Clinical Reviews in Allergy 9 Copyright 1991 by The Humana Press Inc. 0731-8235/91/339-355/$3.40
Allergy to Protamine Michael E. Weiss 1 and N. Franklin Adkinson, Jr.*,2 ~Assistant Clinical Professor of Medicine, University of Washington Hospital, Seattle, Washington; and2Professor of Medicine, Johns Hopkins University School of Medicine, Division of Clinical Immunology at the Johns Hopkins Asthma and Allergy Center, Baltimore, MD
INTRODUCTION
Protsm~nes are foreign proteins that, in nature, are bound to nuclear material in fish sperm heads (1). They were first discovered and named in 1868 by Friedrick Miescher during his investigations of the cell nucleus. Subsequent studies showed that protamines were simple proteins, and using prot~mines as prototypes, Miescher and his colleagues helped establish the fundamentals of protein chemistry (2). ProtAm~nes are polycationic, containing multiple positively charged arginine residues. In fact, nearly two-thirds of the amino acid composition of the 4500 dalton tool wt molecule is arginine (1). The multiple positive charges on prot~m~ne (arginine residues) associate with the negatively charged phosphate groups in sperm DNA to form the nucleoprotAmine complex (Fig. 1). At present, commercially available prot~mine is prepared from salmon m~lt, which has been defatted, precipitated with alcohol and sodium chloride, and then depyrogenated. In the late 1930s, the two major medicinal uses of protsmine were established. Hagedorn et al. discovered that combining protamine with insulin retarded the absorption of insulin (3). This allowed insulin-dependent diabetic patients to achieve euglycemia *Authorto whom all correspondenceand reprint requestsshould be addressed.
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Fig. 1. Schematic representