2) to summarize some of the data showing the efficacy of the drug given in a variety of cardiac, vascular, and transplantation procedures. This section also includes a discussion of issues related to the dose regimen; (3) to review some of the more recent data concerning the problem of the potential mecha-
From the Department of Anesthesia, Harefield Hospital, Harefield, UK. Address reprint requests to David Royston, FFARCS, Department of Anaesthesia, Harefield Hospital, Harefield, Middlesex, UB9 6JH, United Kingdom. Copyright 0 I992 by W B. Saunders Company 1053~0770l9210601-0019$03.00/O 76
nism(s) of action of this agent; and (4) to discuss various safety issues that have been raised with this therapy. BACKGROUND
Chemistry Aprotinin is a basic (pKa 10) polypeptide comprised of 58 amino acid residues with a molecular weight of 6,512 D. The amino acid sequence, biochemical structure, and biophysical characteristics have been described and categorized.’ Aprotinin was independently discovered and isolated from bovine lymph nodes by Kraut et al in 1930,” who identified it as a kallikrein “inactivator,” and by Kunitz and Northrop in 1936,’ who defined it as a trypsin inhibitor in a preparation obtained from bovine pancreas. Aprotinin is a member of a family of serpins (serine protease inhibitors), which are found in all aspects of nature and, as the name implies, are able to inhibit a range of proteases that have serine residues at their active site. This inhibition is provided by inactivation of the active serine of the protease by the lysine residue at position 15 of the aprotinin molecule. Because aprotinin is a small, soluble, and stable polypcptide whose sequence and tertiary structure are well known, it has been a favorite molecule for investigations by protein chemists for many years.h There have been extensive nuclear magnetic resonance studies of the molecule and a great interest in the protein folding (related to the time course of formation of disulphide bridges). Despite these intensive investigations, the physiological role of aprotinin is unknown. Aprotinin appears to be localized in the mast cells in bovine tissue.’ A number of other low molecular weight serpins similar to aprotinin have been detected in a wide range of species including the snail’ and sea anemoneY and in human and bovine plasma.‘“.” Of interest is the fact that these homologous inhibitors are also found in a number of venoms from snakes.” The activity of aprotinin is expressed in various ways. Historically, kallikrein inactivator units (KIU) and trypsin inhibitory units (TIU) have been commonly used. These units rely on measurements of biological potency of aprotinin in fixed analytical systems. Over the years, the manufacturing and purification processes of this drug have improved, and it is now possible to discuss the amount of drug in terms of weight of protein (mg) or concentration of solution (FM). The conversion factors are that 1 mg of
Journalof Cardiothoracic
and
Vascular Anesthesia,Vol
6, No 1 (February),
1992: pp 76.100
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
protein is equivalent to 7,143 KIU or 100,000 KIU is 14 mg of protein. A l+M solution of aprotinin contains 46.5 KIU/mL. Because the majority of the data published before 1986 use the KIU as the unit of measurement, it has been used in this review. However, for some of the discussion on the pharmacological effects of this agent, the weight of substance (mg) or strength of solution (PM) has been used. Native aprotinin has its inhibitory effect on target serine protease by forming reversible stoichometric enzymeinhibitor complexes. This may be easy to read, but to most anesthesiologists, who have no doctorate in chemistry, it is impossible to translate into a clinical situation. In simplified terms, the stability of the enzyme-aprotinin complex is expressed as an equilibrium constant. In its most basic form, this constant can be thought of as a quantifier of the formation of a protease-aprotinin complex (PA) and its breakdown or dissociation from and into the protease (P) and aprotinin (A) components. The equation of this equilibrium constant K is similar to that of the law of mass action associated with acid-base status:
K = (P)
x (A)/(PA)
It can be noted that the smaller the K the greater the affinity of aprotinin for that protease (PA will increase and P will decrease). However, this equation and the resulting K cannot tell what concentrations of aprotinin are clinically necessary to provide a substantial inhibitory effect on a specific protease. For this to occur, it is necessary to know both the concentration of aprotinin and its target. In the presence of increasing A, there will be a relative increase in PA and, therefore, a decrease in P until eventually all of the P is complexed with A. In the case of certain proteases, this may require A levels to be so high that a toxic threshold for aprotinin would be exceeded. In addition, nature has added her own complications to the equation. There are many naturally occurring inhibitors of serine proteases circulating in the plasma. Therefore, any additional functional inhibitory effects of aprotinin will depend on both the concentration and K of the other inhibitors. In pure chemical systems (those without other plasma proteins), the concentration of aprotinin required to inhibit serine proteases that occur in nature such as trypsin, plasmin, or tissue and plasma kallikrein, varies for each of the enzymes with concentrations of approximately 50 KIU/mL (= 1 PM) required to inhibit plasmin and approximately 200 KIU/mL ( = 4 uM) to inhibit plasma kallikrein. These concentrations have been defined from theoretical calculations following experiments performed in pure chemical systems under laboratory conditions.‘.‘2”3 Therefore, it may be that such concentrations may be inadequate to inhibit the enzyme in question in vitro or, conversely, that in combination with naturally occurring inhibitors of these various serine proteases, these concentrations of aprotinin may be in excess of that required to fully inhibit these target enzymes in vivo. These complications confound the issue of defining a specific inhibitory concentration of aprotinin for a specific target protease in any patient. The fact is that the spectrum of inhibition by aprotinin is
77
growing as novel serine proteases are either “discovered” or their importance related to the hemostatic process is studied. In this regard, aprotinin has been shown to inhibit Activated Protein C (APC), a vital protease in feedback control of the coagulation cascade, at relatively high concentrations.14 However, the inhibitory effects of aprotinin on APC have been demonstrated at much lower concentrations in the presence of heparin.15 In contrast, the activator of plasminogen derived from endothelium (tissue-type plasminogen activator or t-PA) is also a serine protease, but is not inhibited by aprotinin in concentrations up to 500 l.~M.l~ Single-chain urokinase is inhibited, but at extremely high concentrations of aprotinin that are not likely to be found clinically. This is also the case for elastase and thrombin. Aprotinin also has no reported inhibitory actions on metallo-proteases and no discernible activity against calcium-dependent thio-proteases such as calpain.6,‘718 As a polypeptide, aprotinin is inactive when given orally and needs to be given intravenously. The half-life in the plasma is biphasic with an initial elimination half-life of approximately 1 hour. It follows that the compound has to be given by continuous infusion if the plasma concentration is to be maintained. Aprotinin is highly resistant to proteolysis and processes associated with moderate chemical degradation. One of the noteworthy aspects of the pharmacokinetics of aprotinin is the affinity of the drug for renal tissue, especially brush border” and proximal convoluted tubule. This may in part be caused by the neuraminic acid content of the brush border in the kidney together with the basic nature of the drug. After injection into rats, approximately 90% of the drug is deposited in the kidney within a few hours.” The drug is freely filtered by the glomeruli, but is not excreted in the urine as an active drug in any significant amount. The predilection of aprotinin to stay in the kidney has led to some concerns regarding the effects of the current dose regimen on renal function. Aprotinin is available for human use as a preservativefree solution of 10,000 KIU/mL in ampules of 50 mL from various manufacturers in Europe. In the studies performed, the agent used was Trasylol (Bayer AG, Leverkusen, Germany). Miles, Inc. (New Haven, CT) is a subsidiary of the Bayer AG company, and is currently investigating the use of this drug in North America. BACKGROUND
AND RESULTS OF PILOT STUDY
As outlined in the introduction, the dramatic effects of aprotinin on bleeding was a chance observation. The use of aprotinin during cardiac surgery was intended to prevent the damaging effects of extracorporeal circulation on certain tissues and organs, specifically the lungs and pulmonary circulation. In particular, an attempt was made to reduce cell activation during the period of extracorporeal circulation. In previous studies, it was shown that it was possible to demonstrate an increase in solute flux into and from the lungs of patients having cardiac surgery.Z’,Z2 In addition, there was evidence for increased oxygen-derived free radical activity, probably derived from neutrophils,*” and that there was a significant relationship between the degree of free radical activity produced from the pulmonary
78
DAVID ROYSTON
circulation and an index of protein leak into the lung.24 At a scientific meeting held in Luxembourg in May 1984, it was suggested that some of the damaging effects of cardiac surgery related to this cell activation could be reduced or abolished by the use of serine protease inhibitors. The rationale for this idea is shown diagramatically in Fig 1. The contact of blood with the foreign surface of the oxygenator stimulates a large number of “inflammatory cascades” that can act through humoral or cellular mechanisms, but that are ultimately controlled by amplification cascades of proteolytic enzymes; the vast majority of inflammatory cascades are serine proteases. The cumulative effect of this amplification is to produce a “whole-body inflammatory response.” This concept was developed by Kirklin et al whose focus of attention had been the role of complement activation in this process.” During the period of extracorporeal circulation, the only inhibition of these potentially deleterious cascades is the routine administration of heparin given to inhibit the intrinsic pathway and prevent blood clotting. Heparin achieves this effect by activating the naturally occurring serine protease inhibitor antithrombin III. The hypothesis originally tested was that the other limbs of complement, fibrinolysis, and kallikrein activation could also be inhibited
Foreign
Intrinsic system
(
by giving reasonable amounts of an appropriate antiprotease. Aprotinin was used for two reasons. First, it was known to have actions to inhibit kallikrein and plasmin at doses that were potentially possible to achieve clinically. Second, it was the only serine protease inhibitor available at that time in the UK that had a sufficiently low toxicity to allow its use in humans at the required concentrations. The concentration of aprotinin thought necessary to block the actions of kallikrein was approximately 4 uM (= 200 KIUimL of plasma), and plasmin was approximately 1 FM (~50 KIUlmL). Based on studies of the use of continuous infusions of aprotinin in multiple trauma patients, members of the biochemistry department in Munich, Germany formulated a dosage schedule for use in cardiac surgery aimed at achieving a concentration of 200 KIU/mL throughout extracorporeal circulation.‘(’ The dose regimen suggested was a 2 x 10”KIU (280 mg) loading dose given over a 20-minute period after induction of anesthesia, followed by a continuous infusion of 500,000 KIU (70 mg) per hour until the patient was returned to the intensive cart unit (ICU). To overcome the dilution effect of the 2 L of crystalloid prime in the oxygenator, a dose of 1 x IO” KIU (140 mg) was added to this prime volume. The study was designed as a pilot investigation; therefore, it was neither
negative charge
>
surface
Plasmin
t
(
Inflammation Post perfusion syndrome
ve tfments
1
Fibrinolysis
Thrombin
_
)
Fig 1. A schematic diagram showing the pathways of activation of white blood cells and platelets following activation at a foreign surface. HMWK, high molecular weight kininogen.
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
randomized nor blinded to the observers. All the patients had coronary artery bypass grafts (CABG) performed by one surgeon with the author as the anesthesiologist.27 At the time of surgery, it was obvious that despite any alterations in biochemical and hematological variables, the most striking effect of the aprotinin therapy was a reduction in bleeding that was, until then, a normal consequence of cardiopulmonary bypass (CPB). Some results of this study have been published:’ and have demonstrated a significant reduction in the blood lost in the postoperative period in patients given the trial drug. The reduction was from a mean of 674 mL in 11 patients who were designated as the control population to a mean of 357 mL in 11 patients who were given aprotinin treatment. Analysis of plasma samples for aprotinin concentrations showed that the target plasma concentration of approximately 200 KIU/mL was not achieved during the time of bypass,’ and that there was a greater than predicted decrease in the plasma aprotinin concentration shortly after the start of the bypass period. To overcome this, it was decided that the addition to the prime volume should be increased by a further 1 x lo6 KIU (140 mg) to give a final total of 2 x lo6 KIU (280 mg) of aprotinin added to the oxygenator prime. This is the dose regimen used in all the subsequent studies. Over the past 4 years, this regimen has been described as the Royston regimen, the London regimen, and, most recently, as the Hammersmith regimen. Apart from the author’s description of the regimen, while holding an honorary contract at the Hammersmith Hospital, the administration schedule itself owes nothing to the author personally nor the Hammersmith Hospital, and is more correctly attributed to the biochemists from Munich. Based on a large number of published and unpublished studies and extensive personal experience, it is believed that the dose regimen for the use of aprotinin can and will be altered to improve efficacy. The optimum dose regimen will depend on a number of factors related to the type of surgery and the patient being given the therapy. EFFICACY
OF APROTININ
THERAPY
The review of the studies of efficacy are divided into 6 broad categories: (1) reoperations through a previous sternotomy; (2) primary surgery for myocardial revascularization in patients who are or are not taking platelet-active agents; specifically aspirin; (3) patients having major vascular surgery; (4) patients with sepsis and endocarditis at the time of surgery; (5) pediatric patients with congenital heart disease; and (6) patients having cardiac or cardiothoracic transplantation. Blood Loss Reduction
Historically, the group of patients having Reoperutions. reoperations was the first in whom the dose regimen currently recommended was described,’ therefore, they will be discussed first. Patients having repeat surgery through a prior median sternotomy are known to be at greater risk for perioperative bleeding and, thereby, an increased requirement for donor blood transfusions when compared with
79
patients having primary operations. The original study of this group showed that the non-treated, control population of 11 patients had a blood loss of 1,509 r 388 mL (mean ? SD) and these patients all required blood transfusions. A total of 41 U of donor blood was given. In striking contrast, the 11 treated patients had a postoperative blood loss of 286 f 48 mL; only 4 of 11 patients received a total of 5 U of donor blood. This reduction in blood use was achieved without postoperative anemia. The venous hemoglobin in the treated patients on the seventh postoperative day was 11.9 ? 0.6 g/dL vs 12.1 ? 0.5 g/dL in the control population. Subsequent use of aprotinin in patients requiring repeat surgery has confirmed these results.” Aprotinin has been available for clinical use in the United Kingdom for compassionate use and, subsequently, was used in an open study in patients at increased risk of bleeding.29 The criteria for aprotinin use were that the patient should be at greater risk of bleeding because they had (1) repeat surgery, (2) endocarditis, or (3) a bleeding diathesis or coagulopathy. In the efficacy data from 451 patients reported, approximately 25% were having repeat CABG, 45% repeat valve replacement, and the remaining 30% were having more complex procedures such as replacement of infected values, double- or triple-valve replacements, replacement or re-replacement of one or both ventricular outflow tracts, or heart and heart-lung transplantation (HLT,). In these patients, the median blood loss in the first 24 hours, postoperatively, was 400 mL. Donor blood use was not strictly controlled. Furthermore, data for blood use were skewed by the inclusion of more complex operations such as reoperation root replacement and HLT,, which often have as their primary bleeding problem massive hemorrhage caused by disruptions of vascular and cardiac integrity rather than an abnormality of hemostatic function. As highlighted, transfusion policy can differ widely between centers, including those with a high commitment to blood conservation programs3 In the UK, there currently is great variability in transfusion practices and a less aggressive approach to blood conservation than is present in most North American and European countries. Despite these caveats in this open study, the median donor blood use was 2 U; 25% of patients received no blood transfusions. These results in high-risk patients are superior to the overall median blood use of 5 U per patient reported for UK cardiac surgery centers3’ Analysis of the safety data from the first 671 patients treated with aprotinin in this study has produced some interesting and intriguing observations, in particular, the reported incidence of events likely to be related to increased thrombosis, ie, perioperative infarction of the heart or neural tissue. Speculation has always existed that if hemostasis was improved by a drug, it must inevitably lead to an increased thrombosis risk. The inconsistencies of this concept are discussed later in this review. Of the patients in the UK open study, 216 had CABG (148 were reoperations), and there were 3 reports of acute postoperative graft occlusion. None of these were attributed to the administration of aprotinin by the clinician in charge of the case. In addition, there were four (1 hemorrhage and 3 thrombotic)
80
DAVID
ROYSTON
Table 1. Blood Loss in the First 24 Hours, Postoperatively* London
Placebo
347 + 103
716 ? 248
270 - 930
210 - 690
295
n = 37 Aprotinin
Amsterdam
Giessen
575 2 164
308 + 132 130 ~ 830 n = 40
n = 30
1,215
380
n = 38
Freiburg
500 - 3,200
n = 35
257 k 69 90
Munich 1,185 r 601 ” = 20
984 k 516 220 - 2,430 n = 38
507 + 206
548 2 290
488 k 297
220 -- 1,075
210
140 ~ 1,350
n = 35
1,400
n = 19
n = 38
Postoperative Donor Blood Transfusionst Placebo Aprotinin
203
155
91
240
179
33
32
40
63
47
*Data presented as mean 2 SD, range, and number of patients. tDonor units given (normalized to U/l00
patients [actual patient numbers are given in above section])
cases reported of neurological complications. This incidence of = 0.5% of neurological episodes in patients having reoperations is remarkably low when compared with the 2.5% to 6% reported in reoperation patients in a recent report?’ It is unlikely that this was achieved by the under-reporting of adverse events as the wound infection rates reported in the UK open study were comparable to the 1.5% to 4% recorded in the US studies. Primary myocardial revascularization: No aspirin. The use of aprotinin therapy has been reported in less complex primary surgery, typically in patients requiring myocardial revascularization.27~*X~‘4~3H These reports have confirmed the efficacy of the new regimen. In addition, there have been five further placebo-controlled, randomized, double-blinded studies in patients having primary surgery for myocardial revascularization. The data for blood loss and blood use from these studies have been reviewed previously (Table 1).24.40 The overall results from these studies in Europe have shown that in approximately 900 patients, who received aprotinin therapy, there was a reduction in the postoperative loss from the pericardial drains of approximately 40% to 50% when compared with 1,000 nontreated patients, who have made up the various control populations. One surprising observation was that there was no direct correlation between the reductions of blood loss and the proportion of patients receiving donor blood. These two aspects of the data from the five randomized controlled
studies are shown in Fig 2. The data show that the proportion of patients transfused in Giessen and London fell by a much greater percentage than in the patients studied in Freiburg and Munich. This is despite the fact that the actual blood loss was reduced by larger amounts in real terms in the latter two centers. Why this effect was observed was difficult to explain at first because all of the studies had similar protocols. The data from the Amsterdam study are difficult to interpret because many of the patients received intraoperative blood transfusions, and the figures shown are only for postoperative blood transfusions and losses. This is probably the reason why the number of postoperative blood transfusions was lower than in the other study centers (Table 1). The additional intraoperative events, which are not shown in the Amsterdam data, would alter the relationship between blood losses and transfusions. In addition to the absolute reduction in the numbers of patients receiving donor blood and the total donor blood transfusions, there was a further observation in those patients who received aprotinin therapy. If the amount of blood given to only the patients who received a blood transfusion (expressed as mean units per patient) are compared, then it can be observed that in the patients receiving aprotinin therapy there were also reductions in the blood used per patient (Table 2). The likely explanation for the apparent anomaly between the donor blood requirements and the postoperative blood losses is based on observations of blood loss and hemoglobin loss into that drainage volume.” It showed in the randomized placebo-controlled, double-blind study that there was a threefold reduction in hemoglobin loss from 38 to 12 g, but blood volume loss was reduced by one-half from 570 to 310 mL (Fig 3). The data suggest that aprotinin was
Table 2. Units of Blood Given Per Patient Study Center
Placebo
Aprotinin
Primary CABG
Fig 2. Differences between placebo and aprotinin therapy for blood losses and proportion of patients receiving donor blood from five placebo-controlled studies in five European centers.
London
2.14
1.65
Giessen
2.36
1.48
Munich
3.2
1.71
Freiburg
2.72
1.78
Reoperations London
3.72
1.25
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
Drains loss
Hb loss
Fig 3. Data shown are for blood loss and the total hemoglobin loss into that volume from a randomized placebo-controlled study in patients having primary CABG.= Data are mean + SEM and show that there was a halving of the volume loss with aprotinin therapy, but this therapy had a greater effect in reduction of hemoglobin loss.
able to reduce the bleeding, but probably had no effect on the drainage of serosanguinous exudate that occurred in the few hours before the chest tubes were removed. It is well recognized and described by the originators of the chest tube reinfusion technique of blood conservation that the hemoglobin content of the reinfused pericardial drainage fluid is highest in the hours immediately after surgery and becomes progressively less in the later postoperative period.“’ None of the patients in the primary revascularization studies was anemic on the seventh postoperative day or before leaving the hospital, which agrees with the studies in patients having more complex repeat surgery. The mean discharge hemoglobin concentration was 12.5 g/dL in the treated group.= This relatively high hemoglobin concentration at discharge from the hospital probably reflected the perioperative “intent-to-transfuse” policy to a level of 10 g/dL. Because of this policy, certain patients in the study received 1 U of blood, or only part of 1 unit. With further experience and modifications of the transfusion policy to allow a lower hematocrit in the immediate perioperative period, it has been possible to tell patients that, excluding a major catastrophe, they will not need blood for surgery or for anemia. This is also true for the majority of patients having reoperations. Primary myocardial revascularization: Aspirin pretreatment. Preoperative aspirin therapy is associated with an improved early graft patency rate and, therefore, is recommended in aspirin ingestion is patients having CABG.4’ However, associated with an increased incidence and degree of to postoperative bleeding,43,44 which has been reported increase the likelihood of the need for a same day reoperation by a twofold factor in aspirin pretreated patients.45 Although aspirin therapy does not universally produce a prolonged bleeding time, a prolongation of this variable has been suggested as a means to determine patients at greater risk of postoperative bleeding.46 However, this concept is not universally accepted.47 Nevertheless, Ferraris et al have suggested that a bleeding time of more than 9 minutes
81
represents a relative contraindication to operation.46 Therefore, such patients should represent a considerable challenge to the therapeutic effectiveness of aprotinin and might also provide some insight into its mechanism of action. Therefore, studies were conducted to establish the efficacy of the current aprotinin regimen in a group of 19 patients who were taking aspirin up to the day of surgery. The entry criterion for the study was a bleeding time of more than 9 minutes.48.49 The postoperative blood loss in the control patients (n = 10) of 2,070 f 280 mL was significantly greater (P < 0.001) than the 290 ? 4.5 mL in the aprotinin-treated patients (n = 9). Only 1 patient in the treated group received donor blood (1 U) compared with all patients in the control group (42 U). Four of 10 control patients and none of the aprotinin-treated patients had a reoperation for bleeding. There are two further reports of the use of aprotinin in aspirin-treated patients. In the first of these, 544 patients scheduled for nonurgent CABG were allocated to receive (26 patients) or not to receive (18 patients) aprotinin. Blood losses were 1,393 t 979 mL in control patients and 352 2 138 mL in aprotinin-treated patients. Total blood use was 49 U in the control group and 21 U in the aprotinintreated patients. However, in the aprotinin-treated group, 11 patients received single-unit transfusions, again highlighting the problems of a rigid intent-to-transfuse policy. In the second report from the Amsterdam group, 40 patients were randomly allocated to treatment and placebo groups.5’ The treated group did not receive the currently recommended dose of aprotinin, but instead were given only 2 x lo6 KIU (280 mg) of aprotinin into the prime of the oxygenator. Postoperative blood loss fell from 1,096 +- 121 mL in 20 control patients to 672 2 65 mL in 20 aprotinintreated patients. Total postoperative red blood cell (RBC) transfusions were 14 U in the control group and 8 U in the aprotinin-treated patients. As previously mentioned, the intraoperative blood use data are not available from these studies, which makes their interpretation difficult. It is estimated that approximately 40% of all patients with ischemic heart disease awaiting surgery are currently taking aspirin. In the current climate of increasing occurence of urgent/emergency surgery, with the attendant and associated scheduling difficulties and pressures on bed allocations, it is not always feasible to allow the routine discontinuation of aspirin therapy 10 to 14 days before surgery. Therefore, in this regard, aprotinin therapy offers a safe and tested treatment to prevent any diffuse bleeding problems. This approach will not only reduce the need for donor blood exposure, but should also reduce the likelihood of prolonged stay in the ICU or the hospital, which is associated with the need for reoperations and larger blood transfusions.45 A second approach may be to administer aspirin only after the patient has returned from surgery. A recently published report from Gavaghan et al has shown that aspirin therapy given within 1 hour of the completion of surgery preserved graft patency.52 This practice was not associated with any increase in the tendency for these
DAVID ROYSTON
82
patients to bleed. The data from this type of study may lead eventually to a reduction in the number of patients taking platelet-activating drugs before surgery. Vascular surgery. Pertinent to this review is a recent preliminary report of the use of the currently recommended higher dose of aprotinin therapy in patients having major vascular surgery. In a pilot study of 32 patients having surgery for aorto-iliac bypass grafting, 10 patients were allocated to receive an aprotinin loading dose of 2 x lo6 KIU (280 mg), followed by a continuous infusion of 500,000 KIU (70 mg) per hour. These aprotinin-treated patients had an intraoperative blood loss of 735 mL (440 to 1,174); (median and interquartile range) compared with the control patient blood losses of 1,300 mL (778 to 2,340). This reduction in operative losses and the provision of a “dry field” with aprotinin therapy was reflected in the median donor blood use of 3 U in the nontreated patients and 0 U in patients allocated to aprotinin therapy. Patients having cardiac and major vascular surgery make up the majority of patients who can benefit from aprotinin therapy. However, they are the relatively easily categorized and controlled patient populations. The groups of patients described in the next three categories are more difficult. This is primarily because they represent subgroups who come for urgent, if not emergency, surgery. They are also patients who have a range of diffuse problems. Therefore, these are also the patients who represent the greatest challenge. Patients with sepsis and endocarditis at the time of surgery. As in the case of the aspirin-treated patients, this group is well recognized to be at great risk of bleeding. Many are having repeat surgery because they have developed infections on a prosthetic valve. In addition, the inflammatory processes activated during septicemia often lead to deranged hemostasis and coagulation. Aprotinin was administered to 1.5patients who had active endocarditis at the time of surgery.’ The absolute blood loss in these 15 patients (13 were reoperations) showed a mean of 300 mL with a range of 140 to 870 mL. A total of 14 U of blood were given to these patients in the first 72 hours, postoperatively. This compares with a total of 93 U given to 9 patients operated on by the same surgeons at the same hospital in the previous 12 months. When the data are normalized to appear as if there were 100 patients in each group, it is easy to see an approximately lo-fold reduction in donor blood requirements in these patients (Fig 4). These figures, although extremely good, hide the pattern of transfusions in these patients. Seven of 15 patients received no donor blood. Five patients received a total of 8 U before and during surgery to maintain a hematocrit of > 22%. An additional 6 U were transfused into 4 patients postoperatively. Pediatric surgery. Before the author’s description of the effects of aprotinin in reoperations, the drug had been championed for some time in Germany to reduce bleeding in pediatric cardiac surgery.53.54 Of interest from these early studies are the results in neonates and small children (weighing 2 to 8 kg). Those given aprotinin in divided doses to a total dosage of 45,000 KIU/kg over the period of
No apdinin
Apmtinin
Fig 4. Data shown for the total WC units administered to patients having surgery with active endocardiiis. The data have been normalized to produce a total use for every 100 patients in each group. The no-aprotinin figures are from patients operated on by the same surgical team in the 12 months before the availability of aprotinin therapy.
surgery had approximately a 60% reduction in blood loss and transfusion needs compared with pediatric patients given nothing at the time of surgery. This former group also had a reduction in mortality, with a group mortality rate of 14.7% compared with 23.6% in the group without intraoperative intervention. However, the anecdotal nature of the reports and their relevance to the current practice of pediatric cardiac surgery make interpretation of certain aspects of the data difficult. Despite this, there seems a place for aprotinin in pediatric practice. With this in mind, the dose regimen has been modified; the present suggestion is to give the currently recommended adult dose to children with a body surface area of > 1.16 m’. In children with a smaller body surface area, 240 mg/m’ is given as the loading dose; this amount is also added to the pump prime. The infusion rate is maintained at 56 mg/m*/hour. This regimen was designed to administer a final total dosage of approximately 75,000 KIU/kg over the time of surgery, which is similar to the adult figure. Currently, this dosing regimen is being administered in a randomized double-blind study being conducted at the Hospital for Sick Children in London. Preliminary data from that center showed that one interesting aspect of the therapy was the effect on length of time for chest closure.” Because of the improvement in hemostasis, there was a threefold reduction in the length of time for chest closure and return to the intensive care unit (Fig 5). The reduction in the total time in the operating room produced by this reduced time for closure has allowed more efficient use of these facilities. More procedures can now be performed in the same operating rooms during the routine working day (M. Elliott personal communication, August, 1991). Patients requiring heart transplantaHeart and HLTx. tion for end-stage cardiac failure can also benefit from aprotinin therapy for two reasons. First, patients with an ischemic cardiomyopathy have often had previous cardiac surgery. These patients who require reoperations obtain the same beneficial effect with aprotinin therapy as in other reoperations. Aprotinin is now routinely used on this group
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
83
No aprotinin
24O3000
la0 eQ0.
,,,, ;:;:;:;: I',',',' ,','/'/' ,',',x/' ,','/'/x t',',',' t',',',' ,'/'/'I' 1','/'/' ....
1
E3Chest
Fig 5. Data (mean + SD) showing time for chest closure in neonates having arterial switch for transposition of the great vessels. The data show that with the dry field produced by aprotinin, the time for chest closure is substantially reduced. (Reprinted with permission.“)
of patients at Harefield Hospital, where approximately 150 heart transplantations are performed each year. Second, another reason for wanting to avoid blood transfusions in these patients is to avoid any blood-borne infection from being given to an immunosuppressed patient. In this regard, cytomegalovirus (CMV) is one of the key problems. With large blood losses and in patients with uncommon blood groups, it may not be possible to guarantee unlimited supplies of CMV-negative blood. HLTx is an effective means of treatment for a number of otherwise lethal diagnoses. A number of problems including bleeding can lead to a stormy early postoperative period following HLTx. In one survey of the results of HLTx, there was a 26% early mortality.56 Bleeding was reported to account for 50% of the deaths. In the 12 months from January 1990 to February 1991, there were 44 HLTx at Harefield Hospital in patients older than 16 years of age. Included in the data were 24 patients having HLTx because of end-stage respiratory failure caused by cystic fibrosis (CF). Nine of the patients received aprotinin therapy. The mean cumulative losses into the pericardial and chest tubes for the first 48 hours postoperatively for the treated and untreated CF patients are shown in Fig 6. The median figures for blood product replacement in the nontreated patients in the immediate postoperative period were 7 U of red blood cells, 6 U of fresh frozen plasma, and 6 U of platelets. The corresponding figures for the patients receiving aprotinin therapy were 3 RBCs, 0 fresh frozen plasma, and 0 platelets. Additional benefits of aprotinin therapy in these patients were noted when the gas exchange of the lungs was considered. Using the PaO,/F,O, ratio as the index of oxygen transport, it can be observed that there was better gas exchange in the aprotinin-treated patients with a higher PaO, at the same or lower inspired oxygen (Fig 7). As the data in Fig 6 shows, the majority of the postoperative bleeding is from the chest tubes. It is well recognized that the major sites of hemorrhage in these patients tend to be from the posterior mediastinum.57 The benefits of aprotinin therapy in this group of patients is that a larger
3000
? ?Pericardial
O-12
Apmtinin
Aprotinin
12-24
24-48
1
Chest W Pericardial
O-12
12-24
24-48
Fig 6. Data (median) at 12, 24, and 46 hours, postoperatively, for cumulative blood loss from thoracic and pericardial drains in patients having HLTx for end-stage CF. The left panel shows data from 15 patients operated on without aprotinin therapy and the right panel shows data from 9 patients operated on with aprotinin. The figure shows that the majority of the postoperative blood losses are from the thoracic tubes, and the considerable effect of aprotinin therapy in reducing this loss.
amount of the capillary bleeding is prevented; this allows the surgeons to more accurately define the major bleeding sites and provide good hemostasis either by electrocoagulation or local application of fibrin or tissue glue. One further finding that emerged from the data in these patients was that donor blood administration over the ensuing period of hospital stay following transplantation and in the subsequent months was independent of whether aprotinin therapy was given. This is probably caused by the fact that patients with CF and those having HLTx have a poorly understood chronic anemia, which requires blood transfusions. With the development and interest in the use of recombinant erythropoetin in this kind of anemia, it will hopefully be possible in the not too distant future to obviate the need for these added blood transfusions in this group of patients. These preliminary data suggest that aprotinin therapy may be of benefit in HLTx. However, as in the case of
Fig 7. Data (mean f SEM) for PaO,/F,O, ratio in the patients whose blood losses are shown in Fig 6. The avoidance of large blood transfusions in the patients receiving aprotinin therapy was reflected in more stable and better gas exchange in this group in the early postoperative period.
DAVID ROYSTON
84
pediatric surgery, it may be benefits blood conservation, part in reducing operating improving the postoperative
that aprotinin therapy not only but may also play a significant and critical care times and in well-being of these patients.
Alterations in the Dose Regimen Before a discussion of the mode of action of aprotinin, the dose regimen now used and the rationale for future modifications and manipulations of the total dose and timing of administration of this agent will be discussed. Analysis of plasma samples taken during the double-blind studies in primary surgery showed that the median aprotinin concentration achieved was 5.5 PM (258 KIU/mL) at the start of surgery and 3.9 ~.LM(181 KIU/mL) at the end of bypass.** Therefore, the concentrations required theoretically to inhibit plasmin and kallikrein were achieved during the entire bypass period. Further analysis of the data has shown that the elimination half-life of the drug was 44 minutes.5H This additional evaluation also raised two other points in relation to the currently recommended dose regimen. First, the actual concentration of aprotinin achieved in the plasma following the original bolus varied markedly among patients. This variability was not related to the size of the patients, as might have been expected from the use of a fixed-dose regimen whatever the patient’s weight and sex (Fig 8). Second, the only significant relationship between the plasma concentration of aprotinin and its effect to prevent bleeding was between the postoperative hemoglobin loss and the aprotinin concentration at the end of bypass (Fig 9). Although this association was statistically significant (P < 0.02) it was not very tight (r = 0.56). There was no significant relationship between the concentration of aprotinin at the start of bypass and any of the variables measured; the data shown in Fig 10 relate the aprotinin concentration achieved after 10 minutes of bypass to the postoperative hemoglobin loss. These data suggest that the maintenance of a high aprotinin concentration toward the end of the period of extracorporeal circulation may be more important than having a high concentration at the start. A more logical approach to the dose regimen may be to give a relatively large bolus infusion before the incision, to add a modest bolus to the pump prime to help maintain adequate
30-
* 0 0 0
.o 0
10-
ti
?? 0
0
.
0
?? .a .
8.;
0..
??
.
0
I
I
i
loo
200
300
400
Apotinin concentration( KIU / ml ) Fig 9. Plot of plasma aprotinin concentration at the end of bypass compared with the postoperative hemoglobin loss from 37 patients in the randomized, placebo-controlled study in primary CABG patients.= The relationship is not tight (r = 0.96). but is statistically significant (P < 0.02).
plasma concentrations throughout bypass, and to give a final bolus immediately before the completion of bypass. This pattern of administration is used in a number of hospitals in Europe.S’ Aprotinin has been given to stop excessive diffuse bleeding in postoperative cardiac surgical patients in the ICU. Angelini et al described the results of administration of a bolus of 2 x 10h KIU of aprotinin followed by a continuous infusion of 500,000 KIU/hour in six patients who were bleeding after cardiac surgery.h” The effects of this therapy were to decrease the rate of blood loss from 490 (250 to 1,146) ml/hour (mean [range]) to 90 (29 to 193) mL/hour during and up to 6 hours from therapy. In this regard, Bidstrup et al also recommended continuing the administra-
loo
&thin Fig 8. Plots of plasma aprotinin concentration against time from 18 individual patients studied in randomized placebo-controlled studies in the UK.” The data show the threefold differences in plasma concentration achieved following a standard bolus loading dose, and the great individual variability in the disappearance of the drug.
0
al-
zoo
300
400
concentdion
(
500
600
KIU / ml )
Fig 10. Plot of plasma aprotinin concentration at the commencement of bypass compared with the postoperative hemoglobin loss from 37 patients in the randomized placebo-controlled study in primary CABG patients. *’ There is no relationship between the variables (/ = 0.07).
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
tion of aprotinin therapy into the postoperative period in patients at particular risk of bleeding, and especially in patients with deranged hemostatic mechanisms as a result of infections and sepsis at the time of surgery.6’ Previous reports of the use of aprotinin administered at the end of CPB used a dose of 10,000 KIU/kg, but did not measure efficacy in reducing bleeding.‘j* Therefore, it would seem reasonable to suggest studies of the use of aprotinin to prevent bleeding unrelated to the period of extracorporeal support. The earliest reports on the use of aprotinin commented on the dry operating field achieved prior to heparin administration.27 Therefore, it follows that in certain cases, such as reoperations or where both internal thoracic arteries are used as conduits for myocardial revascularization, the benefit of aprotinin therapy in producing a dry field during a difficult and possibly prolonged dissection before the period of CPB can be obtained. The optimum dose and timing of the administration of aprotinin will depend greatly on the length of surgery. If it is prolonged by extending the time before establishing CPB and/or by the need for a prolonged period of CPB, then the concentration of aprotinin will decline to low levels toward the end of bypass. This may severely limit the observed efficacy of the therapy. Another confounding influence on the benefits of the therapy and strategies to maintain a constant plasma concentration is if some form of hemofiltration system is used during the period of extracorporeal support. For example, if such a system is used to remove excess fluid at the end of the bypass period to increase the hematocrit, then aprotinin will be lost from the plasma. The mechanism of this loss is twofold: (1) the highly basic molecule will adhere to the dialysis membranes, and (2) with a molecular weigh of 6,512, aprotinin is filtered by the system and discarded. This latter effect may explain some of the preliminary pediatric cardiac surgical data previously cited. At the center performing these studies, it is their practice to allow profound hemodilution during the period of extracorporeal circulation and to return the hematocrit to normal by a period of ultrafiltration before decannulation.“3 In contrast to the administration of the currently recommended high dose, it has been argued that a much smaller dose is of equal benefit. The rationale for this is difficult to define from the published literature in humans, but does follow the adage that a smaller dose for the same efficacy should produce fewer adverse effects. In one study, aprotinin, 2 x lo6 KIU, was given into the oxygenator prime as the only therapy.@ The effects of this regimen were compared with the currently recommended dose in patients having primary surgery. The end-points for efficacy were hemoglobin loss for the perioperative period together with postoperative transfusions of RBCs and FFP. The perioperative hemoglobin loss showed mean values of 110 g in the control group, 65 g in the group given the dose currently recommended, and 74 g in those given aprotinin only into the pump prime. The total postoperative blood product use for these patients was 30 U of RBC and FFP in the 30 nontreated patients; 11 U of RBC and 11 U of FFP in the 30 patients given aprotinin therapy as the current regimen,
a5
and 9 units of RBC and 20 units of FFP in the 22 patients given aprotinin into the oxygenator prime. The authors commented that the increased use of FFP in these latter patients was “because of a longer postoperative bleeding time,” although their published data do not support this statement. Earlier reports have also reported investigation of the efficacy of aprotinin in a total dosage of approximately 2 x lo6 KIU during surgery.65 In this study by Hannekum et al in patients having primary surgery for valve replacement, the mean loss postoperatively decreased from 596 mL to 443 mL, but this difference failed to reach statistical significance.6’ However, the donor blood used in this study was significantly lower, but blood product administration was not completely abolished in the aprotinin-treated patients. In the section dealing with the use of aprotinin in patients taking aspirin, there is also a report on the use of only an oxygenator prime dose that showed efficacy. The mean blood loss decreased from approximately 1,096 mL to 672 mL. This reduction to approximately 60% of the control group loss was statistically significant (P < 0.05). Similarly, there have been reports of the use of half of the presently recommended dose in patients having repeat cardiac surgery.“6 Statistically significant efficacy was achieved with this lower dose. The mean postoperative blood loss decreased from 1,429 mL to 948 mL (P < 0.05). The data from these two low-dose studies are compared with that from separate studies in patients having reoperations’ and those taking aspirin” and shown in Figs 11 and 12. While accepting that it is possibly not appropriate to compare studies from different centers, the data are intriguing and do imply strongly that the higher the dose the more beneficial are the effects of aprotinin. As will be discussed later, there appears to be no evidence for any detrimental toxic effects of the currently recommended regimen, thus the use of a lower dose regimen seems totally illogical. If this lower dose is administered routinely, then a proportion of the patients will bleed sufficiently to require donor blood transfusions, even though they have been challenged by a drug that may have antigenic potential. The end result of this technique may compromise the chance of using aprotinin for some future procedure while failing to prevent the need for donor blood or blood product transfusion. There is a dose-response effect for this agent, which is different for various discrete procedures. The currently recommended regimen will reduce bleeding to the point in which the need for donor blood is prevented in all patients having primary surgery, the majority of reoperation patients, and patients taking aspirin. However, this dose regimen will not totally prevent bleeding in combined HLTx and in certain other situations. In these patients, a larger amount of the drug than is described in the current regimen is needed. This approach has been used on a number of occasions in patients having reoperations for the third or fourth time, while taking aspirin f coumadin-type anticoagulants, and also in patients who are of the Jehovah’s Witness faith.
86
DAVID ROYSTON
18CO-
Tabuchietal E3iietal
-C-
+
0 0
1
I
I
I
I
140
230
420
560
700
Total aprotinindose ( mg )
0
U’
0
ldo
2iO
420
560
700
associated with cardiac surgery. The first relates to establishing a consensus view on what is the definition of excess versus normal blood loss; thus far, virtually all studies of this issue have concentrated on identifying the cause of “abnormal” bleeding associated with a period of extracorporeal support without defining what is abnormal. The difficulties in choosing an acceptable degree of “normal” bleeding are highlighted when, for example, the results for blood loss shown in Table 1 for the double-blind-studies of the efficacy of aprotinin are compared. The patients in the control group in the Geissen study had losses of 347 -t 103; 210 to 690 mL (mean -+ SD; range); whereas those in Munich were 1185 t 601; 500 to 3200 mL. Both centers obviously regard these losses as normal despite the threcfold difference in their magnitude. Similarly, in North America the losses recently reported by Goodenough et alZU in patients having primary surgery showed a range from 672 + 54 mL to 1,445 k 109 mL in the 18 centers with blood conservation programs. Without a consensus on what is considered as abnormal, it is obviously difficult to delineate those factors that are specifically associated with a tendency to increase bleeding. The second problem associated with studies of the action of aprotinin stems from the fact that the vast preponderance of the research has been performed in patients during cardiac surgery. This has inevitably led to studies focused on measuring factors and effects of aprotinin at the time of extracorporeal circulation. These studies have produced conflicting data and hypotheses and little, if any, data that may prove valuable in answering the question of the mode of action of the drug in the patient. Aprotinin therapy acts to improve hemostasis and prevent bleeding before any period of extracorporeal support. It is the author’s belief that the primary action of aprotinin owes nothing to its effects to prevent the damaging effects of CPB. Nonetheless, the preponderance of mechanism of action studies
Total aprotinindose ( mg ) Fig 11. Data for blood loss (actual loss [mean t SEMI in top panel and expressed as percentage of control in lower panel) from two studies of the efficacy of aprotininin patients taking aspirin.“,5’ In the study of Tabuchi et aP 280 mg of aprotinin was added to the oxygenator prime. In the Bidstrup et al report60 the total dose was the currently recommended dose. While both therapies produced significant reductions in losses, the lower dose appears to be less effective.
MECHANISMS
0
Roiystonetal Scott&Au
??
UKopenW
.
0
OF ACTION
The mechanism of action of aprotinin to reduce bleeding is of great importance primarily because there are a number of modified and genetically engineered forms of aprotinin available that can be produced commercially by recombinant technologies. By identifying the mode of action, a more specific and potent agent could be developed. Another reason to define the mode of action of aprotinin is to allay anxieties that if a drug profoundly improves capillary hemostasis, then it must induce clot formation, and thereby adversely influence early graft patency. There are two major problems in attempting to identify the mode of action of aprotinin in reducing the bleeding
U
0
700
Total aprotin?dose ( mg ) Fig 12. Data for postoperative losses (median) from three reports of the use of aprotinin in patients having reoperations.‘“~5* The patients in the report by Scott and ALP received half of the currently recommended regimen. An apparent effect of dose on losses can be observed.
APROTININ TO PREVENT BLEEDING: THE FfRST 5 YEARS
conducted principally during the period of extracorporeal support have been directed nut toward the issue of how aprotinin works to improve hemostasis, but instead have been focused on addressing the question of how aprotinin protects the extracorporeal system. There are a number of agents that do this task far more efficiently than aprotinin, but not all of these drugs prevent bleeding. The problems with investigations of the mode of action of aprotinin that center on the period of bypass are most easily illustrated when the data for one aspect of the damaging effects of extracorporeal support are evaluated. Three studies have reported the effects, of aprotinin therapy on complement activation; measured using the generation of the active fragments C3a and C4a. These studies have shown that there are no effects of aprotinin,” that aprotinin therapy reduced complement activation6’ and, most recently, a further study showed aprotinin administration to be associated with an increased complement activation6’ The inconsistency in reported data probably reflects the huge variability in perioperative techniques. The oxygenator system, pumping system, and type of flow, temperature of the patient and perfusion system, acid-base control, as well as the type and volume of the prime for a particular operation vary widely throughout the world. Similarly, the period of each segment of a specific operation differs between centers and surgical teams. Add to this list the differences that perioperative pharmacological intervention may produce, and it is not difficult to see that much of the data related to the mode of action of aprotinin can only be applied to one specific trial at one time; therefore, it may not be possible to generalize any conclusions from such an investigation. Despite these caveats and before considering aprotinin’s actions, it seems reasonable to briefly review the normal hemostatic and coagulation systems and the various parts of this process, which may in some way be affected by aprotinin therapy, based on current knowledge of its spectrum of inhibitory activity. NORMAL
HEMOSTATIC
FUNCTIONS
From an anatomical point of view, it is possible to differentiate bleeding from arteries, veins, and capillaries. Aprotinin has its main effects by reducing the capillary ooze associated with major tissue injury and surgery. Bleeding from capillaries stops mainly by alterations at the microvascular endothelium. The hemostatic process progresses in several interconnected phases. The primary process is that the platelets plug the holes.@ This is achieved by platelet adhesion, aggregation, and release of granules and other products leading to the production of a hemostatic plug. This initiation of the hemostatic process is followed by the activation of the clotting process to produce fibrin. Before describing certain aspects of these processes, it is worthwhile to point out that the platelet, for its size, has a remarkable array of biochemical functions. These are controlled and activated by an equally impressive array of receptors and response systems that allow the platelets to perform their hemostatic function. Not all of the mecha-
87
nisms are fully understood. In si,mple terms, the normal platelet is able to adhere to a damaged endothelial cell or the subendothelial layer. This adherence is achieved by the platelet sticking to collagen, basal membrane structures, and microfibrillae of subendothelial tissues. The interaction is supported by a bridge of the multimeric form of the von Willebrand factor from the endothelium/subendothelium to attach to the platelet at the glycoprotein Ib (Gp Ib) receptor site. Until recently, it was thought that these Gp Ib receptors were always exposed on the platelet surface. Then the platelet can undergo a shape change and spread over the injured microvascular surface with exposure of different glycoproteins, such as the Gp IIb/IIIa complex, which can bind to fibrinogen (the activated IIb/IIIa COMplex is termed the “fibrinogen receptor”), and Gp IV, which can bind to collagen (as can Gp IIb/IIIa, but not Gp Ib). The Gp IIb/IIIa complex is interesting as it has an amino acid residue sequence with homology to a number of other adhesive glycoproteins found on the surface of many types of cells, particularly on neutrophils. Because the platelet allows the exposure’ or development of the Gp IIb/IIIa complex, there is release of platelet a-Granule materials such as adenosine diphosphate (ADP), l3-Thromboglobulin, and platelet fibrinogen. Advances in the techniques of molecular biology have led to a greater understanding of the type and role of the platelet glycoprotein receptor. The relative frequency of antigenically available glycoprotein receptor sites can now be measured with comparative ease. However, the techniques used are complicated, and there is considerable debate as to the optimum antibody to use to detect these sites and as to the methods used to prepare the platelets before measuring. There are a number of people who have a genetic defect that results in the inability to produce or express these glycoprotein receptors on the platelets. Subjects in whom there is an absence of the Gplb receptor have BernardSoullier syndrome. In these patients, there is a prolongation of the skin bleeding time. The platelets respond normally to aggregation with ADP, and there is a reduced, but not absent response to stimulation with the synthetic antibiotic ristocetin. Patients failing to express the Gp IIb/IIIa receptor have the Glanzmann-Nageli syndrome. Once again, bleeding time is prolonged. The platelets from these patients no longer respond to ADP or collagen with a second phase or aggregation response. Fibrinogen is an important cofactor in platelet adhesion and is currently thought to be essential for the process of platelet-to-platelet binding, which occurs during irreversible aggregation. During this secondary event, there is a release of material including thromboxane A,, a very potent vasoconstrictor and platelet aggregating agent, which is synthesized in the platelet. A further protein complex called “thrombospondin” is also released at this time and binds between the newly exposed fibrinogen receptors and the fibrinogen molecules bridging platelets to stabilize the aggregate. The platelet also contains mitochondria and
88
DAVID ROYSTON
contractile elements, which are sensitive to a number of different stimuli, such as an increase in calcium flux. In addition to the exposure of the various glycoprotein receptors at the time of initial activation, the platelet also exposes various membrane-derived phospholipids. These are thought to be necessary for regulation of the coagulation cascade. These membrane components can serve as activating agents for factor XII. In addition, other factors are able to bind and transform other parts of the coagulation cascade. Factor V of the coagulation cascade binds to platelet lipids and, in a complex with collagen and factor X, can act to release activated factor X. In this way, the final common pathway of coagulation is stimulated. The thrombin generated is able to act as a positive feedback to initiate further platelet aggregation and to stimulate the release of further ADP and thromboxane. Damaged endothelium can also produce tissue factor and tissue thromboplastin to stimulate coagulation by the extrinsic or factor VII-dependent process. The initial hemostatic function of the endothelium can be thought of as a process of initiation of hemostasis and subsequent amplification of the coagulation cascade. It is obvious that this process would carry on unabated to produce unchecked clot formation unless there were equally effective methods of controlling these processes. In this regard, thrombin acts to stimulate release of various substances from the endothelium. Of particular relevance is the production of prostacyclin and an endothelial-derived relaxing factor (EDRF), which are both well recognized as potent antiaggregatory agents and will inhibit or limit the formation of platelet aggregates. Thrombin also acts on the microvascular cells to induce the production of tissue plasminogen activator (t PA), initiating the conversion of plasminogen to plasmin, and thus activation of the process of fibrinolysis. These actions of thrombin limit platelet aggregation, initiate the dissolution of the blood clot, and also reverse the vasoconstrictor response fundamental to the hemostatic process. Finally, thrombin can bind to thrombomodulin on the endothelial cell surface, and this thrombin-thrombomodulin complex can activate protein C (produced in the liver) to produce activated protein C (APC). This APC is a serine protease, which can constrain the rate of thrombus formation by its own inhibitory actions on factors V and VIII in the intrinsic coagulation cascade.
MECHANISMS
RELATED TO BLEEDING AFTER
CARDIOPULMONARY
BYPASS
Activation of Platelets During Extracorporeal Circulation There is excellent evidence of abnormalities in platelet numbers and function associated with CPB: (1) there is rapid consumption of platelets, which is most obvious during the first few minutes of bypass’“,‘l; (2) there is decreased reactivity to known agonists72.73; (3) there is an increase in the concentration of o-granule compounds such as @Thromboglobulin in the plasma”,7s; (4) there is an increase in the concentration of thromboxane B, (the stable metabolite of thromboxane AZ) presumed to be released from aggregating platelets76.77; and (5) the bleeding time is
prolonged following a period of extracorporeal circulation and the magnitude of the prolongation has been directly related to the time on bypass.” Platelet Numbers There are two views on the significance of the immediate fall in platelet numbers at the start of bypass. One view is that this represents activation and loss of the platelet from the circulating pool, which is best prevented by inhibition of the contact activation phase of the system. The second view is that this loss represents only a minor alteration of platelet function and activation is not the principal problem. In support of the first concept are studies that have shown that expression and subsequent loss of platelet surface glycoprotein receptors are associated with the reduction in absolute platelet count in the first few minutes of extracorporeal circulation. Furthermore, this platelet loss from the circulation was attributable specifically to the expression and subsequent loss of the Gp IIb/IIIa complex.” This was demonstrated by the fact that only blood from normal donors and patients with Bernard-Soulier syndrome (platelets deficient in Ib but sufficient in Gp IIb/IIIa receptors) was lost, and that the proteins that remained adherent to the extracorporeal system were primarily made up of fragments of the Gp IIb receptor.-” These observations strongly support the concept that the Gp IIb/IIIa complex is the adhesive glycoprotein most affected by the extracorporeal system and that platelet loss is related to activation by this pathway. The second view is supported by the studies of Zilla et al who argued that the role of early contact in platelet activation is not a major problem or mechanism for the platelet 10~s.~”Using morphological methods, they showed that, although there was a reduction in active platelets during the first few minutes of bypass, there was, at the same time, an increase in the number of platelets showing a shape change. Throughout the period of bypass, there was a reduction in the obvious detrimental aspects of extracorporeal circulation on platelet morphology; there appeared to be a reversal of adverse effects noted at the start of bypass. They also provided evidence, based on the correlation between factors associated with cell death such as plasmafree hemoglobin and lactate dehydrogenase concentrations, and the increase in plasma concentration of a-Granule products, such as platelet factor 4 and p-Thromboglobulin, to show that the platelet was not undergoing activation but lysis. Zilla et al have suggested that any platelet stimulation associated with the initial phase of bypass is of little consequence, and that with modern CPB systems only a portion of the platelets proceed beyond the step of reversible platelet aggregation. Support for the argument that activation of glycoprotein receptors may not play an important part in the loss of platelets comes from the studies of George et al.” They used highly specific antibodies and demonstrated that following CPB there was a significant reduction in the binding of antibodies to both Gplb and the total Gp IIbiIIIa complex in platelet suspensions that had been immediately tixed in glutaraldehyde. Although the reduc-
APROTININ
89
TO PREVENT BLEEDING: THE FIRST 5 YEARS
tion in the number of antibody binding sites and, therefore, presumably receptors was highly significant, (8’ < 0.02 or better), the actual fall in receptor number was 20% for the GpIb and 10% for Gp IIb binding. In none of the patients studied during cardiac surgery was there a decrease in the binding sites for antibody outside the 95% confidence interval for the laboratory concerned. In this study, the authors did not attribute any clinical significance to their observations of glycoprotein receptor binding in terms of the increased likelihood of bleeding. In contrast, they argued the opposite case. Subjects heterozygous for Bernard-Soulier and Glanzmanns thrombasthenia with one-half the normal concentrations of Gp Ib and Gp IIb/IIIa, respectively, have normal platelet function and no excessive bleeding.82 These observations imply that a severe deficit in membrane receptors must exist before there are major bleeding complications. The confusion produced by this contradictory evidence of the nature and importance of platelet activation to bleeding problems is only compounded by interpretation of other data related to platelet function.
Platelet Activation
There is evidence for the release of various components of platelet granules during extracorporeal circulation. This effect is usually demonstrated by measuring increases in plasma concentrations of these granule products. However, the actual amount of activation needed to produce this effect is only slight. For example, a 60-fold increase in plasma concentrations of platelet factor 4 requires only approximately 1% of the platelets to be activated.83 In studies using the ex vivo system, the increases in B-Thromboglobulin concentrations were similar despite threefold differences between adhesion and activation of platelets from patients with various glycoprotein abnormalities.‘l Two studies have failed to demonstrate a-Granule release from stimulated whole blood.8’,84 Therefore, these data do not support the evidence of Harker et al.”
Bleeding Time
The measurement of bleeding time is thought to be an expression of platelet-microvascular reactions that were unaffected by heparin administration. This measurement has been shown to increase in duration as the period of CPB is prolonged and to revert rapidly back to normal when the extracorporeal support is discontinued.‘* However, recent evidence has suggested that heparin can affect the bleeding time:’ and that the bleeding time can be significantly prolonged by processes not directly related to the platelet numbers or possibly their function. For example, the bleeding time is prolonged following the administration of the fibrinolytic agent rt-PA.86 Given this confusion regarding suitable methodologies and models along with the data generated from such systems, it is impossible yet to define precisely the role of the platelet in postoperative bleeding.
Fibrinolysis
There is clear evidence that the concentration of fibrinogen/fibrin degradation products increases during the period of extracorporeal circulation.87’88Although the mechanism of this increase is not fully elucidated, there are recent studies that show that the concentration of plasminogen activators increases during the period of extracorporeal circulation.89.90 As in the case of the platelet mechanism, the question is to relate this evidence for activation of fibrinolysis to postoperative bleeding. There are reports that show that there is a significant correlation between indices of fibrinolytic activity and the volume of losses into the chest drains.91-93 Equally, other studies have not shown any relationship between concentrations of fibrin split products and increased postoperative losses.” A further difficulty in determining the role of fibrinolysis in the bleeding defect is the actual end-point chosen to define the extent of fibrinolysis. For example, an increase in the plasma concentration of fibrin(ogen)-split products can be demonstrated in all patients having cardiac surgery. However, using end-points such as the euglobin clot lysis time or the whole blood clot lysis of the thromboelastogram, there are transient abnormalities in approximately 1O%87 and 6% to 8%95 of patients, respectively. These abnormalities are obvious immediately following protamine administration and rapidly disappear. Despite these difficulties in defining fibrinolysis, there is some evidence to suggest that the administration of antifibrinolytics to patients having cardiac surgery reduces postoperative bleeding.96,97However, the reductions have thus far been relatively modest and there is no evidence of efficacy in patients having surgery in which blood loss may be excessive, eg, in reoperations and in patients taking aspirin. EFFECTS OF APROTININ
ON PLATELETS AND
FIBRINOLYSIS
Platelets As previously discussed, a defect in platelet function has been suggested as the major cause of bleeding in the postoperative period. Indeed, pharmacological interventions to prevent bleeding have been effective when plateletactive agents such as prostacyclin9* and dipyridamole99 have been used. All of the serine protease inhibitors have activity to inhibit various aspects of platelet function.6 Aprotinin is no exception, and has been shown to have a large number of direct and indirect actions on platelets. The second phase response of platelet aggregation (associated with the release of thromboxane A, and serotonin) following stimulation with ADP is effectively inhibited by aprotinin at concentrations of approximately 100 to 400 KIU/mL.‘W~‘o’ This effect has been used to great effect in preserving platelets in donor blood. In addition, there are also reports of the use of an intravenous bolus dose of 20,000 KIU/kg to prevent platelet aggregation, and thereby reduce the incidence of venous thrombosis in major surgery. This dose
90
significantly reduced the platelet :adhesion and aggregation associated with hip replacement ~o,per-ation~.‘~*~‘~~ Studies in ,Germany’” and, more recently, in the United States,‘“’ have shown a highly s&r&cant preservation of platelet nmnbers and function in stored blood when aprotinin was added to the system The exact mechanism of the protectionof stored ,plat&ts is not clear. A major determinant of cell survival in the circulation is thought to be related to the,surface sialic acid content. In platelets, this is found mainly in the GpIb receptor.“’ The GpIb receptor can be hydrolyzed and degraded ‘by plasmin in vitro.iO’ However, separate studies have shown that pro,tease irrhibitors such as leupeptin and aprotmin, despite being potent inhibitors of plasmin, ,did not prevent the loss of ,GpIb in stored blood.‘” There are three points on the effects (of :aprotinin on platelets, which have been activated by foreign surface contact within the extracorporeal system. The most important point concerns the skin bleeding time post-bypass. It has been shown that aprotinin in .the present dose regimen is able to prevent the increased bleeding time normally observed in patients after .a period of CPB.n~W This implies that aprotinin is acting to improve ‘the ,normal plate’let to microvascular reactions. Current understanding is that the platelet adheres to the endothelium,and subendothelium,by way of a bridge of multimeric von Willebrand factor attached to the platelet GpIb receptor. There is evidence to suggest that aprotinin administration is associated with a preservation of this receptor ‘during extracorporeal circulation using a .hollow-fiber type of membrane oxygenator.“4 The mechanism of this preservation is thought to be the aprotinin inhibition of the plasmin that subsequently digests the GpIb receptor.“’ There is also preliminary evidence to show that during bypass the aggregation response to ristocetin, which acts via GpIb, is maintained at or near normal in ,aprotinin-treated patients, but decreases by approximately ,40% in nontreated patients.“’ The relevance of this observation to the mechanism of postoperative bleeding is not clear. In one report, the loss of the aggaegatory response to ristocetin was related to the degree of postoperative bleeding.“” However, the patients in this study with .the greatest reduction in response, and thus the most likelihood of bleeding, also had abnormally low platelet counts. In a second study, Laze&y et al were able to significantly improve the reduced,platelet response to ristocetin with desmopressin (DDAVP).“’ Nevertheless, there was no effects on postoperative blood loss with this therapy. These data related to the preserving effects of aprotinin on the GpIb receptor have also been brought into question for a number of technical reasons. First, they were performed on platelets that had been washed and/or filtered before study; therefore, the observed changes may have been artifactually produced during the preparation of the samples. Second, donor blood was occasionally added to the extracorporeal system during bypass in these studies. Third, recent data have shawn thatthe inability to detect antibody binding sites on the platelet surface to the Gp Ib
receptor does not necessarily mean that this glycopmtein has been degraded and lost. There is evidence to show that the GpIb receptor can be internalized following stimulation with plasmin.“‘* Redistribution of intrapiatelet stores of GpIb to the platelet surface”’ has been suggested as a potential mechanism to explain the rapid return of the bleeding tie to normal after a period of extracorporeal support. Other factors 3 IU/mL) and, therefore, anticoagulation, the ACT probably needs to be maintained at >750 seconds when aprotinin therapy is being administered. Using this approach in 17 patients having repeat cardiac surgery, it was not necessary to administer extra protamine nor was the efficacy of aprotinin lost by maintaining this prolonged ACT.lm Other Possible Unwanted Effects Effects on Renal Function
As aprotinin is selectively taken up by renal tissues, there is a possibility that kidney function might be affected using the high doses currently recommended. In the original studies, it was demonstrated that the urine output was increased in aprotinin-treated patients.28 This effect was seen both during bypass and also in the immediate postbypass period (Fig 18). The increased urine output was associated with an increase in the excretion of sodium ions. A further study focused on investigating the effects of aprotinin on renal function, and confirmed that there was a greater urine output with aprotinin associated with an increase in sodium excretion.67 In this randomized doubleblind study in 60 patients reported by Fraedrich et al, there was an increase in the excretion of -certain proteins in the aprotinin-treated patients. In particular, there was a significantly greater excretion of a-1-microglobulin and aminopep-
VIIa
0 IIa
Fig 17. A simplified representation of the factors involved in the intrinsic and extrinsic coagulation cascades. The activated factors shown enclosed by circles can be partially or completely inhibited by the heparin-antithrombin Ill complex. Activated factors shown within the box can be partially inhibited by aprotinin.
n
Fig 18. Data (mean 2 SEM) for urine output during and in the first 6 hours after surgery for myocardial revasculariration. Results are from patients in a randomized placebo-controlfed studp and show that the urine output was significantly greater (P i 0.0s) in the aprotinin-treated patients during and immediately after surgery,
94
DAVID ROYSTON
tidase in the aprotinin-treated patients. This effect was transient and returned to normal in the early postoperative period. Despite these biochemical indices, which imply tubular overload and dysfunction with aprotinin therapy, there were no effects on creatinine clearance or plasma creatinine concentrations between the two groups. In the UK studies of patients at high risk of bleeding, there was also no evidence of deleterious effects on plasma creatinine or renal function.’ This is in contrast to reports of increased creatinine concentrations in patients having reoperations who had major bleeding problems and thus required large volumes of blood product support.“” It has also been shown that aprotinin therapy is effective and safe when given to patients with established renal dysfunction or failure.ti,4y In these studies, in both the control and treated patients, there was a postoperative increase in the plasma creatinine concentration. This increase tended to reach a peak on the third or fourth postoperative day before returning to near-preoperative values by the seventh to tenth postoperative day. The data for the perioperative creatinine concentrations in the five aprotinin-treated patients are shown in Fig 19. Vascular Occlusion Risk WithAprotinin Therapy (Especially the Patency of the Conduit Following CABG) The issue that has been raised many times, especially by surgeons, is whether a drug can prevent bleeding without an increased risk of thrombotic occlusions, especially in the saphenous vein used for CABG. There are numerous anecdotal stories of graft occlusion or perioperative myocardial infarction in patients who have received aprotinin therapy. However, based on current evidence, aprotinin therapy does not increase the risk of graft occlusions and thrombotic episodes but may reduce them. This drug has been
*1
-2
2
6
-
Ptl
-
Ft2
-
Pt3
-
Pt4
10
l4
Time in days Fig 19. Individual data for postoperative creatinine concentrations from five patients with established renal failure who had cardiac surgery with aprotinin therapy. The data show that renal function was not further compromised by the use of aprotinin. Patient 1 died on the third postoperative day from an acute myocardial infarction.
used routinely for all patients in certain centers in Germany for the past few years. Aprotinin therapy in the currently recommended high dose has been given to more than 10,000 patients having CABG. Therefore, it would seem logical to assume that if there had been any real impact on graft patency, this issue would have been more widely debated than seems the case from the anecdotal stories. Firmer evidence is found in the data from five randomized placebo-controlled studies conducted in Europe.3’,“’ In these studies, the incidence of complications related to the cardiovascular system was no different between the two groups. Eleven of 176 patients allocated to the placebo group and 14 of 176 patients in the group receiving aprotinin therapy had adverse cardiovascular events. At later follow-up, there were no differences between the groups studied in terms of late mortality or the recurrence of angina associated with the need for nitrate therapy.“’ Other data related to occlusive events in the central nervous system have been outlined previously.” The reported incidence of postoperative neurological sequellac related to thrombotic episodes ( ~0.5%) was far lower in the patients who received aprotinin therapy than that predicted from previous reports.“.” Similarly, Dietrich et al14’ reported that 6% of 152 patients given aprotinin therapy had transient neurological deficits postoperatively. This was approximately one-half of the 11% incidence of transient deficits in 317 patients who had operations without aprotinin therapy. There are a number of factors known to affect patency rates, such as surgical skill and judgment, the size of the vessel to be grafted, the distal vessel run off from the anastomoses. and the need for an endarterectomy. One further confounding issue to determine if a drug has an adverse effect on patency is that the graft patency rates arc only measured at the end of the first postoperative week. Therefore, it is unknown precisely when the grafts occlude. This l-week patency rate can be improved by the use of agents that act as antiplatelet + antithrombotic drugs.“’ More commonly, antiplatelet drugs are given and typically reduce the failure rate from approximately 10% to 2% to 4%. The initiation of high shear rate graft occlusion is thought to be by a platelet-dependent process. As discussed previously, the role of the platelet is to plug the holes in the microvasculature. This is achieved by the process of adherence, aggregation, and granule release. The activated platelet can then initiate clot formation. Currently, there are no published data to suggest that aprotinin will promote adhesion, aggregation, or granule release from platelets. On the contrary, there is evidence to show that aprotinin administration leads to an inhibition of the adhesion of platelets to glass beads.“13.“‘4 Aprotinin will also inhibit the adhesion of platelets to thrombin-stimulated human endothelium.14’ Other evidence shows that aprotinin acts in the same way as aspirin to inhibit aggregation and the release of thromboxane. On a more speculative note, there is currently interest in the development of a group of compounds from viper venoms that are able to protect the Gp IIb/IIIa complex.“”
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
These agents are being studied as a means of preventing platelet activation and thereby preventing reocclusion of coronary vessels after successful angioplasty. If aprotinin is shown to protect the Gp IIb/IIIa complex from attack by elastase or free radicals, then it would be acting in a similar way and should protect graft patency. Aprotinin therapy would seem to protect against platelet-dependent processes leading to graft occlusions. As discussed previously in the section dealing with interactions with heparin, aprotinin is well recognized as an inhibitor of certain factors of the coagulation cascade. This inhibition is easily shown by the increase in the activated partial thromboplastin time (APTT). The increased duration of the APTI is typically approximately twice control values perioperatively with the currently recommended is similar to that dose regimen.37,67 This prolongation achieved when low-dose heparin therapy is used to prevent thrombus formation during such procedures as dialysis or in patients with critical ischemia. Inhibition of the intrinsic clotting process will reduce the likelihood of thrombus generation initiated by contact of blood with negatively charged surfaces, such as collagen and similar subendothelial matrix substances. However, the spectrum of inhibitory activity does not include factor Xa or thrombin with the current regimen. If factor Xa is generated by a factor VII-dependent process, then this process will not be inhibited by aprotinin. The modified aprotinin with an arginine residue at the active site at position 15 (“Arg aprotinin) has considerably more anti-Xa activity and also more potent antikallikrein activity than native aprotinin. The spectrum of inhibition of “Arg aprotinin is more akin to other serine proteases, which are true anticoagulants,‘47 but are also reported to prevent bleeding associated with the extracorporeal system used for dialysis.‘4K The only relevant problem with aprotinin related to thrombus formation would be if there was an activation of thrombin formation via a factor VIIa, and, therefore, tissue factor-dependent process, or if thrombin in the form of fibrin glue was inadvertently added to the system. Any intravascular thrombus formed in this way will be less likely to undergo lysis in the early postoperative period because of the antifibrinolytic action of aprotinin. Currently there are conflicting data whether human saphenous vein endothelium can express and secrete tissue factor. The most recent evidence suggests that this endothelium does not have this capability.‘4Y Therefore, in this case it is difficult to envision a scenario in which aprotinin would actively promote graft thrombosis. The data suggest that aprotinin has no actions to increase platelet activity or to be a prothrombotic agent. Indeed, the evidence is to the contrary that aprotinin is more likely to reduce thrombosis of vessels and especially veins. Indeed, because of its antiplatelet action, aprotinin was used as a prophylaxis to prevent deep venous thrombosis in patients having hip surgery.“‘,‘04 There are a number of studies currently underway that are specifically aimed at addressing the issue of the effects
95
of aprotinin therapy on graft patency. One potential problem with interpreting the data from these studies is that graft patency is being assessed by minimally invasive imaging techniques. In the UK the imaging technique uses magnetic resonance; in the USA, ultrafast tine-computed tomography. Therefore, the data collected may not be directly comparable to the data obtained by catheter studies. Nonetheless, the preliminary report of the UK data shows no effects of aprotinin in patients having primary CABG at 7 to 8 days postoperatively.‘5” The results from the studies in North America include patients having primary and second-time operations for myocardial revascularization, and preliminary results from these studies should be available by the early part of 1992.
Allergic Phenomena Because aprotinin is a polybasic polypeptide that is derived from bovine organs, there is always the possibility of an adverse reaction to the agent. Anaphylactoid reactions are not mediated by the immune system and are thought to be caused by the direct action of the agent on the effector system. Infusions of aprotinin in patients in large doses were shown not to release histamine, and possibly to prevent its release.15’ Similarly, in this regard aprotinin does not induce complement activation. The effects of aprotinin on the rate of complement activation that occurs during the period of extracorporeal support are not clear. There have been reports to show either a reduction in generation of C3a,6’ no effect on this variable;’ or an increase in C3a concentrationZ8 in patients receiving aprotinin therapy. True anaphylactic reactions require previous exposure to the drug and the generation of a specific IgE immunoglobulin. It is well known that approximately 40% of patients will develop some antibody of the IgG and IgM class to drugs given around the time of surgery,‘52 and in this regard aprotinin is no exception.” However, it is pertinent to emphasize two aspects of this antibody response. First, none of the drug assays using radioimmune assay or enzyme-linked immunoabsorbance assays are possible without such an antibody being formed. Second, the relevance of IgG or IgM antibodies to adverse reactions with a subsequent drug exposure remains to be proven. In studies of multiple administrations of aprotinin over a prolonged period to patients with pancreatitis, the incidence of adverse reactions is reported to be 1% or less.ls3 In the context of patients receiving aprotinin therapy on more than one occasion for cardiac surgery, this figure is considerably less than the likelihood of reactions to radiographic contrast materials. Despite these theoretical reasons to suggest that aprotinin therapy can be given on more than one occasion, there might still be some risk of a serious allergic reaction. In patients known to have had previous aprotinin therapy, the author performs a prick test before infusion of aprotinin. In addition, if the recommended dosing regimen is followed, the original loading dose should be given over a 20 to 30 minute period. With this slow infusion rate, the first 1 mL or 2 mL of solution infused act as a “test dose” of the agent.
96
DAVID ROYSTON
Before this infusion, the patient has intravenous access with a wide-bore cannula, and intraarterial pressure monitoring is established and monitored closely over the first few minutes of the infusion to ensure that any adverse events are rapidly recognized. The author has given aprotinin to 6 patients known to have had the same therapy in the preceding 3 to 6 months with no ill effects. CONCLUSION
It is true that until 1987 aprotinin was a drug looking for a disease. The discovery of the profound hemostatic properties of this compound have given it a new lease on life and have led to a resurgence of research interest into the use of serine protease inhibitors in clinical practice. Although the
efficacy of the drug, when given in sufficient dose, is now without question, there are still a large number of unanswered questions; the principal one is what the actual role of aprotinin is in nature, and will this lead to the discovery of the mechanism of its action? This, in turn, will lead to the production of more specific and potent compounds. In the meantime, there are a number of important issues to address related to the current drug. In particular, the dose and timing of administration of aprotinin therapy arc still controversial. The effects on contact systems lead to the need to find ways to more accurately control anticoagulation during a period of extracorporeal circulation. There is also a need to investigate more thoroughly aspects of safety. such as the issue of graft patency and the multiple use of the drug in the same patient.
REFERENCES
1. Royston D: The serine antiprotease
aprotinin (Trasylol): A bleeding. Blood Coag
novel approach to reducing postoperative Fibrin01 155-69, 1990 2. Royston D, Bidstrup BP, Taylor KM, Sapsford RN: Effect of aprotinin on the need for blood transfusion after repeat open heart surgery. Lancet 2:1289-1291,1987 3. Fritz H, Wunderer G: Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs. Arzneimittelforsch 33:479-494, 1983 4. Kraut E, Frey EK, Werle E: uber die lnaktivierung des Kallikreins. Hoppe-Seyler’s Zeitschrift fiir Physiologische Chemie 192:1-21, 1930 5. Kunitz M, Northrop JH: Isolation from beef pancreas of crystalline trypsinogen, trypsin, atrypsin inhibitor, and an inhibitor trypsin compound. J Gen Physiol 19:991-1007, 1936 6. Laskowski M, Kato I: Protein inhibitors of proteinases. Ann Rev Biochem 49:593-626,198O 7. Fritz H, Kruck H, Riisse J, et al: Immunofluorescent studies indicate that the basic trypsin-Kallikrein inhibitor of bovineorgans (Trasylol) originates from mast cells. Hoppe-Seyler’s Z Physiol Chem 360:437-444,1979 8. Diet1 T, Tschesche H: Trypsin-Kallikrein isoenzyme K (type Kunitz) from snails (Helix pomatia): Purification and characterization. Eur J Biochem 58:453-460,1975 9. Wunderer G, Beress L, Machleidt W, et al: Broad specifiity inhibitors from sea anemones. Methods Enzymol45:881-888, 1976 10. Fioretti E, Angeletti M, Citro G, et al: Kunitz type inhibitors in human serum. Identification and characterization. J Biol Chem 262:3586-3589, 1987 11. Fioretti E, Binotti I, Barra D, et al: Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs. Eur J Biochem 130:13-18,1983 12. Phillipe E: Calculations and hypothetical considerations on the inhibition of plasmin and plasma kallikrein by Trasylol, in Davidson JF, Rowan RM, Samoma MM, Desnoyers PC, (eds): Progress in Chemical Fibrinoiysis and Thrombolysis, (vol 3). New York, NY, Raven, 1978, pp 291-298 13. Markwardt F: Naturally occurring inhibitors of fibrinolysis. Handbook Exp Pharmacol46:487-509,1977 14. Taby 0, Chabbat J, Steinbuch M: Inhibition of activated protein C by aprotinin and the use of the insolubilized inhibitor for its purification. Thrombosis Res 59:27-35,199O 15. Espafia F, Estelles A, Griffin JH, et al: Aprotinin (Trasylol) is a competitive inhibitor of activated protein C. Thrombosis Res 56:751-756,1989 16. Lottenberg G, Sjak-Shie N, Fazleabas AT, et al: Aprotinin
inhibits urokinase but not tissue-Type plasminogen activator. Thrombosis Res 49:549-556, 1988 17. Puri RN, Zhou FX, Bradford H, et al: Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain. Arch Biochem Biophys 271:356-358, 1989 18. Murphy WG, Moore JC, Kelton JG: Calcium-dependent cysteine protease activity in the sera of patients with thrombotic thrombocytopenic purpura. Blood 70:1683-1687,1987 19. Habermann E, Arnts D, Just M, et al: Das Verhalten des Trasylol im Organismus als Model1 fuer die Pharmakokinetik basischer Polypeptide. Med Welt 24:1163-l 167, 1973 20. Fischer JH: Trasylol-Effeckte an der Neire-Temperatur und Dosisabhaengigkeit, in Dudziak R, Kirchhoff PG, Reuter HD, Schumann F. (eds): Proteolyse und Proteinaseninhibition in de1 Herz und Gefasschirurgie. New York, NY, Schattaer-Verlag, 1985. pp 127-135 21. Royston D, Minty BD, Wallwork J, et al: The effects of surgery with cardiopulmonary bypass on alveolar-capillary barrier function in man. Ann Thorac Surg 40:133-142, 1985 22. Royston D, Braude S, Nolop KB, Hughes JMB: 113m lndium protein flux does not reflect degree or outcome in respiratoIy failure. Am Rev Respir Dis 139:A380, 1989 23. Royston D, Fleming JS, Desai JB, et al: Increased peroxide product generation associated with open heart surgery; evidence for free radical generation. J Thorac Cardiovasc Surg 91:759-766. 1986 24. Braude S, Nolop KB, Fleming JB, et al: Increased pulmonary transvascular protein flux after canine cardiopulmonary bypass. Association with lung neutrophil sequestration and tissue peroxidation. Am Rev Respir Dis 134:867-872, 1986 25. Kirklin JK, Westaby S, Blackstone EH, et al: Complement and the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 86:845-852, 1983 26. Clasen C, Jochum M, Mueller-Ester1 W: Feasibility study of very high dose aprotinin in polytrauma patients, in Schlag G, Red1 H (eds): First Vienna Shock Forum. Pathophysiological Role of Mediators and Mediator Inhibitors in Shock. New York, NY, Liss, 1987, pp 175-183 27. van Oeveren W, Jansen NJ, Bidstrup BP, et al: Effects of aprotinin on haemostatic mechanisms during cardiopulmonary bypass. Ann Thorac Surg 44:640-645,1987 28. Bidstrup BP, Royston D, Taylor KM, Sapsford RN: Reduction in blood loss and blood use after cardiopulmonary bypass with high dose aprotinin (Trasylol). J Thorac Cardiovasc Surg 97:364372,1989 29. Bidstrup BP, Harrison J, Royston D, et al: Aprotinin therapy
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
in cardiac surgery: A report on use in 42 UK cardiac centres. Lancet 1991 (In press) 30. Goodnough LT, Johnston MF, Toy PTCY: The variability of transfusion practice in coronary artery bypass surgery. JAMA 265:86-90,199l 31. Russell GN, Peterson S, Harper SJ, Fox MA: Homologous blood use and conservation techniques in cardiac surgery in the United Kingdom. Br Med J 297:1390-1391,1988 32. Jones EL, Weintraub WS, Craver JM, et al: Coronary bypass surgery: Is the operation different today? J Thorac Cardiovasc Surg 101:108-115, 1991 33. Lytle BW, Loop FD, Cosgrove DM, et al: Coronary reoperations: Results and determinants of early and late survival. J Thorac Cardiovasc Surg 93:874-895,1987 34. Freidrich G, Weber C, Bernard C, et al: Reduction of blood transfusion requirement in open heart surgery by administration of high dose aprotinin-Preliminary results. Thorac Cardiovasc Surgeon 37:89-91,1989 35. Dietrich W, Barankay A, Dilthey G, et al: Reduction of homologous blood requirements in cardiac surgery by intraoperative aprotinin application: Clinical experience in 152 cardiac surgical patients. Thorac Cardiovasc Surg 37:29-98,1989 36. Alajmo F, Calamai G, Perna AM, et al: High-dose aprotinin: Hemostatic effects in open-heart operations. Ann Thorac Surg 48:536-539, 1989 37. Dietrich W, Spannagl M, Jochum M, et al: Influence of high-dose aprotinin treatment on blood loss and coagulation patterns in patients undergoing myocardial revascularization. Anesthesiology 73:1119-1126, 1990 38. Dietrich W, Hahnel C, Richter JA: Routine application of high-dose aprotinin in open heart surgery-A study in 1,784 patients. Anesthesiology 73:A146, 1990 39. Royston D: Aprotinin in open heart surgery; Background and results in patients having aortocoronary bypass grafts. Perfusion 5:63-72,199O (suppl) 40. Royston D: Clinical effectiveness of aprotinin in heart surgery, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 208220 41. Von der Emde J, Mahmoud FO, Esperer HD: Retransfusion of Postoperative Drainage Blood, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 129-132 42. Chesbro JH, Clements IP, Fuster V, et al: A plateletinhibitory drug trial in coronary artery bypass operations: benefits of perioperative dipyridamole and aspirin therapy on early postoperative vein-graft patency. N Engl J Med 307:73-78,1982 43. Ferraris VA, Ferraris SP, Lough FC, Berry W: Preoperative aspirin ingestion increases operative blood loss after coronary artery bypass grafting. Ann Thorac Surg 4.5:71-74, 1988 44. Sethi GK, Copeland JG, Goldman S, et al: Implications of preoperative administration of aspirin in patients undergoing coronary artery bypass grafting. J Am Co11 Cardiol1.5:15-20,199O 45. Bashein G, Nessly ML, Rice AL, et al: Preoperative aspirin therapy and reoperation for bleeding after coronary artery bypass surgery. Arch Int Med 151:89-93, 1991 46. Ferraris VA, Gildengorn V: Predictors of excessive blood use after coronary artery bypass grafting. J Thorac Cardiovasc Surg 98:492-497, 1989 47. Gluszko PR, Maring JK, Edmunds LH: Invited letter concerning bleeding time test. J Thorac Cardiovasc Surg 101:173-174, 1991 48. Royston D, Bidstrup BP, Taylor KM, et al: Aprotinin reduces bleeding after open heart surgery in patients taking aspirin and those with renal failure. Anesthesiology 71:A6, 1989
97
49. Royston D, Bidstrup BP, Taylor KM, et al: Reduction of bleeding after open heart surgery with aprotinin (Trasylol): Beneficial effects in patients taking aspirin and in those with renal failure, in Birnbaum DE, Hoffmeister HE (eds): Blood Saving in Open Heart Surgery. New York, NY, Shattauer, 1990, pp 66-75 50. Bidstrup BP, Royston D, McGuiness C, et al: Aprotinin in aspirin treated patients. Perfusion 5:77-81, 1990 51. Tabuchi N, van Oeveren W, Eijsman L, et al: Preserved hemostasis during the combined use of aprotinin and aspirin in CABG operations, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 245-251 52. Gavaghan TP, Gebski V, Baron DW: Immediate postoperative aspirin improves vein graft patency early and late after coronary artery bypass graft surgery. Circulation 83:1526-1533, 1991 53. Popov-Cenic S, Murday H, Kirchoff PG, et al: Anlage und zusammenfassendes Ergebnis einer klinischen Doppelblindstudie bei aorto-koronaren Bypass-Operationen, in Dudziak R, Kirchhoff PG, Reuter HD, Schumann F, (eds): Proteolyse und ProteinasenInhibition in der Herz und Gefasschirurgie. New York, NY, Schattauer, 1985, pp 171-186 54. Murday H, Orellano L, Fenyes J, et al: Sind postoperative Blutungeb in der Herzchirurgie durch behandlung mit Aprotinin bzw. Cl inhibitor vermeidbar? Thorac Cardiovasc Surg 44:88, 1984 55. Elliot MJ, Allen A: Aprotinin in paediatric cardiac surgery. Perfusion 5:73-76,199O (suppl) 56. Dawkins KD, Jamieson SW, Hunt SA, et al: Long term results, haemodynamics, and complications after combined heartlung transplantation. Circulation 71:919-929,1985 57. Novick RJ, Menkis FN, Reid KR, et al: Reduction in bleeding after heart-lung transplantation: the importance of posterior mediastinal haemostasis. Chest 98:1383-1387, 1990 58. Brier M, Royston D, Bidstrup BP, Aronoff G: Kinetics of aprotinin during open heart surgery. J Pharmacol Therap 47:183, 1990 59. Struck E: Blood saving strategies in cardiac surgery, in Birnbaum DE, Hoffmeister HE (eds): Blood Saving in Open Heart Surgery. New York, NY, Schattauer, 1990, pp 16-28 60. Angelini GD, Cooper GJ, Lamarra M, Bryan AJ: Unorthodox use of aprotinin to control life-threatening bleeding after cardiopulmonary bypass. Lancet 1:799-800, 1990 61. Bidstrup BP, Royston Sapsford RN, Taylor KM: Effect of aprotinin on need for blood transfusions in patients with endocarditis having open heart surgery. Lancet 1:366-367, 1988 62. Koestering H, Kirchoff PG, Voelker P, et al: Untersuchungen der Blutgerinnungsveraenderungen wahrend und nach Operationen mit Hilfe der Herz-Lungen - Maschine. Thoraxchirurge 21:534-543, 1978 63. Elliot MJ: Perfusion for pediatric open heart surgery. Sem Thorac Cardiovasc Surg 2:332-340, 1990 64. van Oeveren W, Harden MP, Roozendaal KJ, et al: Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovas Surg 99:788-797,199O 65. Hannekum A, Reuter HD, Dalichau H, et al: Anlage und zussammenfassendes Ergebnis einer Klinischen Doppelblindstudie bei Operationen am offenen HerzenEinfluss von Aprotinin auf Thrombozytensahl und -funktion, in Dudziak R, Kirchhoff PG, Reuter HD, Schumann F (eds): Proteolyse und Proteinaseninhibition in der Herz - und Gefasschirurgie. New York, NY, Schattauer, 1985, pp 222-233 66. Scott D, Au J: The Edinburgh experience-Low dose Trasylol, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag. 1991, pp 263-265
98
67. Fraedrich G, Neukamm K, Schneider T, et al: Safety and risk/benefit assessment of aprotinin in primary CABG, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 221-231 68. Blauhut B, Gross C, Necek S, et al: Effects of high-dose aprotinin on blood loss, platelet function, fibrinolysis, complement, and renal function after cardiopulmonary bypass. J Thorac Cardiovas Surg 101:958-967,1991 69. Roth GJ: Developing relations; Arterial platelet adhesion, Glycoprotein Ib, and leucine rich glycoproteins. Blood 77:5-19, 1991 70. Hennessy VL, Hicks RE, Niewiarowski S, et al: Function of human platelets during extracorporeal circulation. Am J Physiol 232:H622-H628,1977 71. Glusko P, Rucinski B, Musial J, et al: Fibrinogen receptors in platelet adhesion to surfaces of extracorporeal circuit. Am J Physiol252:H615-H621,1987 72. Salzman EW: Blood platelets and extracorporeal circulation. Transfusion 3:274-277,1963 73. McKena R, Bachmann F, Whittacker BB, et al: The hemostatic defect after open heart surgery. II. Frequency of abnormal platelet functions during and after extracorporeal circulation. J Thorac Cardiovasc Surg 70:298-308, 1975 74. Cella G, Vittadello 0, Galluci, et al: The release of platelet factor 4 and beta-thromboglobulin during extracorporeal circulation for open heart surgery. Eur J Clin Invest 11:165-169,198l 75. Mezzano D, Aranda E, Urzua, et al: Changes in platelet b thromboglobulin, fibrinogen, albumin, 5-hydroxyttyptamine ATP and ADP during and after surgery with extracorporeal circulation in man. Am J Hematol22:133-142, 1986 76. Watkins WD, Peterson MB, Kong DL, et al: Thromboxane and prostacyclin changes during cardiopulmonary bypass with pulsatile perfusion. J Thorac Cardiovasc Surg 84:250-256, 1982 77. Ditter H, Heinrich D, Matthias FR, et al: Effects of prostacyclin during cardiopulmonary bypass in man on plasma levels of beta-thromboglobulin, platelet factor 4, thromboxane B2, 6-keto-prostaglandin Fl alpha and heparin. Thromb Res 32:393408,1983 78. Harker LA, Malpass TW, Branson HE, et al: Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass; aquired transient platelet defect associated with selective alpha granule release. Blood 56:824-834,198O 79. Musial J, Niewiarowski S, Hershock, et al: Loss of fibrinogen receptors from the platelet surface during stimulated extracorporeal circulation. J Lab Clin Med 105:514-526,1985 80. Zilla P, Faso1 R, Groscurth P, et al: Blood platelets in cardiopulmonary bypass operations. J Thorac Cardiovasc Surg 97:379-388, 1989 81. George JN, Pickett EB, Saucerman S, et al: Platelet surface glycoproteins; Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 78:340-348,1986 82. George JN, Nurden AT, Phillips DR: Molecular defects in interactions of platelets with the vessel wall. N Engl J Med 311:1084-1098,1984 83. Levine SP, Krentz LS: Development of a radioimmunoassay for human platelet factor 4. Thromb Res 11:673-686,1977 84. Abrams CS, Ellison N, Budzynski A, Shattil SJ: Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 75:128-138, 1987 85. Schulman S, Johnsson H: Heparin, DDAVP and the Bleeding Time. Thomb Haem 65:242-244,199l 86. Gimple LW, Gold HK, Leinbach RC, et al: Correlation between template bleeding times and spontaneous bleeding during
DAVID ROYSTON
treatment of acute myocardial infarction with recombinant tissue type plasminogen activator. Circulation 80:581-585,1989 87. Bick R: Hemostasis defects associated with cardiac surgery. prosthetic devices, and other extracorporeal cicuits. Semin Thromb Hemost 11:249-280,1985 88. Mammen E, Kocts MH, Washington DC, et al: Hemostasis changes during cardiopulmonary bypass surgery. Semin Thromb Hemost 11:281-292,1985 89. Stibbe J, Kluft C, Brommer EJP, et al: Enhanced fibrinolytic activity during cardiopulmonary bypass in open heart surgery in man is caused by extrinsic (tissue type) plasminogen activator. Eur J Clin Invest 14:375-382,1984 90. Tanaka K, Takao M, Yada I, et al: Alterations in coagulation and fibrinolysis associated with cardiopulmonary bypassnduring open heart surgery. J Cardiothorac Anesth 3:181-188,1989 91. Holloway DS, Summaria L, Sanesara J, et al: Decreased platelet numbers and function and increased fibrinolysis contribute to postoperative bleeding in cardiopulmonary bypass patients. Thromb Haemost 59:62-67,198s 92. Van Riper DF, Horrow JC, Osborne D: Is the thromboelastograph a clinically useful predictor of blood loss after bypass? Anesthesiology 73:3A, A1206, 1990 93. Gram J, Janetzko T. Jespersen J, et al: Enhanced etfectivr fibrinolysis following the neutralization of heparin in open heart surgery increases the risk of post-surgical bleeding. Thromb Haemost 63:241-245, 1990 94. Marengo-Rowe AJ, Levenson JE: Fibrinolysis: A frequent cause of bleeding, in Ellison N, Jobes DR (eds): Effective Hemostasis in Cardiac Surgery. Philadelphia. PA, Saunders, 1988, pp 41-55 95. Spiess BD, Tuman KJ, McCarthy RJ, et al: Thromboelastographic diagnosis of coagulopathies in patients undergoing cardiopulmonary bypass. Anesth Analg 68:53-72, 1989 96. Horrow J, Hlavacek J, Strong MD, et al: Prophylactic tranexamic acid decreases bleeding after cardiac operations. J Thorac Cardiovasc Surg 99:70-74, 1990 97. Van der Salm TJ, Ansell JE, O’Kike ON, et al: The role ot epsilon aminocaproic acid in reducing bleeding after cardiac operations; a double blind randiomized study. J Thorac Cardiovasc Surg 96:538-540,198s 98. Fish KJ, Sarnquist FH, van Steennis C, et al: A prospective, randomized study of the effects of prostacyclin on platelets and blood loss during coronary bypass operations. J Thorac Cardiovasc Surg 91:436-442,1986 99. Teoh KH, Cristakis GT. Weisel RD, et al: Dipyridamole preserved platelets and reduced blood loss after cardiopulmonary bypass. J Thorac Cardiovasc Surg 96:332-341, 1988 100. Harke H, Gennrich M: Aprotinin-ACD Blut. I. Experimentelle Untersuchungen uber den Einfluss von Aprotinin auf die plasmatische und thrombozytare. Gerinnun Anesthetist 29:266276,198O 101. Aoki N, Yoshida N: Inhibition of platelet aggregation by protease inhibitors. Possible involvement of proteases in platelet aggregation. Blood 52:1-12, 1978 102. Ketterl R, Haas S, Lechner F, et al: Wirkung von Aprotinin auf die Thrombozytenfunktion wahrendHtift-Totalendoprosthesenoperation. Med Welt 31:1239-1240, 1980 103. Ketterl R, Haas S, Heiss A. et al: Zur Wirkung des natiirlichen Proteinaseninhibitors Aprotinin auf die Plattchenfunktion beim alloarthroplastischen Huftgelenkersatz. Med Welt 33:480486,1982 104. Harke H, Steinen G, Rahman S, et al: Aprotinin ACD Blut II: Der Einfluss von Aprotinin auf die Freisetzung zellularere Medioraten un Enzyme im Konservenblut. Anaesthetist 31:165171,1982 105. Bode AP, Miller DT: The use of thrombin inhibitors and
99
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
aprotinin in the preservation of platelets stored for transfusion. J Lab Chn Med 113:753-758,1989 106. Okumura T, Jam&on GA: Platelet glycocalicin 1: Orientation of glyoproteins of the human platelet surface. J Biol Chem 251:5944-5949,1976 107. Adelman B, Michelson AD, Loscalzo J, et al: Plasmin effect on platelet glycoprotein lb-von Willebrand factor interactions. Blood 65:32-40, 1985 108. Bode AP, Knupp CL, Miller DT: Effect of platelet activation inhibitors on the loss of glycoprotein Ib during storage of platelet concentrates. J Lab Clin Med 115:669-679, 1990 109. Commin PL, Lu H, Soria C, et al: Mechanisms of bleeding induced by aprotinin during cardiopulmonary bypass: A controlled study. Anesthesiology 73:3A, A1205, 1990 110. Mohr R, Golan operations on platelets.
M, Martinowitz U, et al: Effect of cardiac J Thorac Cardiovas Surg 92:434-441, 1986
111. Lazenby WI), Russo I, Zadeh BJ, et al: Treatment with desmopressin acetate in routine coronary artery bypass surgery to improve postoperative haemostasis. Circulation 82:IV 413-419, 1990 (suppl5) 112. Cramer ER, Lu H, Caen JP, et al: Differential redistribution of platelet glycoproteins Ib and IIb/IIIa after plasmin stimulation. Blood 77~694-699, 1991 113. Michelson AD, Adelman B, Bernard MR, et al: Platelet storage results in a redistribution of glycoprotein Ib molecules; evidence for a large intraplatelet pool of glycoprotein IB. J Clin Invest 81:1734-1740, 1988 114. Colman RW, Scott CF, Schmier AU, et al: Initiation of blood coagulation at artificial surfaces. Ann NY Acad Sci 56:253267,1987 115. Miles gen interacts nisms. J Clin 116. Miles of the platelet
LSA, Ginsberg MA, White JG, Plow EF: Plasminowith human platelets through two distinct mechaInvest 77:2001-2009, 1986 LA, Plow EF: Binding and activation of plasminogen surface. J Biol Chem 260:4303-4311,1985
117. Parf S, Harker LA, Marzec UM, Levin EG: Demonstration of single chain urokinase type plasminogen activator on human platelet membrane. Blood 73:1421-1425,1989 118. Kluft C, Dooijewaard G, Emeis JJ: Role of the contactsystern in fibrinolysis. Semin Thromb Haemost 13:50-68, 1987 119. Kowalski E, Kopec M, Wegrzynowicz Z: Influence of fibrinogen degradation products (FDP) on platelet aggregation, adhesiveness and viscous metamorphosis. Thromb Diath Haemorrh 10:406-413, 1963 120. de Bono DP, Pringle S, Underwood I: Differential effects of aprotinin and tranexamic acid on cerebral bleeding and cutaneous bleeding time during rt-PA infusion. Thromb Res 61:159-163,199l 121. Kornecki E, Ehrlich YH, de Mars DD, Lenox RH: Exposure of fibrinogen receptors in human platelets by surface proteolysis with elastase. J Clin Invest 77:750-756, 1986 122. Royston D: Free radicals; formation, function and potential relevance in anaesthesia. Anaesthesia 43:315-320, 1988 123. Henson P: Mechanisms of release of constituents from rabbit platelets by antigen-antibody complexes: II. Interactions between platelets and neutrophils. J Immunol X:490-498, 1970 124. Clark RA, Klebanoff SJ: Neutrophil platelet interactions are mediated by myeloperoxidase or hydrogen peroxide. J Immunol 124:399-407, 1980 125. Wachtfogel YT, Kucich U, James HL, et al: Human plasma kallikrein releases neutrophil elastase during blood coagulation. J Clin Invest 72:1672-1677, 1983 126. Zimmerli W, Huber I, Bouma BN, Lammle B: Purified human plasma kallikrein does not stimulate but primes neutrophils for superoxide production. Thromb Haemost 62:1121-1125,1989
127. Francis JL, Lord RAM, Roath OS: The effect of aprotinin on haemostasis and white cell function in aortic surgery. Thromb Haemost (In press) 128. Royston D, Bidstrup BP, Fleming JS: Platelet preservation reduces production of lipid peroxidation during cardiopulmonary bypass in humans. Am Rev Resp Dis 135:A13,1987 129. Ruggerio M, Lepetina EG: Protease inhibitors synergistically prevent activation Proc Nat1 Acad Sci USA 83:3456-3459, 1986
and cyclooxygenase of human platelets.
130. Ruggerio M, Lepetina EG: Antipain and leupeptin in combination with aspirin or indomethacin synergistically inhibit platelet activation by thrombin and trypsin. Adv Prost Thrombox Leukotr Res 17A:563-568,1987 131. Royston D: Effects of aprotinin lysis associated with open heart surgery. (In press)
on the whole blood clot Blood Coag Fibrin01 1991
132. Spiess BD, Tuman KJ, McCarthy RJ, et al: Thromboelastography as an indicator of post-cardiopulmonary bypass coagulopathies. J Clin Monit 3:25-30, 1987 133. Kang Y, Lewis JM, Navalgund A, et al: Epsilon amino caproic acid for the treatment of fibrinolysis during liver transplantation. Anesthesiology 66:766-773, 1987 134. Clozel JP, Banken L, Roux S: Aprotinin: An antidote for recombinant tissue-type plasminogen activator (rt-PA) active in vivo. J Am Co11 Carcliol16:507-510, 1990 135. Wiman B: On the streptokinase complex with Thromb Res 17:143-152,198O
reaction aprotinin
of plasmin or plasminor alpha 2 antiplasmin.
136. Gverlack A, Stumpe KO, Kuehnert M, et al: Evidence for participation of kinins in the antihypertensive effect of converting enzyme inhibition. Klin Wochenschr 59:69-74, 1981 137. Kurusz M, Schneide B, Brown BP, et al: Filtration during open heart surgery: Devices, techniques, opinions, and complications. Proc Am Acad Cardiovasc Perf 4:123-129, 1983 138. Royston D, Bidstrup BP, Sapsford RN, Taylor KM: Reduced blood loss after open heart surgery with aprotinin is associated with an increase in the activated clotting time (ACT). J Cardiothorac Anesth 3:80,1989 139. de Smet AAEA, Increased anticoagulation tinin. J Thorac Cardiovasc
Joen MCN, van Oeveren W, et al: during cardiopulmonary bypass by aproSurg 100:520-527, 1990
140. Royston D: A prolonged activated clotting time unreliable indicator of thrombus formation during aprotinin apy. J Thorac Cardiovasc Surg (In press) 141. Pratt CW, Church FC: Antithrombin: tion. Semin Hematol28:3-9,199l
Structure
is an ther-
and func-
142. Ross DN, Simpson JC: Aprotinin; Effect on redo surgery, in Freidel N, Hetzer R, Royston D, (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 260-262 143. Dietrich W, Barankay A, Niekau E, et al: High-dose aprotinin in cardiac surgery. Old drug-new aspects of homologous blood requirement, in Birnbaum DE, Hoffmeister HE (eds): Blood Saving in Open heart Surgery. New York, NY, Schattauer, 1990, pp 76-82 144. Henderson WG, Goldman S, Copeland JG, et al: Antiplatelet or anticoagulant therapy after coronary artery bypass surgery: A meta-analysis of clinical trials. Ann Int Med 111:743-750,1989 145. Royston BD, Royston platelet adhesion to thrombin Thromb Haemost (In press)
D, Pearson JD: Aprotinin inhibits stimulated human endothelial cells.
146. Gould RJ, Gan Z-R, Polokoff MA, et al: Disintegrins; A family of integrin inhibitory proteins from viper venoms. Faseb J (In press)
100
147. Ohno H, Kambayashi J, Chang SW, Kosaki G: Foy: [Ethyl-p (guanidinohexanyloxy) benzoate] methylsulfonate as a serine protease inhibitor. II In Vivo effect on coagulo-fibrinolytic system in comparison with heparin and aprotinin. Thromb Res 24:445-452,198l 148. Taenaka N, Shimada Y, Hirata T, et al: New approach to regional anticoagulation in haemodialysis. Crit Care Med 10:773175,1982 149. Solbereg S, 0sterud B, Larsen T, Sorlied D: Lack of ability to synthesize tissue factor by endothelial cells from intact saphenous vein. Blood Coag Fibrinol 1991 (In press)
DAVID ROYSTON
150. Bidstrup BP: Aprotinin therapy has no effects on early graft patency. Ldncet (In press) 151. Harke H, Rahman S: Clinical relevance of the membrane protective action of aprotinin on the intraoperative histamine liberation. Prog Clin Biol Res 308:959-963, 1989 152. Weiss ME, Adkinson NF, Hirshman CA: Evaluation of allergic drug reactions in the perioperative period. Anesthesiology 71:483-486, 1989 153. Freeman JG, Turner GA, Venables CW, et al: Serial use 01 aprotinin and incidence of allergic reactions. Curr Med Res Opin 8:559-561. 1983
High-Dose
Aprotinin
Therapy:
A Review
of the First Five Years’ Experience
David Royston, FFARCS “A Scientist must be like a child. If he sees a thing he must say that he sees it, whether he thought he was going to see it or not. Otherwise you only see what you are expecting (most scientists forget that). See first, think later, then test.” So Long, and thanks for ail the fish Douglas Adams
T
HE ROLES OF HEPARIN and protamine in manipulating anticoagulation are well known, but their actions were discovered and recognized by accident rather than by design. Heparin was shown to be a potent anticoagulant while investigators were looking for a procoagulant in ether extracts from various tissues including the liver. The activity of protamine to reverse the anticoagulant action of heparin was recognized during studies investigating whether protamine would prolong the duration of action of heparin. In the same way, serendipity, and not pure scientific reasoning, led to the use of the serine protease inhibitor aprotinin to substantially reduce operative and postoperative bleeding in a variety of operations, but specifically during cardiac and vascular surgery. Historically, a novel and relatively high-dose regimen for this agent was used in the pivotal early study conducted by Royston et al not to prevent bleeding, but in an attempt to inhibit cell activation, thereby preventing tissue injury that may occur during and after cardiac surgery.’ The original high dose used in the first study was further increased, and the dose regimen now recommended was subsequently administered to patients having reoperations.* It produced dramatic reductions in bleeding and the need for donor blood transfusions. Since the original observations published in 1987, there has been considerable interest and enthusiasm for the use of aprotinin in Europe, and now North America, to prevent bleeding associated with major operations, and particularly during cardiac surgery. The aims of this review are (1) to discuss briefly the chemistry of aprotinin and the background to the use of the dose regimen currently recommended; (2) to summarize some of the data showing the efficacy of the drug given in a variety of cardiac, vascular, and transplantation procedures. This section also includes a discussion of issues related to the dose regimen; (3) to review some of the more recent data concerning the problem of the potential mecha-
From the Department of Anesthesia, Harefield Hospital, Harefield, UK. Address reprint requests to David Royston, FFARCS, Department of Anaesthesia, Harefield Hospital, Harefield, Middlesex, UB9 6JH, United Kingdom. Copyright 0 I992 by W B. Saunders Company 1053~0770l9210601-0019$03.00/O 76
nism(s) of action of this agent; and (4) to discuss various safety issues that have been raised with this therapy. BACKGROUND
Chemistry Aprotinin is a basic (pKa 10) polypeptide comprised of 58 amino acid residues with a molecular weight of 6,512 D. The amino acid sequence, biochemical structure, and biophysical characteristics have been described and categorized.’ Aprotinin was independently discovered and isolated from bovine lymph nodes by Kraut et al in 1930,” who identified it as a kallikrein “inactivator,” and by Kunitz and Northrop in 1936,’ who defined it as a trypsin inhibitor in a preparation obtained from bovine pancreas. Aprotinin is a member of a family of serpins (serine protease inhibitors), which are found in all aspects of nature and, as the name implies, are able to inhibit a range of proteases that have serine residues at their active site. This inhibition is provided by inactivation of the active serine of the protease by the lysine residue at position 15 of the aprotinin molecule. Because aprotinin is a small, soluble, and stable polypcptide whose sequence and tertiary structure are well known, it has been a favorite molecule for investigations by protein chemists for many years.h There have been extensive nuclear magnetic resonance studies of the molecule and a great interest in the protein folding (related to the time course of formation of disulphide bridges). Despite these intensive investigations, the physiological role of aprotinin is unknown. Aprotinin appears to be localized in the mast cells in bovine tissue.’ A number of other low molecular weight serpins similar to aprotinin have been detected in a wide range of species including the snail’ and sea anemoneY and in human and bovine plasma.‘“.” Of interest is the fact that these homologous inhibitors are also found in a number of venoms from snakes.” The activity of aprotinin is expressed in various ways. Historically, kallikrein inactivator units (KIU) and trypsin inhibitory units (TIU) have been commonly used. These units rely on measurements of biological potency of aprotinin in fixed analytical systems. Over the years, the manufacturing and purification processes of this drug have improved, and it is now possible to discuss the amount of drug in terms of weight of protein (mg) or concentration of solution (FM). The conversion factors are that 1 mg of
Journalof Cardiothoracic
and
Vascular Anesthesia,Vol
6, No 1 (February),
1992: pp 76.100
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
protein is equivalent to 7,143 KIU or 100,000 KIU is 14 mg of protein. A l+M solution of aprotinin contains 46.5 KIU/mL. Because the majority of the data published before 1986 use the KIU as the unit of measurement, it has been used in this review. However, for some of the discussion on the pharmacological effects of this agent, the weight of substance (mg) or strength of solution (PM) has been used. Native aprotinin has its inhibitory effect on target serine protease by forming reversible stoichometric enzymeinhibitor complexes. This may be easy to read, but to most anesthesiologists, who have no doctorate in chemistry, it is impossible to translate into a clinical situation. In simplified terms, the stability of the enzyme-aprotinin complex is expressed as an equilibrium constant. In its most basic form, this constant can be thought of as a quantifier of the formation of a protease-aprotinin complex (PA) and its breakdown or dissociation from and into the protease (P) and aprotinin (A) components. The equation of this equilibrium constant K is similar to that of the law of mass action associated with acid-base status:
K = (P)
x (A)/(PA)
It can be noted that the smaller the K the greater the affinity of aprotinin for that protease (PA will increase and P will decrease). However, this equation and the resulting K cannot tell what concentrations of aprotinin are clinically necessary to provide a substantial inhibitory effect on a specific protease. For this to occur, it is necessary to know both the concentration of aprotinin and its target. In the presence of increasing A, there will be a relative increase in PA and, therefore, a decrease in P until eventually all of the P is complexed with A. In the case of certain proteases, this may require A levels to be so high that a toxic threshold for aprotinin would be exceeded. In addition, nature has added her own complications to the equation. There are many naturally occurring inhibitors of serine proteases circulating in the plasma. Therefore, any additional functional inhibitory effects of aprotinin will depend on both the concentration and K of the other inhibitors. In pure chemical systems (those without other plasma proteins), the concentration of aprotinin required to inhibit serine proteases that occur in nature such as trypsin, plasmin, or tissue and plasma kallikrein, varies for each of the enzymes with concentrations of approximately 50 KIU/mL (= 1 PM) required to inhibit plasmin and approximately 200 KIU/mL ( = 4 uM) to inhibit plasma kallikrein. These concentrations have been defined from theoretical calculations following experiments performed in pure chemical systems under laboratory conditions.‘.‘2”3 Therefore, it may be that such concentrations may be inadequate to inhibit the enzyme in question in vitro or, conversely, that in combination with naturally occurring inhibitors of these various serine proteases, these concentrations of aprotinin may be in excess of that required to fully inhibit these target enzymes in vivo. These complications confound the issue of defining a specific inhibitory concentration of aprotinin for a specific target protease in any patient. The fact is that the spectrum of inhibition by aprotinin is
77
growing as novel serine proteases are either “discovered” or their importance related to the hemostatic process is studied. In this regard, aprotinin has been shown to inhibit Activated Protein C (APC), a vital protease in feedback control of the coagulation cascade, at relatively high concentrations.14 However, the inhibitory effects of aprotinin on APC have been demonstrated at much lower concentrations in the presence of heparin.15 In contrast, the activator of plasminogen derived from endothelium (tissue-type plasminogen activator or t-PA) is also a serine protease, but is not inhibited by aprotinin in concentrations up to 500 l.~M.l~ Single-chain urokinase is inhibited, but at extremely high concentrations of aprotinin that are not likely to be found clinically. This is also the case for elastase and thrombin. Aprotinin also has no reported inhibitory actions on metallo-proteases and no discernible activity against calcium-dependent thio-proteases such as calpain.6,‘718 As a polypeptide, aprotinin is inactive when given orally and needs to be given intravenously. The half-life in the plasma is biphasic with an initial elimination half-life of approximately 1 hour. It follows that the compound has to be given by continuous infusion if the plasma concentration is to be maintained. Aprotinin is highly resistant to proteolysis and processes associated with moderate chemical degradation. One of the noteworthy aspects of the pharmacokinetics of aprotinin is the affinity of the drug for renal tissue, especially brush border” and proximal convoluted tubule. This may in part be caused by the neuraminic acid content of the brush border in the kidney together with the basic nature of the drug. After injection into rats, approximately 90% of the drug is deposited in the kidney within a few hours.” The drug is freely filtered by the glomeruli, but is not excreted in the urine as an active drug in any significant amount. The predilection of aprotinin to stay in the kidney has led to some concerns regarding the effects of the current dose regimen on renal function. Aprotinin is available for human use as a preservativefree solution of 10,000 KIU/mL in ampules of 50 mL from various manufacturers in Europe. In the studies performed, the agent used was Trasylol (Bayer AG, Leverkusen, Germany). Miles, Inc. (New Haven, CT) is a subsidiary of the Bayer AG company, and is currently investigating the use of this drug in North America. BACKGROUND
AND RESULTS OF PILOT STUDY
As outlined in the introduction, the dramatic effects of aprotinin on bleeding was a chance observation. The use of aprotinin during cardiac surgery was intended to prevent the damaging effects of extracorporeal circulation on certain tissues and organs, specifically the lungs and pulmonary circulation. In particular, an attempt was made to reduce cell activation during the period of extracorporeal circulation. In previous studies, it was shown that it was possible to demonstrate an increase in solute flux into and from the lungs of patients having cardiac surgery.Z’,Z2 In addition, there was evidence for increased oxygen-derived free radical activity, probably derived from neutrophils,*” and that there was a significant relationship between the degree of free radical activity produced from the pulmonary
78
DAVID ROYSTON
circulation and an index of protein leak into the lung.24 At a scientific meeting held in Luxembourg in May 1984, it was suggested that some of the damaging effects of cardiac surgery related to this cell activation could be reduced or abolished by the use of serine protease inhibitors. The rationale for this idea is shown diagramatically in Fig 1. The contact of blood with the foreign surface of the oxygenator stimulates a large number of “inflammatory cascades” that can act through humoral or cellular mechanisms, but that are ultimately controlled by amplification cascades of proteolytic enzymes; the vast majority of inflammatory cascades are serine proteases. The cumulative effect of this amplification is to produce a “whole-body inflammatory response.” This concept was developed by Kirklin et al whose focus of attention had been the role of complement activation in this process.” During the period of extracorporeal circulation, the only inhibition of these potentially deleterious cascades is the routine administration of heparin given to inhibit the intrinsic pathway and prevent blood clotting. Heparin achieves this effect by activating the naturally occurring serine protease inhibitor antithrombin III. The hypothesis originally tested was that the other limbs of complement, fibrinolysis, and kallikrein activation could also be inhibited
Foreign
Intrinsic system
(
by giving reasonable amounts of an appropriate antiprotease. Aprotinin was used for two reasons. First, it was known to have actions to inhibit kallikrein and plasmin at doses that were potentially possible to achieve clinically. Second, it was the only serine protease inhibitor available at that time in the UK that had a sufficiently low toxicity to allow its use in humans at the required concentrations. The concentration of aprotinin thought necessary to block the actions of kallikrein was approximately 4 uM (= 200 KIUimL of plasma), and plasmin was approximately 1 FM (~50 KIUlmL). Based on studies of the use of continuous infusions of aprotinin in multiple trauma patients, members of the biochemistry department in Munich, Germany formulated a dosage schedule for use in cardiac surgery aimed at achieving a concentration of 200 KIU/mL throughout extracorporeal circulation.‘(’ The dose regimen suggested was a 2 x 10”KIU (280 mg) loading dose given over a 20-minute period after induction of anesthesia, followed by a continuous infusion of 500,000 KIU (70 mg) per hour until the patient was returned to the intensive cart unit (ICU). To overcome the dilution effect of the 2 L of crystalloid prime in the oxygenator, a dose of 1 x IO” KIU (140 mg) was added to this prime volume. The study was designed as a pilot investigation; therefore, it was neither
negative charge
>
surface
Plasmin
t
(
Inflammation Post perfusion syndrome
ve tfments
1
Fibrinolysis
Thrombin
_
)
Fig 1. A schematic diagram showing the pathways of activation of white blood cells and platelets following activation at a foreign surface. HMWK, high molecular weight kininogen.
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
randomized nor blinded to the observers. All the patients had coronary artery bypass grafts (CABG) performed by one surgeon with the author as the anesthesiologist.27 At the time of surgery, it was obvious that despite any alterations in biochemical and hematological variables, the most striking effect of the aprotinin therapy was a reduction in bleeding that was, until then, a normal consequence of cardiopulmonary bypass (CPB). Some results of this study have been published:’ and have demonstrated a significant reduction in the blood lost in the postoperative period in patients given the trial drug. The reduction was from a mean of 674 mL in 11 patients who were designated as the control population to a mean of 357 mL in 11 patients who were given aprotinin treatment. Analysis of plasma samples for aprotinin concentrations showed that the target plasma concentration of approximately 200 KIU/mL was not achieved during the time of bypass,’ and that there was a greater than predicted decrease in the plasma aprotinin concentration shortly after the start of the bypass period. To overcome this, it was decided that the addition to the prime volume should be increased by a further 1 x lo6 KIU (140 mg) to give a final total of 2 x lo6 KIU (280 mg) of aprotinin added to the oxygenator prime. This is the dose regimen used in all the subsequent studies. Over the past 4 years, this regimen has been described as the Royston regimen, the London regimen, and, most recently, as the Hammersmith regimen. Apart from the author’s description of the regimen, while holding an honorary contract at the Hammersmith Hospital, the administration schedule itself owes nothing to the author personally nor the Hammersmith Hospital, and is more correctly attributed to the biochemists from Munich. Based on a large number of published and unpublished studies and extensive personal experience, it is believed that the dose regimen for the use of aprotinin can and will be altered to improve efficacy. The optimum dose regimen will depend on a number of factors related to the type of surgery and the patient being given the therapy. EFFICACY
OF APROTININ
THERAPY
The review of the studies of efficacy are divided into 6 broad categories: (1) reoperations through a previous sternotomy; (2) primary surgery for myocardial revascularization in patients who are or are not taking platelet-active agents; specifically aspirin; (3) patients having major vascular surgery; (4) patients with sepsis and endocarditis at the time of surgery; (5) pediatric patients with congenital heart disease; and (6) patients having cardiac or cardiothoracic transplantation. Blood Loss Reduction
Historically, the group of patients having Reoperutions. reoperations was the first in whom the dose regimen currently recommended was described,’ therefore, they will be discussed first. Patients having repeat surgery through a prior median sternotomy are known to be at greater risk for perioperative bleeding and, thereby, an increased requirement for donor blood transfusions when compared with
79
patients having primary operations. The original study of this group showed that the non-treated, control population of 11 patients had a blood loss of 1,509 r 388 mL (mean ? SD) and these patients all required blood transfusions. A total of 41 U of donor blood was given. In striking contrast, the 11 treated patients had a postoperative blood loss of 286 f 48 mL; only 4 of 11 patients received a total of 5 U of donor blood. This reduction in blood use was achieved without postoperative anemia. The venous hemoglobin in the treated patients on the seventh postoperative day was 11.9 ? 0.6 g/dL vs 12.1 ? 0.5 g/dL in the control population. Subsequent use of aprotinin in patients requiring repeat surgery has confirmed these results.” Aprotinin has been available for clinical use in the United Kingdom for compassionate use and, subsequently, was used in an open study in patients at increased risk of bleeding.29 The criteria for aprotinin use were that the patient should be at greater risk of bleeding because they had (1) repeat surgery, (2) endocarditis, or (3) a bleeding diathesis or coagulopathy. In the efficacy data from 451 patients reported, approximately 25% were having repeat CABG, 45% repeat valve replacement, and the remaining 30% were having more complex procedures such as replacement of infected values, double- or triple-valve replacements, replacement or re-replacement of one or both ventricular outflow tracts, or heart and heart-lung transplantation (HLT,). In these patients, the median blood loss in the first 24 hours, postoperatively, was 400 mL. Donor blood use was not strictly controlled. Furthermore, data for blood use were skewed by the inclusion of more complex operations such as reoperation root replacement and HLT,, which often have as their primary bleeding problem massive hemorrhage caused by disruptions of vascular and cardiac integrity rather than an abnormality of hemostatic function. As highlighted, transfusion policy can differ widely between centers, including those with a high commitment to blood conservation programs3 In the UK, there currently is great variability in transfusion practices and a less aggressive approach to blood conservation than is present in most North American and European countries. Despite these caveats in this open study, the median donor blood use was 2 U; 25% of patients received no blood transfusions. These results in high-risk patients are superior to the overall median blood use of 5 U per patient reported for UK cardiac surgery centers3’ Analysis of the safety data from the first 671 patients treated with aprotinin in this study has produced some interesting and intriguing observations, in particular, the reported incidence of events likely to be related to increased thrombosis, ie, perioperative infarction of the heart or neural tissue. Speculation has always existed that if hemostasis was improved by a drug, it must inevitably lead to an increased thrombosis risk. The inconsistencies of this concept are discussed later in this review. Of the patients in the UK open study, 216 had CABG (148 were reoperations), and there were 3 reports of acute postoperative graft occlusion. None of these were attributed to the administration of aprotinin by the clinician in charge of the case. In addition, there were four (1 hemorrhage and 3 thrombotic)
80
DAVID
ROYSTON
Table 1. Blood Loss in the First 24 Hours, Postoperatively* London
Placebo
347 + 103
716 ? 248
270 - 930
210 - 690
295
n = 37 Aprotinin
Amsterdam
Giessen
575 2 164
308 + 132 130 ~ 830 n = 40
n = 30
1,215
380
n = 38
Freiburg
500 - 3,200
n = 35
257 k 69 90
Munich 1,185 r 601 ” = 20
984 k 516 220 - 2,430 n = 38
507 + 206
548 2 290
488 k 297
220 -- 1,075
210
140 ~ 1,350
n = 35
1,400
n = 19
n = 38
Postoperative Donor Blood Transfusionst Placebo Aprotinin
203
155
91
240
179
33
32
40
63
47
*Data presented as mean 2 SD, range, and number of patients. tDonor units given (normalized to U/l00
patients [actual patient numbers are given in above section])
cases reported of neurological complications. This incidence of = 0.5% of neurological episodes in patients having reoperations is remarkably low when compared with the 2.5% to 6% reported in reoperation patients in a recent report?’ It is unlikely that this was achieved by the under-reporting of adverse events as the wound infection rates reported in the UK open study were comparable to the 1.5% to 4% recorded in the US studies. Primary myocardial revascularization: No aspirin. The use of aprotinin therapy has been reported in less complex primary surgery, typically in patients requiring myocardial revascularization.27~*X~‘4~3H These reports have confirmed the efficacy of the new regimen. In addition, there have been five further placebo-controlled, randomized, double-blinded studies in patients having primary surgery for myocardial revascularization. The data for blood loss and blood use from these studies have been reviewed previously (Table 1).24.40 The overall results from these studies in Europe have shown that in approximately 900 patients, who received aprotinin therapy, there was a reduction in the postoperative loss from the pericardial drains of approximately 40% to 50% when compared with 1,000 nontreated patients, who have made up the various control populations. One surprising observation was that there was no direct correlation between the reductions of blood loss and the proportion of patients receiving donor blood. These two aspects of the data from the five randomized controlled
studies are shown in Fig 2. The data show that the proportion of patients transfused in Giessen and London fell by a much greater percentage than in the patients studied in Freiburg and Munich. This is despite the fact that the actual blood loss was reduced by larger amounts in real terms in the latter two centers. Why this effect was observed was difficult to explain at first because all of the studies had similar protocols. The data from the Amsterdam study are difficult to interpret because many of the patients received intraoperative blood transfusions, and the figures shown are only for postoperative blood transfusions and losses. This is probably the reason why the number of postoperative blood transfusions was lower than in the other study centers (Table 1). The additional intraoperative events, which are not shown in the Amsterdam data, would alter the relationship between blood losses and transfusions. In addition to the absolute reduction in the numbers of patients receiving donor blood and the total donor blood transfusions, there was a further observation in those patients who received aprotinin therapy. If the amount of blood given to only the patients who received a blood transfusion (expressed as mean units per patient) are compared, then it can be observed that in the patients receiving aprotinin therapy there were also reductions in the blood used per patient (Table 2). The likely explanation for the apparent anomaly between the donor blood requirements and the postoperative blood losses is based on observations of blood loss and hemoglobin loss into that drainage volume.” It showed in the randomized placebo-controlled, double-blind study that there was a threefold reduction in hemoglobin loss from 38 to 12 g, but blood volume loss was reduced by one-half from 570 to 310 mL (Fig 3). The data suggest that aprotinin was
Table 2. Units of Blood Given Per Patient Study Center
Placebo
Aprotinin
Primary CABG
Fig 2. Differences between placebo and aprotinin therapy for blood losses and proportion of patients receiving donor blood from five placebo-controlled studies in five European centers.
London
2.14
1.65
Giessen
2.36
1.48
Munich
3.2
1.71
Freiburg
2.72
1.78
Reoperations London
3.72
1.25
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
Drains loss
Hb loss
Fig 3. Data shown are for blood loss and the total hemoglobin loss into that volume from a randomized placebo-controlled study in patients having primary CABG.= Data are mean + SEM and show that there was a halving of the volume loss with aprotinin therapy, but this therapy had a greater effect in reduction of hemoglobin loss.
able to reduce the bleeding, but probably had no effect on the drainage of serosanguinous exudate that occurred in the few hours before the chest tubes were removed. It is well recognized and described by the originators of the chest tube reinfusion technique of blood conservation that the hemoglobin content of the reinfused pericardial drainage fluid is highest in the hours immediately after surgery and becomes progressively less in the later postoperative period.“’ None of the patients in the primary revascularization studies was anemic on the seventh postoperative day or before leaving the hospital, which agrees with the studies in patients having more complex repeat surgery. The mean discharge hemoglobin concentration was 12.5 g/dL in the treated group.= This relatively high hemoglobin concentration at discharge from the hospital probably reflected the perioperative “intent-to-transfuse” policy to a level of 10 g/dL. Because of this policy, certain patients in the study received 1 U of blood, or only part of 1 unit. With further experience and modifications of the transfusion policy to allow a lower hematocrit in the immediate perioperative period, it has been possible to tell patients that, excluding a major catastrophe, they will not need blood for surgery or for anemia. This is also true for the majority of patients having reoperations. Primary myocardial revascularization: Aspirin pretreatment. Preoperative aspirin therapy is associated with an improved early graft patency rate and, therefore, is recommended in aspirin ingestion is patients having CABG.4’ However, associated with an increased incidence and degree of to postoperative bleeding,43,44 which has been reported increase the likelihood of the need for a same day reoperation by a twofold factor in aspirin pretreated patients.45 Although aspirin therapy does not universally produce a prolonged bleeding time, a prolongation of this variable has been suggested as a means to determine patients at greater risk of postoperative bleeding.46 However, this concept is not universally accepted.47 Nevertheless, Ferraris et al have suggested that a bleeding time of more than 9 minutes
81
represents a relative contraindication to operation.46 Therefore, such patients should represent a considerable challenge to the therapeutic effectiveness of aprotinin and might also provide some insight into its mechanism of action. Therefore, studies were conducted to establish the efficacy of the current aprotinin regimen in a group of 19 patients who were taking aspirin up to the day of surgery. The entry criterion for the study was a bleeding time of more than 9 minutes.48.49 The postoperative blood loss in the control patients (n = 10) of 2,070 f 280 mL was significantly greater (P < 0.001) than the 290 ? 4.5 mL in the aprotinin-treated patients (n = 9). Only 1 patient in the treated group received donor blood (1 U) compared with all patients in the control group (42 U). Four of 10 control patients and none of the aprotinin-treated patients had a reoperation for bleeding. There are two further reports of the use of aprotinin in aspirin-treated patients. In the first of these, 544 patients scheduled for nonurgent CABG were allocated to receive (26 patients) or not to receive (18 patients) aprotinin. Blood losses were 1,393 t 979 mL in control patients and 352 2 138 mL in aprotinin-treated patients. Total blood use was 49 U in the control group and 21 U in the aprotinintreated patients. However, in the aprotinin-treated group, 11 patients received single-unit transfusions, again highlighting the problems of a rigid intent-to-transfuse policy. In the second report from the Amsterdam group, 40 patients were randomly allocated to treatment and placebo groups.5’ The treated group did not receive the currently recommended dose of aprotinin, but instead were given only 2 x lo6 KIU (280 mg) of aprotinin into the prime of the oxygenator. Postoperative blood loss fell from 1,096 +- 121 mL in 20 control patients to 672 2 65 mL in 20 aprotinintreated patients. Total postoperative red blood cell (RBC) transfusions were 14 U in the control group and 8 U in the aprotinin-treated patients. As previously mentioned, the intraoperative blood use data are not available from these studies, which makes their interpretation difficult. It is estimated that approximately 40% of all patients with ischemic heart disease awaiting surgery are currently taking aspirin. In the current climate of increasing occurence of urgent/emergency surgery, with the attendant and associated scheduling difficulties and pressures on bed allocations, it is not always feasible to allow the routine discontinuation of aspirin therapy 10 to 14 days before surgery. Therefore, in this regard, aprotinin therapy offers a safe and tested treatment to prevent any diffuse bleeding problems. This approach will not only reduce the need for donor blood exposure, but should also reduce the likelihood of prolonged stay in the ICU or the hospital, which is associated with the need for reoperations and larger blood transfusions.45 A second approach may be to administer aspirin only after the patient has returned from surgery. A recently published report from Gavaghan et al has shown that aspirin therapy given within 1 hour of the completion of surgery preserved graft patency.52 This practice was not associated with any increase in the tendency for these
DAVID ROYSTON
82
patients to bleed. The data from this type of study may lead eventually to a reduction in the number of patients taking platelet-activating drugs before surgery. Vascular surgery. Pertinent to this review is a recent preliminary report of the use of the currently recommended higher dose of aprotinin therapy in patients having major vascular surgery. In a pilot study of 32 patients having surgery for aorto-iliac bypass grafting, 10 patients were allocated to receive an aprotinin loading dose of 2 x lo6 KIU (280 mg), followed by a continuous infusion of 500,000 KIU (70 mg) per hour. These aprotinin-treated patients had an intraoperative blood loss of 735 mL (440 to 1,174); (median and interquartile range) compared with the control patient blood losses of 1,300 mL (778 to 2,340). This reduction in operative losses and the provision of a “dry field” with aprotinin therapy was reflected in the median donor blood use of 3 U in the nontreated patients and 0 U in patients allocated to aprotinin therapy. Patients having cardiac and major vascular surgery make up the majority of patients who can benefit from aprotinin therapy. However, they are the relatively easily categorized and controlled patient populations. The groups of patients described in the next three categories are more difficult. This is primarily because they represent subgroups who come for urgent, if not emergency, surgery. They are also patients who have a range of diffuse problems. Therefore, these are also the patients who represent the greatest challenge. Patients with sepsis and endocarditis at the time of surgery. As in the case of the aspirin-treated patients, this group is well recognized to be at great risk of bleeding. Many are having repeat surgery because they have developed infections on a prosthetic valve. In addition, the inflammatory processes activated during septicemia often lead to deranged hemostasis and coagulation. Aprotinin was administered to 1.5patients who had active endocarditis at the time of surgery.’ The absolute blood loss in these 15 patients (13 were reoperations) showed a mean of 300 mL with a range of 140 to 870 mL. A total of 14 U of blood were given to these patients in the first 72 hours, postoperatively. This compares with a total of 93 U given to 9 patients operated on by the same surgeons at the same hospital in the previous 12 months. When the data are normalized to appear as if there were 100 patients in each group, it is easy to see an approximately lo-fold reduction in donor blood requirements in these patients (Fig 4). These figures, although extremely good, hide the pattern of transfusions in these patients. Seven of 15 patients received no donor blood. Five patients received a total of 8 U before and during surgery to maintain a hematocrit of > 22%. An additional 6 U were transfused into 4 patients postoperatively. Pediatric surgery. Before the author’s description of the effects of aprotinin in reoperations, the drug had been championed for some time in Germany to reduce bleeding in pediatric cardiac surgery.53.54 Of interest from these early studies are the results in neonates and small children (weighing 2 to 8 kg). Those given aprotinin in divided doses to a total dosage of 45,000 KIU/kg over the period of
No apdinin
Apmtinin
Fig 4. Data shown for the total WC units administered to patients having surgery with active endocardiiis. The data have been normalized to produce a total use for every 100 patients in each group. The no-aprotinin figures are from patients operated on by the same surgical team in the 12 months before the availability of aprotinin therapy.
surgery had approximately a 60% reduction in blood loss and transfusion needs compared with pediatric patients given nothing at the time of surgery. This former group also had a reduction in mortality, with a group mortality rate of 14.7% compared with 23.6% in the group without intraoperative intervention. However, the anecdotal nature of the reports and their relevance to the current practice of pediatric cardiac surgery make interpretation of certain aspects of the data difficult. Despite this, there seems a place for aprotinin in pediatric practice. With this in mind, the dose regimen has been modified; the present suggestion is to give the currently recommended adult dose to children with a body surface area of > 1.16 m’. In children with a smaller body surface area, 240 mg/m’ is given as the loading dose; this amount is also added to the pump prime. The infusion rate is maintained at 56 mg/m*/hour. This regimen was designed to administer a final total dosage of approximately 75,000 KIU/kg over the time of surgery, which is similar to the adult figure. Currently, this dosing regimen is being administered in a randomized double-blind study being conducted at the Hospital for Sick Children in London. Preliminary data from that center showed that one interesting aspect of the therapy was the effect on length of time for chest closure.” Because of the improvement in hemostasis, there was a threefold reduction in the length of time for chest closure and return to the intensive care unit (Fig 5). The reduction in the total time in the operating room produced by this reduced time for closure has allowed more efficient use of these facilities. More procedures can now be performed in the same operating rooms during the routine working day (M. Elliott personal communication, August, 1991). Patients requiring heart transplantaHeart and HLTx. tion for end-stage cardiac failure can also benefit from aprotinin therapy for two reasons. First, patients with an ischemic cardiomyopathy have often had previous cardiac surgery. These patients who require reoperations obtain the same beneficial effect with aprotinin therapy as in other reoperations. Aprotinin is now routinely used on this group
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
83
No aprotinin
24O3000
la0 eQ0.
,,,, ;:;:;:;: I',',',' ,','/'/' ,',',x/' ,','/'/x t',',',' t',',',' ,'/'/'I' 1','/'/' ....
1
E3Chest
Fig 5. Data (mean + SD) showing time for chest closure in neonates having arterial switch for transposition of the great vessels. The data show that with the dry field produced by aprotinin, the time for chest closure is substantially reduced. (Reprinted with permission.“)
of patients at Harefield Hospital, where approximately 150 heart transplantations are performed each year. Second, another reason for wanting to avoid blood transfusions in these patients is to avoid any blood-borne infection from being given to an immunosuppressed patient. In this regard, cytomegalovirus (CMV) is one of the key problems. With large blood losses and in patients with uncommon blood groups, it may not be possible to guarantee unlimited supplies of CMV-negative blood. HLTx is an effective means of treatment for a number of otherwise lethal diagnoses. A number of problems including bleeding can lead to a stormy early postoperative period following HLTx. In one survey of the results of HLTx, there was a 26% early mortality.56 Bleeding was reported to account for 50% of the deaths. In the 12 months from January 1990 to February 1991, there were 44 HLTx at Harefield Hospital in patients older than 16 years of age. Included in the data were 24 patients having HLTx because of end-stage respiratory failure caused by cystic fibrosis (CF). Nine of the patients received aprotinin therapy. The mean cumulative losses into the pericardial and chest tubes for the first 48 hours postoperatively for the treated and untreated CF patients are shown in Fig 6. The median figures for blood product replacement in the nontreated patients in the immediate postoperative period were 7 U of red blood cells, 6 U of fresh frozen plasma, and 6 U of platelets. The corresponding figures for the patients receiving aprotinin therapy were 3 RBCs, 0 fresh frozen plasma, and 0 platelets. Additional benefits of aprotinin therapy in these patients were noted when the gas exchange of the lungs was considered. Using the PaO,/F,O, ratio as the index of oxygen transport, it can be observed that there was better gas exchange in the aprotinin-treated patients with a higher PaO, at the same or lower inspired oxygen (Fig 7). As the data in Fig 6 shows, the majority of the postoperative bleeding is from the chest tubes. It is well recognized that the major sites of hemorrhage in these patients tend to be from the posterior mediastinum.57 The benefits of aprotinin therapy in this group of patients is that a larger
3000
? ?Pericardial
O-12
Apmtinin
Aprotinin
12-24
24-48
1
Chest W Pericardial
O-12
12-24
24-48
Fig 6. Data (median) at 12, 24, and 46 hours, postoperatively, for cumulative blood loss from thoracic and pericardial drains in patients having HLTx for end-stage CF. The left panel shows data from 15 patients operated on without aprotinin therapy and the right panel shows data from 9 patients operated on with aprotinin. The figure shows that the majority of the postoperative blood losses are from the thoracic tubes, and the considerable effect of aprotinin therapy in reducing this loss.
amount of the capillary bleeding is prevented; this allows the surgeons to more accurately define the major bleeding sites and provide good hemostasis either by electrocoagulation or local application of fibrin or tissue glue. One further finding that emerged from the data in these patients was that donor blood administration over the ensuing period of hospital stay following transplantation and in the subsequent months was independent of whether aprotinin therapy was given. This is probably caused by the fact that patients with CF and those having HLTx have a poorly understood chronic anemia, which requires blood transfusions. With the development and interest in the use of recombinant erythropoetin in this kind of anemia, it will hopefully be possible in the not too distant future to obviate the need for these added blood transfusions in this group of patients. These preliminary data suggest that aprotinin therapy may be of benefit in HLTx. However, as in the case of
Fig 7. Data (mean f SEM) for PaO,/F,O, ratio in the patients whose blood losses are shown in Fig 6. The avoidance of large blood transfusions in the patients receiving aprotinin therapy was reflected in more stable and better gas exchange in this group in the early postoperative period.
DAVID ROYSTON
84
pediatric surgery, it may be benefits blood conservation, part in reducing operating improving the postoperative
that aprotinin therapy not only but may also play a significant and critical care times and in well-being of these patients.
Alterations in the Dose Regimen Before a discussion of the mode of action of aprotinin, the dose regimen now used and the rationale for future modifications and manipulations of the total dose and timing of administration of this agent will be discussed. Analysis of plasma samples taken during the double-blind studies in primary surgery showed that the median aprotinin concentration achieved was 5.5 PM (258 KIU/mL) at the start of surgery and 3.9 ~.LM(181 KIU/mL) at the end of bypass.** Therefore, the concentrations required theoretically to inhibit plasmin and kallikrein were achieved during the entire bypass period. Further analysis of the data has shown that the elimination half-life of the drug was 44 minutes.5H This additional evaluation also raised two other points in relation to the currently recommended dose regimen. First, the actual concentration of aprotinin achieved in the plasma following the original bolus varied markedly among patients. This variability was not related to the size of the patients, as might have been expected from the use of a fixed-dose regimen whatever the patient’s weight and sex (Fig 8). Second, the only significant relationship between the plasma concentration of aprotinin and its effect to prevent bleeding was between the postoperative hemoglobin loss and the aprotinin concentration at the end of bypass (Fig 9). Although this association was statistically significant (P < 0.02) it was not very tight (r = 0.56). There was no significant relationship between the concentration of aprotinin at the start of bypass and any of the variables measured; the data shown in Fig 10 relate the aprotinin concentration achieved after 10 minutes of bypass to the postoperative hemoglobin loss. These data suggest that the maintenance of a high aprotinin concentration toward the end of the period of extracorporeal circulation may be more important than having a high concentration at the start. A more logical approach to the dose regimen may be to give a relatively large bolus infusion before the incision, to add a modest bolus to the pump prime to help maintain adequate
30-
* 0 0 0
.o 0
10-
ti
?? 0
0
.
0
?? .a .
8.;
0..
??
.
0
I
I
i
loo
200
300
400
Apotinin concentration( KIU / ml ) Fig 9. Plot of plasma aprotinin concentration at the end of bypass compared with the postoperative hemoglobin loss from 37 patients in the randomized, placebo-controlled study in primary CABG patients.= The relationship is not tight (r = 0.96). but is statistically significant (P < 0.02).
plasma concentrations throughout bypass, and to give a final bolus immediately before the completion of bypass. This pattern of administration is used in a number of hospitals in Europe.S’ Aprotinin has been given to stop excessive diffuse bleeding in postoperative cardiac surgical patients in the ICU. Angelini et al described the results of administration of a bolus of 2 x 10h KIU of aprotinin followed by a continuous infusion of 500,000 KIU/hour in six patients who were bleeding after cardiac surgery.h” The effects of this therapy were to decrease the rate of blood loss from 490 (250 to 1,146) ml/hour (mean [range]) to 90 (29 to 193) mL/hour during and up to 6 hours from therapy. In this regard, Bidstrup et al also recommended continuing the administra-
loo
&thin Fig 8. Plots of plasma aprotinin concentration against time from 18 individual patients studied in randomized placebo-controlled studies in the UK.” The data show the threefold differences in plasma concentration achieved following a standard bolus loading dose, and the great individual variability in the disappearance of the drug.
0
al-
zoo
300
400
concentdion
(
500
600
KIU / ml )
Fig 10. Plot of plasma aprotinin concentration at the commencement of bypass compared with the postoperative hemoglobin loss from 37 patients in the randomized placebo-controlled study in primary CABG patients. *’ There is no relationship between the variables (/ = 0.07).
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
tion of aprotinin therapy into the postoperative period in patients at particular risk of bleeding, and especially in patients with deranged hemostatic mechanisms as a result of infections and sepsis at the time of surgery.6’ Previous reports of the use of aprotinin administered at the end of CPB used a dose of 10,000 KIU/kg, but did not measure efficacy in reducing bleeding.‘j* Therefore, it would seem reasonable to suggest studies of the use of aprotinin to prevent bleeding unrelated to the period of extracorporeal support. The earliest reports on the use of aprotinin commented on the dry operating field achieved prior to heparin administration.27 Therefore, it follows that in certain cases, such as reoperations or where both internal thoracic arteries are used as conduits for myocardial revascularization, the benefit of aprotinin therapy in producing a dry field during a difficult and possibly prolonged dissection before the period of CPB can be obtained. The optimum dose and timing of the administration of aprotinin will depend greatly on the length of surgery. If it is prolonged by extending the time before establishing CPB and/or by the need for a prolonged period of CPB, then the concentration of aprotinin will decline to low levels toward the end of bypass. This may severely limit the observed efficacy of the therapy. Another confounding influence on the benefits of the therapy and strategies to maintain a constant plasma concentration is if some form of hemofiltration system is used during the period of extracorporeal support. For example, if such a system is used to remove excess fluid at the end of the bypass period to increase the hematocrit, then aprotinin will be lost from the plasma. The mechanism of this loss is twofold: (1) the highly basic molecule will adhere to the dialysis membranes, and (2) with a molecular weigh of 6,512, aprotinin is filtered by the system and discarded. This latter effect may explain some of the preliminary pediatric cardiac surgical data previously cited. At the center performing these studies, it is their practice to allow profound hemodilution during the period of extracorporeal circulation and to return the hematocrit to normal by a period of ultrafiltration before decannulation.“3 In contrast to the administration of the currently recommended high dose, it has been argued that a much smaller dose is of equal benefit. The rationale for this is difficult to define from the published literature in humans, but does follow the adage that a smaller dose for the same efficacy should produce fewer adverse effects. In one study, aprotinin, 2 x lo6 KIU, was given into the oxygenator prime as the only therapy.@ The effects of this regimen were compared with the currently recommended dose in patients having primary surgery. The end-points for efficacy were hemoglobin loss for the perioperative period together with postoperative transfusions of RBCs and FFP. The perioperative hemoglobin loss showed mean values of 110 g in the control group, 65 g in the group given the dose currently recommended, and 74 g in those given aprotinin only into the pump prime. The total postoperative blood product use for these patients was 30 U of RBC and FFP in the 30 nontreated patients; 11 U of RBC and 11 U of FFP in the 30 patients given aprotinin therapy as the current regimen,
a5
and 9 units of RBC and 20 units of FFP in the 22 patients given aprotinin into the oxygenator prime. The authors commented that the increased use of FFP in these latter patients was “because of a longer postoperative bleeding time,” although their published data do not support this statement. Earlier reports have also reported investigation of the efficacy of aprotinin in a total dosage of approximately 2 x lo6 KIU during surgery.65 In this study by Hannekum et al in patients having primary surgery for valve replacement, the mean loss postoperatively decreased from 596 mL to 443 mL, but this difference failed to reach statistical significance.6’ However, the donor blood used in this study was significantly lower, but blood product administration was not completely abolished in the aprotinin-treated patients. In the section dealing with the use of aprotinin in patients taking aspirin, there is also a report on the use of only an oxygenator prime dose that showed efficacy. The mean blood loss decreased from approximately 1,096 mL to 672 mL. This reduction to approximately 60% of the control group loss was statistically significant (P < 0.05). Similarly, there have been reports of the use of half of the presently recommended dose in patients having repeat cardiac surgery.“6 Statistically significant efficacy was achieved with this lower dose. The mean postoperative blood loss decreased from 1,429 mL to 948 mL (P < 0.05). The data from these two low-dose studies are compared with that from separate studies in patients having reoperations’ and those taking aspirin” and shown in Figs 11 and 12. While accepting that it is possibly not appropriate to compare studies from different centers, the data are intriguing and do imply strongly that the higher the dose the more beneficial are the effects of aprotinin. As will be discussed later, there appears to be no evidence for any detrimental toxic effects of the currently recommended regimen, thus the use of a lower dose regimen seems totally illogical. If this lower dose is administered routinely, then a proportion of the patients will bleed sufficiently to require donor blood transfusions, even though they have been challenged by a drug that may have antigenic potential. The end result of this technique may compromise the chance of using aprotinin for some future procedure while failing to prevent the need for donor blood or blood product transfusion. There is a dose-response effect for this agent, which is different for various discrete procedures. The currently recommended regimen will reduce bleeding to the point in which the need for donor blood is prevented in all patients having primary surgery, the majority of reoperation patients, and patients taking aspirin. However, this dose regimen will not totally prevent bleeding in combined HLTx and in certain other situations. In these patients, a larger amount of the drug than is described in the current regimen is needed. This approach has been used on a number of occasions in patients having reoperations for the third or fourth time, while taking aspirin f coumadin-type anticoagulants, and also in patients who are of the Jehovah’s Witness faith.
86
DAVID ROYSTON
18CO-
Tabuchietal E3iietal
-C-
+
0 0
1
I
I
I
I
140
230
420
560
700
Total aprotinindose ( mg )
0
U’
0
ldo
2iO
420
560
700
associated with cardiac surgery. The first relates to establishing a consensus view on what is the definition of excess versus normal blood loss; thus far, virtually all studies of this issue have concentrated on identifying the cause of “abnormal” bleeding associated with a period of extracorporeal support without defining what is abnormal. The difficulties in choosing an acceptable degree of “normal” bleeding are highlighted when, for example, the results for blood loss shown in Table 1 for the double-blind-studies of the efficacy of aprotinin are compared. The patients in the control group in the Geissen study had losses of 347 -t 103; 210 to 690 mL (mean -+ SD; range); whereas those in Munich were 1185 t 601; 500 to 3200 mL. Both centers obviously regard these losses as normal despite the threcfold difference in their magnitude. Similarly, in North America the losses recently reported by Goodenough et alZU in patients having primary surgery showed a range from 672 + 54 mL to 1,445 k 109 mL in the 18 centers with blood conservation programs. Without a consensus on what is considered as abnormal, it is obviously difficult to delineate those factors that are specifically associated with a tendency to increase bleeding. The second problem associated with studies of the action of aprotinin stems from the fact that the vast preponderance of the research has been performed in patients during cardiac surgery. This has inevitably led to studies focused on measuring factors and effects of aprotinin at the time of extracorporeal circulation. These studies have produced conflicting data and hypotheses and little, if any, data that may prove valuable in answering the question of the mode of action of the drug in the patient. Aprotinin therapy acts to improve hemostasis and prevent bleeding before any period of extracorporeal support. It is the author’s belief that the primary action of aprotinin owes nothing to its effects to prevent the damaging effects of CPB. Nonetheless, the preponderance of mechanism of action studies
Total aprotinindose ( mg ) Fig 11. Data for blood loss (actual loss [mean t SEMI in top panel and expressed as percentage of control in lower panel) from two studies of the efficacy of aprotininin patients taking aspirin.“,5’ In the study of Tabuchi et aP 280 mg of aprotinin was added to the oxygenator prime. In the Bidstrup et al report60 the total dose was the currently recommended dose. While both therapies produced significant reductions in losses, the lower dose appears to be less effective.
MECHANISMS
0
Roiystonetal Scott&Au
??
UKopenW
.
0
OF ACTION
The mechanism of action of aprotinin to reduce bleeding is of great importance primarily because there are a number of modified and genetically engineered forms of aprotinin available that can be produced commercially by recombinant technologies. By identifying the mode of action, a more specific and potent agent could be developed. Another reason to define the mode of action of aprotinin is to allay anxieties that if a drug profoundly improves capillary hemostasis, then it must induce clot formation, and thereby adversely influence early graft patency. There are two major problems in attempting to identify the mode of action of aprotinin in reducing the bleeding
U
0
700
Total aprotin?dose ( mg ) Fig 12. Data for postoperative losses (median) from three reports of the use of aprotinin in patients having reoperations.‘“~5* The patients in the report by Scott and ALP received half of the currently recommended regimen. An apparent effect of dose on losses can be observed.
APROTININ TO PREVENT BLEEDING: THE FfRST 5 YEARS
conducted principally during the period of extracorporeal support have been directed nut toward the issue of how aprotinin works to improve hemostasis, but instead have been focused on addressing the question of how aprotinin protects the extracorporeal system. There are a number of agents that do this task far more efficiently than aprotinin, but not all of these drugs prevent bleeding. The problems with investigations of the mode of action of aprotinin that center on the period of bypass are most easily illustrated when the data for one aspect of the damaging effects of extracorporeal support are evaluated. Three studies have reported the effects, of aprotinin therapy on complement activation; measured using the generation of the active fragments C3a and C4a. These studies have shown that there are no effects of aprotinin,” that aprotinin therapy reduced complement activation6’ and, most recently, a further study showed aprotinin administration to be associated with an increased complement activation6’ The inconsistency in reported data probably reflects the huge variability in perioperative techniques. The oxygenator system, pumping system, and type of flow, temperature of the patient and perfusion system, acid-base control, as well as the type and volume of the prime for a particular operation vary widely throughout the world. Similarly, the period of each segment of a specific operation differs between centers and surgical teams. Add to this list the differences that perioperative pharmacological intervention may produce, and it is not difficult to see that much of the data related to the mode of action of aprotinin can only be applied to one specific trial at one time; therefore, it may not be possible to generalize any conclusions from such an investigation. Despite these caveats and before considering aprotinin’s actions, it seems reasonable to briefly review the normal hemostatic and coagulation systems and the various parts of this process, which may in some way be affected by aprotinin therapy, based on current knowledge of its spectrum of inhibitory activity. NORMAL
HEMOSTATIC
FUNCTIONS
From an anatomical point of view, it is possible to differentiate bleeding from arteries, veins, and capillaries. Aprotinin has its main effects by reducing the capillary ooze associated with major tissue injury and surgery. Bleeding from capillaries stops mainly by alterations at the microvascular endothelium. The hemostatic process progresses in several interconnected phases. The primary process is that the platelets plug the holes.@ This is achieved by platelet adhesion, aggregation, and release of granules and other products leading to the production of a hemostatic plug. This initiation of the hemostatic process is followed by the activation of the clotting process to produce fibrin. Before describing certain aspects of these processes, it is worthwhile to point out that the platelet, for its size, has a remarkable array of biochemical functions. These are controlled and activated by an equally impressive array of receptors and response systems that allow the platelets to perform their hemostatic function. Not all of the mecha-
87
nisms are fully understood. In si,mple terms, the normal platelet is able to adhere to a damaged endothelial cell or the subendothelial layer. This adherence is achieved by the platelet sticking to collagen, basal membrane structures, and microfibrillae of subendothelial tissues. The interaction is supported by a bridge of the multimeric form of the von Willebrand factor from the endothelium/subendothelium to attach to the platelet at the glycoprotein Ib (Gp Ib) receptor site. Until recently, it was thought that these Gp Ib receptors were always exposed on the platelet surface. Then the platelet can undergo a shape change and spread over the injured microvascular surface with exposure of different glycoproteins, such as the Gp IIb/IIIa complex, which can bind to fibrinogen (the activated IIb/IIIa COMplex is termed the “fibrinogen receptor”), and Gp IV, which can bind to collagen (as can Gp IIb/IIIa, but not Gp Ib). The Gp IIb/IIIa complex is interesting as it has an amino acid residue sequence with homology to a number of other adhesive glycoproteins found on the surface of many types of cells, particularly on neutrophils. Because the platelet allows the exposure’ or development of the Gp IIb/IIIa complex, there is release of platelet a-Granule materials such as adenosine diphosphate (ADP), l3-Thromboglobulin, and platelet fibrinogen. Advances in the techniques of molecular biology have led to a greater understanding of the type and role of the platelet glycoprotein receptor. The relative frequency of antigenically available glycoprotein receptor sites can now be measured with comparative ease. However, the techniques used are complicated, and there is considerable debate as to the optimum antibody to use to detect these sites and as to the methods used to prepare the platelets before measuring. There are a number of people who have a genetic defect that results in the inability to produce or express these glycoprotein receptors on the platelets. Subjects in whom there is an absence of the Gplb receptor have BernardSoullier syndrome. In these patients, there is a prolongation of the skin bleeding time. The platelets respond normally to aggregation with ADP, and there is a reduced, but not absent response to stimulation with the synthetic antibiotic ristocetin. Patients failing to express the Gp IIb/IIIa receptor have the Glanzmann-Nageli syndrome. Once again, bleeding time is prolonged. The platelets from these patients no longer respond to ADP or collagen with a second phase or aggregation response. Fibrinogen is an important cofactor in platelet adhesion and is currently thought to be essential for the process of platelet-to-platelet binding, which occurs during irreversible aggregation. During this secondary event, there is a release of material including thromboxane A,, a very potent vasoconstrictor and platelet aggregating agent, which is synthesized in the platelet. A further protein complex called “thrombospondin” is also released at this time and binds between the newly exposed fibrinogen receptors and the fibrinogen molecules bridging platelets to stabilize the aggregate. The platelet also contains mitochondria and
88
DAVID ROYSTON
contractile elements, which are sensitive to a number of different stimuli, such as an increase in calcium flux. In addition to the exposure of the various glycoprotein receptors at the time of initial activation, the platelet also exposes various membrane-derived phospholipids. These are thought to be necessary for regulation of the coagulation cascade. These membrane components can serve as activating agents for factor XII. In addition, other factors are able to bind and transform other parts of the coagulation cascade. Factor V of the coagulation cascade binds to platelet lipids and, in a complex with collagen and factor X, can act to release activated factor X. In this way, the final common pathway of coagulation is stimulated. The thrombin generated is able to act as a positive feedback to initiate further platelet aggregation and to stimulate the release of further ADP and thromboxane. Damaged endothelium can also produce tissue factor and tissue thromboplastin to stimulate coagulation by the extrinsic or factor VII-dependent process. The initial hemostatic function of the endothelium can be thought of as a process of initiation of hemostasis and subsequent amplification of the coagulation cascade. It is obvious that this process would carry on unabated to produce unchecked clot formation unless there were equally effective methods of controlling these processes. In this regard, thrombin acts to stimulate release of various substances from the endothelium. Of particular relevance is the production of prostacyclin and an endothelial-derived relaxing factor (EDRF), which are both well recognized as potent antiaggregatory agents and will inhibit or limit the formation of platelet aggregates. Thrombin also acts on the microvascular cells to induce the production of tissue plasminogen activator (t PA), initiating the conversion of plasminogen to plasmin, and thus activation of the process of fibrinolysis. These actions of thrombin limit platelet aggregation, initiate the dissolution of the blood clot, and also reverse the vasoconstrictor response fundamental to the hemostatic process. Finally, thrombin can bind to thrombomodulin on the endothelial cell surface, and this thrombin-thrombomodulin complex can activate protein C (produced in the liver) to produce activated protein C (APC). This APC is a serine protease, which can constrain the rate of thrombus formation by its own inhibitory actions on factors V and VIII in the intrinsic coagulation cascade.
MECHANISMS
RELATED TO BLEEDING AFTER
CARDIOPULMONARY
BYPASS
Activation of Platelets During Extracorporeal Circulation There is excellent evidence of abnormalities in platelet numbers and function associated with CPB: (1) there is rapid consumption of platelets, which is most obvious during the first few minutes of bypass’“,‘l; (2) there is decreased reactivity to known agonists72.73; (3) there is an increase in the concentration of o-granule compounds such as @Thromboglobulin in the plasma”,7s; (4) there is an increase in the concentration of thromboxane B, (the stable metabolite of thromboxane AZ) presumed to be released from aggregating platelets76.77; and (5) the bleeding time is
prolonged following a period of extracorporeal circulation and the magnitude of the prolongation has been directly related to the time on bypass.” Platelet Numbers There are two views on the significance of the immediate fall in platelet numbers at the start of bypass. One view is that this represents activation and loss of the platelet from the circulating pool, which is best prevented by inhibition of the contact activation phase of the system. The second view is that this loss represents only a minor alteration of platelet function and activation is not the principal problem. In support of the first concept are studies that have shown that expression and subsequent loss of platelet surface glycoprotein receptors are associated with the reduction in absolute platelet count in the first few minutes of extracorporeal circulation. Furthermore, this platelet loss from the circulation was attributable specifically to the expression and subsequent loss of the Gp IIb/IIIa complex.” This was demonstrated by the fact that only blood from normal donors and patients with Bernard-Soulier syndrome (platelets deficient in Ib but sufficient in Gp IIb/IIIa receptors) was lost, and that the proteins that remained adherent to the extracorporeal system were primarily made up of fragments of the Gp IIb receptor.-” These observations strongly support the concept that the Gp IIb/IIIa complex is the adhesive glycoprotein most affected by the extracorporeal system and that platelet loss is related to activation by this pathway. The second view is supported by the studies of Zilla et al who argued that the role of early contact in platelet activation is not a major problem or mechanism for the platelet 10~s.~”Using morphological methods, they showed that, although there was a reduction in active platelets during the first few minutes of bypass, there was, at the same time, an increase in the number of platelets showing a shape change. Throughout the period of bypass, there was a reduction in the obvious detrimental aspects of extracorporeal circulation on platelet morphology; there appeared to be a reversal of adverse effects noted at the start of bypass. They also provided evidence, based on the correlation between factors associated with cell death such as plasmafree hemoglobin and lactate dehydrogenase concentrations, and the increase in plasma concentration of a-Granule products, such as platelet factor 4 and p-Thromboglobulin, to show that the platelet was not undergoing activation but lysis. Zilla et al have suggested that any platelet stimulation associated with the initial phase of bypass is of little consequence, and that with modern CPB systems only a portion of the platelets proceed beyond the step of reversible platelet aggregation. Support for the argument that activation of glycoprotein receptors may not play an important part in the loss of platelets comes from the studies of George et al.” They used highly specific antibodies and demonstrated that following CPB there was a significant reduction in the binding of antibodies to both Gplb and the total Gp IIbiIIIa complex in platelet suspensions that had been immediately tixed in glutaraldehyde. Although the reduc-
APROTININ
89
TO PREVENT BLEEDING: THE FIRST 5 YEARS
tion in the number of antibody binding sites and, therefore, presumably receptors was highly significant, (8’ < 0.02 or better), the actual fall in receptor number was 20% for the GpIb and 10% for Gp IIb binding. In none of the patients studied during cardiac surgery was there a decrease in the binding sites for antibody outside the 95% confidence interval for the laboratory concerned. In this study, the authors did not attribute any clinical significance to their observations of glycoprotein receptor binding in terms of the increased likelihood of bleeding. In contrast, they argued the opposite case. Subjects heterozygous for Bernard-Soulier and Glanzmanns thrombasthenia with one-half the normal concentrations of Gp Ib and Gp IIb/IIIa, respectively, have normal platelet function and no excessive bleeding.82 These observations imply that a severe deficit in membrane receptors must exist before there are major bleeding complications. The confusion produced by this contradictory evidence of the nature and importance of platelet activation to bleeding problems is only compounded by interpretation of other data related to platelet function.
Platelet Activation
There is evidence for the release of various components of platelet granules during extracorporeal circulation. This effect is usually demonstrated by measuring increases in plasma concentrations of these granule products. However, the actual amount of activation needed to produce this effect is only slight. For example, a 60-fold increase in plasma concentrations of platelet factor 4 requires only approximately 1% of the platelets to be activated.83 In studies using the ex vivo system, the increases in B-Thromboglobulin concentrations were similar despite threefold differences between adhesion and activation of platelets from patients with various glycoprotein abnormalities.‘l Two studies have failed to demonstrate a-Granule release from stimulated whole blood.8’,84 Therefore, these data do not support the evidence of Harker et al.”
Bleeding Time
The measurement of bleeding time is thought to be an expression of platelet-microvascular reactions that were unaffected by heparin administration. This measurement has been shown to increase in duration as the period of CPB is prolonged and to revert rapidly back to normal when the extracorporeal support is discontinued.‘* However, recent evidence has suggested that heparin can affect the bleeding time:’ and that the bleeding time can be significantly prolonged by processes not directly related to the platelet numbers or possibly their function. For example, the bleeding time is prolonged following the administration of the fibrinolytic agent rt-PA.86 Given this confusion regarding suitable methodologies and models along with the data generated from such systems, it is impossible yet to define precisely the role of the platelet in postoperative bleeding.
Fibrinolysis
There is clear evidence that the concentration of fibrinogen/fibrin degradation products increases during the period of extracorporeal circulation.87’88Although the mechanism of this increase is not fully elucidated, there are recent studies that show that the concentration of plasminogen activators increases during the period of extracorporeal circulation.89.90 As in the case of the platelet mechanism, the question is to relate this evidence for activation of fibrinolysis to postoperative bleeding. There are reports that show that there is a significant correlation between indices of fibrinolytic activity and the volume of losses into the chest drains.91-93 Equally, other studies have not shown any relationship between concentrations of fibrin split products and increased postoperative losses.” A further difficulty in determining the role of fibrinolysis in the bleeding defect is the actual end-point chosen to define the extent of fibrinolysis. For example, an increase in the plasma concentration of fibrin(ogen)-split products can be demonstrated in all patients having cardiac surgery. However, using end-points such as the euglobin clot lysis time or the whole blood clot lysis of the thromboelastogram, there are transient abnormalities in approximately 1O%87 and 6% to 8%95 of patients, respectively. These abnormalities are obvious immediately following protamine administration and rapidly disappear. Despite these difficulties in defining fibrinolysis, there is some evidence to suggest that the administration of antifibrinolytics to patients having cardiac surgery reduces postoperative bleeding.96,97However, the reductions have thus far been relatively modest and there is no evidence of efficacy in patients having surgery in which blood loss may be excessive, eg, in reoperations and in patients taking aspirin. EFFECTS OF APROTININ
ON PLATELETS AND
FIBRINOLYSIS
Platelets As previously discussed, a defect in platelet function has been suggested as the major cause of bleeding in the postoperative period. Indeed, pharmacological interventions to prevent bleeding have been effective when plateletactive agents such as prostacyclin9* and dipyridamole99 have been used. All of the serine protease inhibitors have activity to inhibit various aspects of platelet function.6 Aprotinin is no exception, and has been shown to have a large number of direct and indirect actions on platelets. The second phase response of platelet aggregation (associated with the release of thromboxane A, and serotonin) following stimulation with ADP is effectively inhibited by aprotinin at concentrations of approximately 100 to 400 KIU/mL.‘W~‘o’ This effect has been used to great effect in preserving platelets in donor blood. In addition, there are also reports of the use of an intravenous bolus dose of 20,000 KIU/kg to prevent platelet aggregation, and thereby reduce the incidence of venous thrombosis in major surgery. This dose
90
significantly reduced the platelet :adhesion and aggregation associated with hip replacement ~o,per-ation~.‘~*~‘~~ Studies in ,Germany’” and, more recently, in the United States,‘“’ have shown a highly s&r&cant preservation of platelet nmnbers and function in stored blood when aprotinin was added to the system The exact mechanism of the protectionof stored ,plat&ts is not clear. A major determinant of cell survival in the circulation is thought to be related to the,surface sialic acid content. In platelets, this is found mainly in the GpIb receptor.“’ The GpIb receptor can be hydrolyzed and degraded ‘by plasmin in vitro.iO’ However, separate studies have shown that pro,tease irrhibitors such as leupeptin and aprotmin, despite being potent inhibitors of plasmin, ,did not prevent the loss of ,GpIb in stored blood.‘” There are three points on the effects (of :aprotinin on platelets, which have been activated by foreign surface contact within the extracorporeal system. The most important point concerns the skin bleeding time post-bypass. It has been shown that aprotinin in .the present dose regimen is able to prevent the increased bleeding time normally observed in patients after .a period of CPB.n~W This implies that aprotinin is acting to improve ‘the ,normal plate’let to microvascular reactions. Current understanding is that the platelet adheres to the endothelium,and subendothelium,by way of a bridge of multimeric von Willebrand factor attached to the platelet GpIb receptor. There is evidence to suggest that aprotinin administration is associated with a preservation of this receptor ‘during extracorporeal circulation using a .hollow-fiber type of membrane oxygenator.“4 The mechanism of this preservation is thought to be the aprotinin inhibition of the plasmin that subsequently digests the GpIb receptor.“’ There is also preliminary evidence to show that during bypass the aggregation response to ristocetin, which acts via GpIb, is maintained at or near normal in ,aprotinin-treated patients, but decreases by approximately ,40% in nontreated patients.“’ The relevance of this observation to the mechanism of postoperative bleeding is not clear. In one report, the loss of the aggaegatory response to ristocetin was related to the degree of postoperative bleeding.“” However, the patients in this study with .the greatest reduction in response, and thus the most likelihood of bleeding, also had abnormally low platelet counts. In a second study, Laze&y et al were able to significantly improve the reduced,platelet response to ristocetin with desmopressin (DDAVP).“’ Nevertheless, there was no effects on postoperative blood loss with this therapy. These data related to the preserving effects of aprotinin on the GpIb receptor have also been brought into question for a number of technical reasons. First, they were performed on platelets that had been washed and/or filtered before study; therefore, the observed changes may have been artifactually produced during the preparation of the samples. Second, donor blood was occasionally added to the extracorporeal system during bypass in these studies. Third, recent data have shawn thatthe inability to detect antibody binding sites on the platelet surface to the Gp Ib
receptor does not necessarily mean that this glycopmtein has been degraded and lost. There is evidence to show that the GpIb receptor can be internalized following stimulation with plasmin.“‘* Redistribution of intrapiatelet stores of GpIb to the platelet surface”’ has been suggested as a potential mechanism to explain the rapid return of the bleeding tie to normal after a period of extracorporeal support. Other factors 3 IU/mL) and, therefore, anticoagulation, the ACT probably needs to be maintained at >750 seconds when aprotinin therapy is being administered. Using this approach in 17 patients having repeat cardiac surgery, it was not necessary to administer extra protamine nor was the efficacy of aprotinin lost by maintaining this prolonged ACT.lm Other Possible Unwanted Effects Effects on Renal Function
As aprotinin is selectively taken up by renal tissues, there is a possibility that kidney function might be affected using the high doses currently recommended. In the original studies, it was demonstrated that the urine output was increased in aprotinin-treated patients.28 This effect was seen both during bypass and also in the immediate postbypass period (Fig 18). The increased urine output was associated with an increase in the excretion of sodium ions. A further study focused on investigating the effects of aprotinin on renal function, and confirmed that there was a greater urine output with aprotinin associated with an increase in sodium excretion.67 In this randomized doubleblind study in 60 patients reported by Fraedrich et al, there was an increase in the excretion of -certain proteins in the aprotinin-treated patients. In particular, there was a significantly greater excretion of a-1-microglobulin and aminopep-
VIIa
0 IIa
Fig 17. A simplified representation of the factors involved in the intrinsic and extrinsic coagulation cascades. The activated factors shown enclosed by circles can be partially or completely inhibited by the heparin-antithrombin Ill complex. Activated factors shown within the box can be partially inhibited by aprotinin.
n
Fig 18. Data (mean 2 SEM) for urine output during and in the first 6 hours after surgery for myocardial revasculariration. Results are from patients in a randomized placebo-controlfed studp and show that the urine output was significantly greater (P i 0.0s) in the aprotinin-treated patients during and immediately after surgery,
94
DAVID ROYSTON
tidase in the aprotinin-treated patients. This effect was transient and returned to normal in the early postoperative period. Despite these biochemical indices, which imply tubular overload and dysfunction with aprotinin therapy, there were no effects on creatinine clearance or plasma creatinine concentrations between the two groups. In the UK studies of patients at high risk of bleeding, there was also no evidence of deleterious effects on plasma creatinine or renal function.’ This is in contrast to reports of increased creatinine concentrations in patients having reoperations who had major bleeding problems and thus required large volumes of blood product support.“” It has also been shown that aprotinin therapy is effective and safe when given to patients with established renal dysfunction or failure.ti,4y In these studies, in both the control and treated patients, there was a postoperative increase in the plasma creatinine concentration. This increase tended to reach a peak on the third or fourth postoperative day before returning to near-preoperative values by the seventh to tenth postoperative day. The data for the perioperative creatinine concentrations in the five aprotinin-treated patients are shown in Fig 19. Vascular Occlusion Risk WithAprotinin Therapy (Especially the Patency of the Conduit Following CABG) The issue that has been raised many times, especially by surgeons, is whether a drug can prevent bleeding without an increased risk of thrombotic occlusions, especially in the saphenous vein used for CABG. There are numerous anecdotal stories of graft occlusion or perioperative myocardial infarction in patients who have received aprotinin therapy. However, based on current evidence, aprotinin therapy does not increase the risk of graft occlusions and thrombotic episodes but may reduce them. This drug has been
*1
-2
2
6
-
Ptl
-
Ft2
-
Pt3
-
Pt4
10
l4
Time in days Fig 19. Individual data for postoperative creatinine concentrations from five patients with established renal failure who had cardiac surgery with aprotinin therapy. The data show that renal function was not further compromised by the use of aprotinin. Patient 1 died on the third postoperative day from an acute myocardial infarction.
used routinely for all patients in certain centers in Germany for the past few years. Aprotinin therapy in the currently recommended high dose has been given to more than 10,000 patients having CABG. Therefore, it would seem logical to assume that if there had been any real impact on graft patency, this issue would have been more widely debated than seems the case from the anecdotal stories. Firmer evidence is found in the data from five randomized placebo-controlled studies conducted in Europe.3’,“’ In these studies, the incidence of complications related to the cardiovascular system was no different between the two groups. Eleven of 176 patients allocated to the placebo group and 14 of 176 patients in the group receiving aprotinin therapy had adverse cardiovascular events. At later follow-up, there were no differences between the groups studied in terms of late mortality or the recurrence of angina associated with the need for nitrate therapy.“’ Other data related to occlusive events in the central nervous system have been outlined previously.” The reported incidence of postoperative neurological sequellac related to thrombotic episodes ( ~0.5%) was far lower in the patients who received aprotinin therapy than that predicted from previous reports.“.” Similarly, Dietrich et al14’ reported that 6% of 152 patients given aprotinin therapy had transient neurological deficits postoperatively. This was approximately one-half of the 11% incidence of transient deficits in 317 patients who had operations without aprotinin therapy. There are a number of factors known to affect patency rates, such as surgical skill and judgment, the size of the vessel to be grafted, the distal vessel run off from the anastomoses. and the need for an endarterectomy. One further confounding issue to determine if a drug has an adverse effect on patency is that the graft patency rates arc only measured at the end of the first postoperative week. Therefore, it is unknown precisely when the grafts occlude. This l-week patency rate can be improved by the use of agents that act as antiplatelet + antithrombotic drugs.“’ More commonly, antiplatelet drugs are given and typically reduce the failure rate from approximately 10% to 2% to 4%. The initiation of high shear rate graft occlusion is thought to be by a platelet-dependent process. As discussed previously, the role of the platelet is to plug the holes in the microvasculature. This is achieved by the process of adherence, aggregation, and granule release. The activated platelet can then initiate clot formation. Currently, there are no published data to suggest that aprotinin will promote adhesion, aggregation, or granule release from platelets. On the contrary, there is evidence to show that aprotinin administration leads to an inhibition of the adhesion of platelets to glass beads.“13.“‘4 Aprotinin will also inhibit the adhesion of platelets to thrombin-stimulated human endothelium.14’ Other evidence shows that aprotinin acts in the same way as aspirin to inhibit aggregation and the release of thromboxane. On a more speculative note, there is currently interest in the development of a group of compounds from viper venoms that are able to protect the Gp IIb/IIIa complex.“”
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
These agents are being studied as a means of preventing platelet activation and thereby preventing reocclusion of coronary vessels after successful angioplasty. If aprotinin is shown to protect the Gp IIb/IIIa complex from attack by elastase or free radicals, then it would be acting in a similar way and should protect graft patency. Aprotinin therapy would seem to protect against platelet-dependent processes leading to graft occlusions. As discussed previously in the section dealing with interactions with heparin, aprotinin is well recognized as an inhibitor of certain factors of the coagulation cascade. This inhibition is easily shown by the increase in the activated partial thromboplastin time (APTT). The increased duration of the APTI is typically approximately twice control values perioperatively with the currently recommended is similar to that dose regimen.37,67 This prolongation achieved when low-dose heparin therapy is used to prevent thrombus formation during such procedures as dialysis or in patients with critical ischemia. Inhibition of the intrinsic clotting process will reduce the likelihood of thrombus generation initiated by contact of blood with negatively charged surfaces, such as collagen and similar subendothelial matrix substances. However, the spectrum of inhibitory activity does not include factor Xa or thrombin with the current regimen. If factor Xa is generated by a factor VII-dependent process, then this process will not be inhibited by aprotinin. The modified aprotinin with an arginine residue at the active site at position 15 (“Arg aprotinin) has considerably more anti-Xa activity and also more potent antikallikrein activity than native aprotinin. The spectrum of inhibition of “Arg aprotinin is more akin to other serine proteases, which are true anticoagulants,‘47 but are also reported to prevent bleeding associated with the extracorporeal system used for dialysis.‘4K The only relevant problem with aprotinin related to thrombus formation would be if there was an activation of thrombin formation via a factor VIIa, and, therefore, tissue factor-dependent process, or if thrombin in the form of fibrin glue was inadvertently added to the system. Any intravascular thrombus formed in this way will be less likely to undergo lysis in the early postoperative period because of the antifibrinolytic action of aprotinin. Currently there are conflicting data whether human saphenous vein endothelium can express and secrete tissue factor. The most recent evidence suggests that this endothelium does not have this capability.‘4Y Therefore, in this case it is difficult to envision a scenario in which aprotinin would actively promote graft thrombosis. The data suggest that aprotinin has no actions to increase platelet activity or to be a prothrombotic agent. Indeed, the evidence is to the contrary that aprotinin is more likely to reduce thrombosis of vessels and especially veins. Indeed, because of its antiplatelet action, aprotinin was used as a prophylaxis to prevent deep venous thrombosis in patients having hip surgery.“‘,‘04 There are a number of studies currently underway that are specifically aimed at addressing the issue of the effects
95
of aprotinin therapy on graft patency. One potential problem with interpreting the data from these studies is that graft patency is being assessed by minimally invasive imaging techniques. In the UK the imaging technique uses magnetic resonance; in the USA, ultrafast tine-computed tomography. Therefore, the data collected may not be directly comparable to the data obtained by catheter studies. Nonetheless, the preliminary report of the UK data shows no effects of aprotinin in patients having primary CABG at 7 to 8 days postoperatively.‘5” The results from the studies in North America include patients having primary and second-time operations for myocardial revascularization, and preliminary results from these studies should be available by the early part of 1992.
Allergic Phenomena Because aprotinin is a polybasic polypeptide that is derived from bovine organs, there is always the possibility of an adverse reaction to the agent. Anaphylactoid reactions are not mediated by the immune system and are thought to be caused by the direct action of the agent on the effector system. Infusions of aprotinin in patients in large doses were shown not to release histamine, and possibly to prevent its release.15’ Similarly, in this regard aprotinin does not induce complement activation. The effects of aprotinin on the rate of complement activation that occurs during the period of extracorporeal support are not clear. There have been reports to show either a reduction in generation of C3a,6’ no effect on this variable;’ or an increase in C3a concentrationZ8 in patients receiving aprotinin therapy. True anaphylactic reactions require previous exposure to the drug and the generation of a specific IgE immunoglobulin. It is well known that approximately 40% of patients will develop some antibody of the IgG and IgM class to drugs given around the time of surgery,‘52 and in this regard aprotinin is no exception.” However, it is pertinent to emphasize two aspects of this antibody response. First, none of the drug assays using radioimmune assay or enzyme-linked immunoabsorbance assays are possible without such an antibody being formed. Second, the relevance of IgG or IgM antibodies to adverse reactions with a subsequent drug exposure remains to be proven. In studies of multiple administrations of aprotinin over a prolonged period to patients with pancreatitis, the incidence of adverse reactions is reported to be 1% or less.ls3 In the context of patients receiving aprotinin therapy on more than one occasion for cardiac surgery, this figure is considerably less than the likelihood of reactions to radiographic contrast materials. Despite these theoretical reasons to suggest that aprotinin therapy can be given on more than one occasion, there might still be some risk of a serious allergic reaction. In patients known to have had previous aprotinin therapy, the author performs a prick test before infusion of aprotinin. In addition, if the recommended dosing regimen is followed, the original loading dose should be given over a 20 to 30 minute period. With this slow infusion rate, the first 1 mL or 2 mL of solution infused act as a “test dose” of the agent.
96
DAVID ROYSTON
Before this infusion, the patient has intravenous access with a wide-bore cannula, and intraarterial pressure monitoring is established and monitored closely over the first few minutes of the infusion to ensure that any adverse events are rapidly recognized. The author has given aprotinin to 6 patients known to have had the same therapy in the preceding 3 to 6 months with no ill effects. CONCLUSION
It is true that until 1987 aprotinin was a drug looking for a disease. The discovery of the profound hemostatic properties of this compound have given it a new lease on life and have led to a resurgence of research interest into the use of serine protease inhibitors in clinical practice. Although the
efficacy of the drug, when given in sufficient dose, is now without question, there are still a large number of unanswered questions; the principal one is what the actual role of aprotinin is in nature, and will this lead to the discovery of the mechanism of its action? This, in turn, will lead to the production of more specific and potent compounds. In the meantime, there are a number of important issues to address related to the current drug. In particular, the dose and timing of administration of aprotinin therapy arc still controversial. The effects on contact systems lead to the need to find ways to more accurately control anticoagulation during a period of extracorporeal circulation. There is also a need to investigate more thoroughly aspects of safety. such as the issue of graft patency and the multiple use of the drug in the same patient.
REFERENCES
1. Royston D: The serine antiprotease
aprotinin (Trasylol): A bleeding. Blood Coag
novel approach to reducing postoperative Fibrin01 155-69, 1990 2. Royston D, Bidstrup BP, Taylor KM, Sapsford RN: Effect of aprotinin on the need for blood transfusion after repeat open heart surgery. Lancet 2:1289-1291,1987 3. Fritz H, Wunderer G: Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs. Arzneimittelforsch 33:479-494, 1983 4. Kraut E, Frey EK, Werle E: uber die lnaktivierung des Kallikreins. Hoppe-Seyler’s Zeitschrift fiir Physiologische Chemie 192:1-21, 1930 5. Kunitz M, Northrop JH: Isolation from beef pancreas of crystalline trypsinogen, trypsin, atrypsin inhibitor, and an inhibitor trypsin compound. J Gen Physiol 19:991-1007, 1936 6. Laskowski M, Kato I: Protein inhibitors of proteinases. Ann Rev Biochem 49:593-626,198O 7. Fritz H, Kruck H, Riisse J, et al: Immunofluorescent studies indicate that the basic trypsin-Kallikrein inhibitor of bovineorgans (Trasylol) originates from mast cells. Hoppe-Seyler’s Z Physiol Chem 360:437-444,1979 8. Diet1 T, Tschesche H: Trypsin-Kallikrein isoenzyme K (type Kunitz) from snails (Helix pomatia): Purification and characterization. Eur J Biochem 58:453-460,1975 9. Wunderer G, Beress L, Machleidt W, et al: Broad specifiity inhibitors from sea anemones. Methods Enzymol45:881-888, 1976 10. Fioretti E, Angeletti M, Citro G, et al: Kunitz type inhibitors in human serum. Identification and characterization. J Biol Chem 262:3586-3589, 1987 11. Fioretti E, Binotti I, Barra D, et al: Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs. Eur J Biochem 130:13-18,1983 12. Phillipe E: Calculations and hypothetical considerations on the inhibition of plasmin and plasma kallikrein by Trasylol, in Davidson JF, Rowan RM, Samoma MM, Desnoyers PC, (eds): Progress in Chemical Fibrinoiysis and Thrombolysis, (vol 3). New York, NY, Raven, 1978, pp 291-298 13. Markwardt F: Naturally occurring inhibitors of fibrinolysis. Handbook Exp Pharmacol46:487-509,1977 14. Taby 0, Chabbat J, Steinbuch M: Inhibition of activated protein C by aprotinin and the use of the insolubilized inhibitor for its purification. Thrombosis Res 59:27-35,199O 15. Espafia F, Estelles A, Griffin JH, et al: Aprotinin (Trasylol) is a competitive inhibitor of activated protein C. Thrombosis Res 56:751-756,1989 16. Lottenberg G, Sjak-Shie N, Fazleabas AT, et al: Aprotinin
inhibits urokinase but not tissue-Type plasminogen activator. Thrombosis Res 49:549-556, 1988 17. Puri RN, Zhou FX, Bradford H, et al: Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain. Arch Biochem Biophys 271:356-358, 1989 18. Murphy WG, Moore JC, Kelton JG: Calcium-dependent cysteine protease activity in the sera of patients with thrombotic thrombocytopenic purpura. Blood 70:1683-1687,1987 19. Habermann E, Arnts D, Just M, et al: Das Verhalten des Trasylol im Organismus als Model1 fuer die Pharmakokinetik basischer Polypeptide. Med Welt 24:1163-l 167, 1973 20. Fischer JH: Trasylol-Effeckte an der Neire-Temperatur und Dosisabhaengigkeit, in Dudziak R, Kirchhoff PG, Reuter HD, Schumann F. (eds): Proteolyse und Proteinaseninhibition in de1 Herz und Gefasschirurgie. New York, NY, Schattaer-Verlag, 1985. pp 127-135 21. Royston D, Minty BD, Wallwork J, et al: The effects of surgery with cardiopulmonary bypass on alveolar-capillary barrier function in man. Ann Thorac Surg 40:133-142, 1985 22. Royston D, Braude S, Nolop KB, Hughes JMB: 113m lndium protein flux does not reflect degree or outcome in respiratoIy failure. Am Rev Respir Dis 139:A380, 1989 23. Royston D, Fleming JS, Desai JB, et al: Increased peroxide product generation associated with open heart surgery; evidence for free radical generation. J Thorac Cardiovasc Surg 91:759-766. 1986 24. Braude S, Nolop KB, Fleming JB, et al: Increased pulmonary transvascular protein flux after canine cardiopulmonary bypass. Association with lung neutrophil sequestration and tissue peroxidation. Am Rev Respir Dis 134:867-872, 1986 25. Kirklin JK, Westaby S, Blackstone EH, et al: Complement and the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 86:845-852, 1983 26. Clasen C, Jochum M, Mueller-Ester1 W: Feasibility study of very high dose aprotinin in polytrauma patients, in Schlag G, Red1 H (eds): First Vienna Shock Forum. Pathophysiological Role of Mediators and Mediator Inhibitors in Shock. New York, NY, Liss, 1987, pp 175-183 27. van Oeveren W, Jansen NJ, Bidstrup BP, et al: Effects of aprotinin on haemostatic mechanisms during cardiopulmonary bypass. Ann Thorac Surg 44:640-645,1987 28. Bidstrup BP, Royston D, Taylor KM, Sapsford RN: Reduction in blood loss and blood use after cardiopulmonary bypass with high dose aprotinin (Trasylol). J Thorac Cardiovasc Surg 97:364372,1989 29. Bidstrup BP, Harrison J, Royston D, et al: Aprotinin therapy
APROTININ
TO PREVENT BLEEDING: THE FIRST 5 YEARS
in cardiac surgery: A report on use in 42 UK cardiac centres. Lancet 1991 (In press) 30. Goodnough LT, Johnston MF, Toy PTCY: The variability of transfusion practice in coronary artery bypass surgery. JAMA 265:86-90,199l 31. Russell GN, Peterson S, Harper SJ, Fox MA: Homologous blood use and conservation techniques in cardiac surgery in the United Kingdom. Br Med J 297:1390-1391,1988 32. Jones EL, Weintraub WS, Craver JM, et al: Coronary bypass surgery: Is the operation different today? J Thorac Cardiovasc Surg 101:108-115, 1991 33. Lytle BW, Loop FD, Cosgrove DM, et al: Coronary reoperations: Results and determinants of early and late survival. J Thorac Cardiovasc Surg 93:874-895,1987 34. Freidrich G, Weber C, Bernard C, et al: Reduction of blood transfusion requirement in open heart surgery by administration of high dose aprotinin-Preliminary results. Thorac Cardiovasc Surgeon 37:89-91,1989 35. Dietrich W, Barankay A, Dilthey G, et al: Reduction of homologous blood requirements in cardiac surgery by intraoperative aprotinin application: Clinical experience in 152 cardiac surgical patients. Thorac Cardiovasc Surg 37:29-98,1989 36. Alajmo F, Calamai G, Perna AM, et al: High-dose aprotinin: Hemostatic effects in open-heart operations. Ann Thorac Surg 48:536-539, 1989 37. Dietrich W, Spannagl M, Jochum M, et al: Influence of high-dose aprotinin treatment on blood loss and coagulation patterns in patients undergoing myocardial revascularization. Anesthesiology 73:1119-1126, 1990 38. Dietrich W, Hahnel C, Richter JA: Routine application of high-dose aprotinin in open heart surgery-A study in 1,784 patients. Anesthesiology 73:A146, 1990 39. Royston D: Aprotinin in open heart surgery; Background and results in patients having aortocoronary bypass grafts. Perfusion 5:63-72,199O (suppl) 40. Royston D: Clinical effectiveness of aprotinin in heart surgery, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 208220 41. Von der Emde J, Mahmoud FO, Esperer HD: Retransfusion of Postoperative Drainage Blood, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 129-132 42. Chesbro JH, Clements IP, Fuster V, et al: A plateletinhibitory drug trial in coronary artery bypass operations: benefits of perioperative dipyridamole and aspirin therapy on early postoperative vein-graft patency. N Engl J Med 307:73-78,1982 43. Ferraris VA, Ferraris SP, Lough FC, Berry W: Preoperative aspirin ingestion increases operative blood loss after coronary artery bypass grafting. Ann Thorac Surg 4.5:71-74, 1988 44. Sethi GK, Copeland JG, Goldman S, et al: Implications of preoperative administration of aspirin in patients undergoing coronary artery bypass grafting. J Am Co11 Cardiol1.5:15-20,199O 45. Bashein G, Nessly ML, Rice AL, et al: Preoperative aspirin therapy and reoperation for bleeding after coronary artery bypass surgery. Arch Int Med 151:89-93, 1991 46. Ferraris VA, Gildengorn V: Predictors of excessive blood use after coronary artery bypass grafting. J Thorac Cardiovasc Surg 98:492-497, 1989 47. Gluszko PR, Maring JK, Edmunds LH: Invited letter concerning bleeding time test. J Thorac Cardiovasc Surg 101:173-174, 1991 48. Royston D, Bidstrup BP, Taylor KM, et al: Aprotinin reduces bleeding after open heart surgery in patients taking aspirin and those with renal failure. Anesthesiology 71:A6, 1989
97
49. Royston D, Bidstrup BP, Taylor KM, et al: Reduction of bleeding after open heart surgery with aprotinin (Trasylol): Beneficial effects in patients taking aspirin and in those with renal failure, in Birnbaum DE, Hoffmeister HE (eds): Blood Saving in Open Heart Surgery. New York, NY, Shattauer, 1990, pp 66-75 50. Bidstrup BP, Royston D, McGuiness C, et al: Aprotinin in aspirin treated patients. Perfusion 5:77-81, 1990 51. Tabuchi N, van Oeveren W, Eijsman L, et al: Preserved hemostasis during the combined use of aprotinin and aspirin in CABG operations, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 245-251 52. Gavaghan TP, Gebski V, Baron DW: Immediate postoperative aspirin improves vein graft patency early and late after coronary artery bypass graft surgery. Circulation 83:1526-1533, 1991 53. Popov-Cenic S, Murday H, Kirchoff PG, et al: Anlage und zusammenfassendes Ergebnis einer klinischen Doppelblindstudie bei aorto-koronaren Bypass-Operationen, in Dudziak R, Kirchhoff PG, Reuter HD, Schumann F, (eds): Proteolyse und ProteinasenInhibition in der Herz und Gefasschirurgie. New York, NY, Schattauer, 1985, pp 171-186 54. Murday H, Orellano L, Fenyes J, et al: Sind postoperative Blutungeb in der Herzchirurgie durch behandlung mit Aprotinin bzw. Cl inhibitor vermeidbar? Thorac Cardiovasc Surg 44:88, 1984 55. Elliot MJ, Allen A: Aprotinin in paediatric cardiac surgery. Perfusion 5:73-76,199O (suppl) 56. Dawkins KD, Jamieson SW, Hunt SA, et al: Long term results, haemodynamics, and complications after combined heartlung transplantation. Circulation 71:919-929,1985 57. Novick RJ, Menkis FN, Reid KR, et al: Reduction in bleeding after heart-lung transplantation: the importance of posterior mediastinal haemostasis. Chest 98:1383-1387, 1990 58. Brier M, Royston D, Bidstrup BP, Aronoff G: Kinetics of aprotinin during open heart surgery. J Pharmacol Therap 47:183, 1990 59. Struck E: Blood saving strategies in cardiac surgery, in Birnbaum DE, Hoffmeister HE (eds): Blood Saving in Open Heart Surgery. New York, NY, Schattauer, 1990, pp 16-28 60. Angelini GD, Cooper GJ, Lamarra M, Bryan AJ: Unorthodox use of aprotinin to control life-threatening bleeding after cardiopulmonary bypass. Lancet 1:799-800, 1990 61. Bidstrup BP, Royston Sapsford RN, Taylor KM: Effect of aprotinin on need for blood transfusions in patients with endocarditis having open heart surgery. Lancet 1:366-367, 1988 62. Koestering H, Kirchoff PG, Voelker P, et al: Untersuchungen der Blutgerinnungsveraenderungen wahrend und nach Operationen mit Hilfe der Herz-Lungen - Maschine. Thoraxchirurge 21:534-543, 1978 63. Elliot MJ: Perfusion for pediatric open heart surgery. Sem Thorac Cardiovasc Surg 2:332-340, 1990 64. van Oeveren W, Harden MP, Roozendaal KJ, et al: Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovas Surg 99:788-797,199O 65. Hannekum A, Reuter HD, Dalichau H, et al: Anlage und zussammenfassendes Ergebnis einer Klinischen Doppelblindstudie bei Operationen am offenen HerzenEinfluss von Aprotinin auf Thrombozytensahl und -funktion, in Dudziak R, Kirchhoff PG, Reuter HD, Schumann F (eds): Proteolyse und Proteinaseninhibition in der Herz - und Gefasschirurgie. New York, NY, Schattauer, 1985, pp 222-233 66. Scott D, Au J: The Edinburgh experience-Low dose Trasylol, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag. 1991, pp 263-265
98
67. Fraedrich G, Neukamm K, Schneider T, et al: Safety and risk/benefit assessment of aprotinin in primary CABG, in Freidel N, Hetzer R, Royston D (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 221-231 68. Blauhut B, Gross C, Necek S, et al: Effects of high-dose aprotinin on blood loss, platelet function, fibrinolysis, complement, and renal function after cardiopulmonary bypass. J Thorac Cardiovas Surg 101:958-967,1991 69. Roth GJ: Developing relations; Arterial platelet adhesion, Glycoprotein Ib, and leucine rich glycoproteins. Blood 77:5-19, 1991 70. Hennessy VL, Hicks RE, Niewiarowski S, et al: Function of human platelets during extracorporeal circulation. Am J Physiol 232:H622-H628,1977 71. Glusko P, Rucinski B, Musial J, et al: Fibrinogen receptors in platelet adhesion to surfaces of extracorporeal circuit. Am J Physiol252:H615-H621,1987 72. Salzman EW: Blood platelets and extracorporeal circulation. Transfusion 3:274-277,1963 73. McKena R, Bachmann F, Whittacker BB, et al: The hemostatic defect after open heart surgery. II. Frequency of abnormal platelet functions during and after extracorporeal circulation. J Thorac Cardiovasc Surg 70:298-308, 1975 74. Cella G, Vittadello 0, Galluci, et al: The release of platelet factor 4 and beta-thromboglobulin during extracorporeal circulation for open heart surgery. Eur J Clin Invest 11:165-169,198l 75. Mezzano D, Aranda E, Urzua, et al: Changes in platelet b thromboglobulin, fibrinogen, albumin, 5-hydroxyttyptamine ATP and ADP during and after surgery with extracorporeal circulation in man. Am J Hematol22:133-142, 1986 76. Watkins WD, Peterson MB, Kong DL, et al: Thromboxane and prostacyclin changes during cardiopulmonary bypass with pulsatile perfusion. J Thorac Cardiovasc Surg 84:250-256, 1982 77. Ditter H, Heinrich D, Matthias FR, et al: Effects of prostacyclin during cardiopulmonary bypass in man on plasma levels of beta-thromboglobulin, platelet factor 4, thromboxane B2, 6-keto-prostaglandin Fl alpha and heparin. Thromb Res 32:393408,1983 78. Harker LA, Malpass TW, Branson HE, et al: Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass; aquired transient platelet defect associated with selective alpha granule release. Blood 56:824-834,198O 79. Musial J, Niewiarowski S, Hershock, et al: Loss of fibrinogen receptors from the platelet surface during stimulated extracorporeal circulation. J Lab Clin Med 105:514-526,1985 80. Zilla P, Faso1 R, Groscurth P, et al: Blood platelets in cardiopulmonary bypass operations. J Thorac Cardiovasc Surg 97:379-388, 1989 81. George JN, Pickett EB, Saucerman S, et al: Platelet surface glycoproteins; Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 78:340-348,1986 82. George JN, Nurden AT, Phillips DR: Molecular defects in interactions of platelets with the vessel wall. N Engl J Med 311:1084-1098,1984 83. Levine SP, Krentz LS: Development of a radioimmunoassay for human platelet factor 4. Thromb Res 11:673-686,1977 84. Abrams CS, Ellison N, Budzynski A, Shattil SJ: Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 75:128-138, 1987 85. Schulman S, Johnsson H: Heparin, DDAVP and the Bleeding Time. Thomb Haem 65:242-244,199l 86. Gimple LW, Gold HK, Leinbach RC, et al: Correlation between template bleeding times and spontaneous bleeding during
DAVID ROYSTON
treatment of acute myocardial infarction with recombinant tissue type plasminogen activator. Circulation 80:581-585,1989 87. Bick R: Hemostasis defects associated with cardiac surgery. prosthetic devices, and other extracorporeal cicuits. Semin Thromb Hemost 11:249-280,1985 88. Mammen E, Kocts MH, Washington DC, et al: Hemostasis changes during cardiopulmonary bypass surgery. Semin Thromb Hemost 11:281-292,1985 89. Stibbe J, Kluft C, Brommer EJP, et al: Enhanced fibrinolytic activity during cardiopulmonary bypass in open heart surgery in man is caused by extrinsic (tissue type) plasminogen activator. Eur J Clin Invest 14:375-382,1984 90. Tanaka K, Takao M, Yada I, et al: Alterations in coagulation and fibrinolysis associated with cardiopulmonary bypassnduring open heart surgery. J Cardiothorac Anesth 3:181-188,1989 91. Holloway DS, Summaria L, Sanesara J, et al: Decreased platelet numbers and function and increased fibrinolysis contribute to postoperative bleeding in cardiopulmonary bypass patients. Thromb Haemost 59:62-67,198s 92. Van Riper DF, Horrow JC, Osborne D: Is the thromboelastograph a clinically useful predictor of blood loss after bypass? Anesthesiology 73:3A, A1206, 1990 93. Gram J, Janetzko T. Jespersen J, et al: Enhanced etfectivr fibrinolysis following the neutralization of heparin in open heart surgery increases the risk of post-surgical bleeding. Thromb Haemost 63:241-245, 1990 94. Marengo-Rowe AJ, Levenson JE: Fibrinolysis: A frequent cause of bleeding, in Ellison N, Jobes DR (eds): Effective Hemostasis in Cardiac Surgery. Philadelphia. PA, Saunders, 1988, pp 41-55 95. Spiess BD, Tuman KJ, McCarthy RJ, et al: Thromboelastographic diagnosis of coagulopathies in patients undergoing cardiopulmonary bypass. Anesth Analg 68:53-72, 1989 96. Horrow J, Hlavacek J, Strong MD, et al: Prophylactic tranexamic acid decreases bleeding after cardiac operations. J Thorac Cardiovasc Surg 99:70-74, 1990 97. Van der Salm TJ, Ansell JE, O’Kike ON, et al: The role ot epsilon aminocaproic acid in reducing bleeding after cardiac operations; a double blind randiomized study. J Thorac Cardiovasc Surg 96:538-540,198s 98. Fish KJ, Sarnquist FH, van Steennis C, et al: A prospective, randomized study of the effects of prostacyclin on platelets and blood loss during coronary bypass operations. J Thorac Cardiovasc Surg 91:436-442,1986 99. Teoh KH, Cristakis GT. Weisel RD, et al: Dipyridamole preserved platelets and reduced blood loss after cardiopulmonary bypass. J Thorac Cardiovasc Surg 96:332-341, 1988 100. Harke H, Gennrich M: Aprotinin-ACD Blut. I. Experimentelle Untersuchungen uber den Einfluss von Aprotinin auf die plasmatische und thrombozytare. Gerinnun Anesthetist 29:266276,198O 101. Aoki N, Yoshida N: Inhibition of platelet aggregation by protease inhibitors. Possible involvement of proteases in platelet aggregation. Blood 52:1-12, 1978 102. Ketterl R, Haas S, Lechner F, et al: Wirkung von Aprotinin auf die Thrombozytenfunktion wahrendHtift-Totalendoprosthesenoperation. Med Welt 31:1239-1240, 1980 103. Ketterl R, Haas S, Heiss A. et al: Zur Wirkung des natiirlichen Proteinaseninhibitors Aprotinin auf die Plattchenfunktion beim alloarthroplastischen Huftgelenkersatz. Med Welt 33:480486,1982 104. Harke H, Steinen G, Rahman S, et al: Aprotinin ACD Blut II: Der Einfluss von Aprotinin auf die Freisetzung zellularere Medioraten un Enzyme im Konservenblut. Anaesthetist 31:165171,1982 105. Bode AP, Miller DT: The use of thrombin inhibitors and
99
APROTININ TO PREVENT BLEEDING: THE FIRST 5 YEARS
aprotinin in the preservation of platelets stored for transfusion. J Lab Chn Med 113:753-758,1989 106. Okumura T, Jam&on GA: Platelet glycocalicin 1: Orientation of glyoproteins of the human platelet surface. J Biol Chem 251:5944-5949,1976 107. Adelman B, Michelson AD, Loscalzo J, et al: Plasmin effect on platelet glycoprotein lb-von Willebrand factor interactions. Blood 65:32-40, 1985 108. Bode AP, Knupp CL, Miller DT: Effect of platelet activation inhibitors on the loss of glycoprotein Ib during storage of platelet concentrates. J Lab Clin Med 115:669-679, 1990 109. Commin PL, Lu H, Soria C, et al: Mechanisms of bleeding induced by aprotinin during cardiopulmonary bypass: A controlled study. Anesthesiology 73:3A, A1205, 1990 110. Mohr R, Golan operations on platelets.
M, Martinowitz U, et al: Effect of cardiac J Thorac Cardiovas Surg 92:434-441, 1986
111. Lazenby WI), Russo I, Zadeh BJ, et al: Treatment with desmopressin acetate in routine coronary artery bypass surgery to improve postoperative haemostasis. Circulation 82:IV 413-419, 1990 (suppl5) 112. Cramer ER, Lu H, Caen JP, et al: Differential redistribution of platelet glycoproteins Ib and IIb/IIIa after plasmin stimulation. Blood 77~694-699, 1991 113. Michelson AD, Adelman B, Bernard MR, et al: Platelet storage results in a redistribution of glycoprotein Ib molecules; evidence for a large intraplatelet pool of glycoprotein IB. J Clin Invest 81:1734-1740, 1988 114. Colman RW, Scott CF, Schmier AU, et al: Initiation of blood coagulation at artificial surfaces. Ann NY Acad Sci 56:253267,1987 115. Miles gen interacts nisms. J Clin 116. Miles of the platelet
LSA, Ginsberg MA, White JG, Plow EF: Plasminowith human platelets through two distinct mechaInvest 77:2001-2009, 1986 LA, Plow EF: Binding and activation of plasminogen surface. J Biol Chem 260:4303-4311,1985
117. Parf S, Harker LA, Marzec UM, Levin EG: Demonstration of single chain urokinase type plasminogen activator on human platelet membrane. Blood 73:1421-1425,1989 118. Kluft C, Dooijewaard G, Emeis JJ: Role of the contactsystern in fibrinolysis. Semin Thromb Haemost 13:50-68, 1987 119. Kowalski E, Kopec M, Wegrzynowicz Z: Influence of fibrinogen degradation products (FDP) on platelet aggregation, adhesiveness and viscous metamorphosis. Thromb Diath Haemorrh 10:406-413, 1963 120. de Bono DP, Pringle S, Underwood I: Differential effects of aprotinin and tranexamic acid on cerebral bleeding and cutaneous bleeding time during rt-PA infusion. Thromb Res 61:159-163,199l 121. Kornecki E, Ehrlich YH, de Mars DD, Lenox RH: Exposure of fibrinogen receptors in human platelets by surface proteolysis with elastase. J Clin Invest 77:750-756, 1986 122. Royston D: Free radicals; formation, function and potential relevance in anaesthesia. Anaesthesia 43:315-320, 1988 123. Henson P: Mechanisms of release of constituents from rabbit platelets by antigen-antibody complexes: II. Interactions between platelets and neutrophils. J Immunol X:490-498, 1970 124. Clark RA, Klebanoff SJ: Neutrophil platelet interactions are mediated by myeloperoxidase or hydrogen peroxide. J Immunol 124:399-407, 1980 125. Wachtfogel YT, Kucich U, James HL, et al: Human plasma kallikrein releases neutrophil elastase during blood coagulation. J Clin Invest 72:1672-1677, 1983 126. Zimmerli W, Huber I, Bouma BN, Lammle B: Purified human plasma kallikrein does not stimulate but primes neutrophils for superoxide production. Thromb Haemost 62:1121-1125,1989
127. Francis JL, Lord RAM, Roath OS: The effect of aprotinin on haemostasis and white cell function in aortic surgery. Thromb Haemost (In press) 128. Royston D, Bidstrup BP, Fleming JS: Platelet preservation reduces production of lipid peroxidation during cardiopulmonary bypass in humans. Am Rev Resp Dis 135:A13,1987 129. Ruggerio M, Lepetina EG: Protease inhibitors synergistically prevent activation Proc Nat1 Acad Sci USA 83:3456-3459, 1986
and cyclooxygenase of human platelets.
130. Ruggerio M, Lepetina EG: Antipain and leupeptin in combination with aspirin or indomethacin synergistically inhibit platelet activation by thrombin and trypsin. Adv Prost Thrombox Leukotr Res 17A:563-568,1987 131. Royston D: Effects of aprotinin lysis associated with open heart surgery. (In press)
on the whole blood clot Blood Coag Fibrin01 1991
132. Spiess BD, Tuman KJ, McCarthy RJ, et al: Thromboelastography as an indicator of post-cardiopulmonary bypass coagulopathies. J Clin Monit 3:25-30, 1987 133. Kang Y, Lewis JM, Navalgund A, et al: Epsilon amino caproic acid for the treatment of fibrinolysis during liver transplantation. Anesthesiology 66:766-773, 1987 134. Clozel JP, Banken L, Roux S: Aprotinin: An antidote for recombinant tissue-type plasminogen activator (rt-PA) active in vivo. J Am Co11 Carcliol16:507-510, 1990 135. Wiman B: On the streptokinase complex with Thromb Res 17:143-152,198O
reaction aprotinin
of plasmin or plasminor alpha 2 antiplasmin.
136. Gverlack A, Stumpe KO, Kuehnert M, et al: Evidence for participation of kinins in the antihypertensive effect of converting enzyme inhibition. Klin Wochenschr 59:69-74, 1981 137. Kurusz M, Schneide B, Brown BP, et al: Filtration during open heart surgery: Devices, techniques, opinions, and complications. Proc Am Acad Cardiovasc Perf 4:123-129, 1983 138. Royston D, Bidstrup BP, Sapsford RN, Taylor KM: Reduced blood loss after open heart surgery with aprotinin is associated with an increase in the activated clotting time (ACT). J Cardiothorac Anesth 3:80,1989 139. de Smet AAEA, Increased anticoagulation tinin. J Thorac Cardiovasc
Joen MCN, van Oeveren W, et al: during cardiopulmonary bypass by aproSurg 100:520-527, 1990
140. Royston D: A prolonged activated clotting time unreliable indicator of thrombus formation during aprotinin apy. J Thorac Cardiovasc Surg (In press) 141. Pratt CW, Church FC: Antithrombin: tion. Semin Hematol28:3-9,199l
Structure
is an ther-
and func-
142. Ross DN, Simpson JC: Aprotinin; Effect on redo surgery, in Freidel N, Hetzer R, Royston D, (eds): Blood Use in Cardiac Surgery. New York, NY, Springer-Verlag, 1991, pp 260-262 143. Dietrich W, Barankay A, Niekau E, et al: High-dose aprotinin in cardiac surgery. Old drug-new aspects of homologous blood requirement, in Birnbaum DE, Hoffmeister HE (eds): Blood Saving in Open heart Surgery. New York, NY, Schattauer, 1990, pp 76-82 144. Henderson WG, Goldman S, Copeland JG, et al: Antiplatelet or anticoagulant therapy after coronary artery bypass surgery: A meta-analysis of clinical trials. Ann Int Med 111:743-750,1989 145. Royston BD, Royston platelet adhesion to thrombin Thromb Haemost (In press)
D, Pearson JD: Aprotinin inhibits stimulated human endothelial cells.
146. Gould RJ, Gan Z-R, Polokoff MA, et al: Disintegrins; A family of integrin inhibitory proteins from viper venoms. Faseb J (In press)
100
147. Ohno H, Kambayashi J, Chang SW, Kosaki G: Foy: [Ethyl-p (guanidinohexanyloxy) benzoate] methylsulfonate as a serine protease inhibitor. II In Vivo effect on coagulo-fibrinolytic system in comparison with heparin and aprotinin. Thromb Res 24:445-452,198l 148. Taenaka N, Shimada Y, Hirata T, et al: New approach to regional anticoagulation in haemodialysis. Crit Care Med 10:773175,1982 149. Solbereg S, 0sterud B, Larsen T, Sorlied D: Lack of ability to synthesize tissue factor by endothelial cells from intact saphenous vein. Blood Coag Fibrinol 1991 (In press)
DAVID ROYSTON
150. Bidstrup BP: Aprotinin therapy has no effects on early graft patency. Ldncet (In press) 151. Harke H, Rahman S: Clinical relevance of the membrane protective action of aprotinin on the intraoperative histamine liberation. Prog Clin Biol Res 308:959-963, 1989 152. Weiss ME, Adkinson NF, Hirshman CA: Evaluation of allergic drug reactions in the perioperative period. Anesthesiology 71:483-486, 1989 153. Freeman JG, Turner GA, Venables CW, et al: Serial use 01 aprotinin and incidence of allergic reactions. Curr Med Res Opin 8:559-561. 1983
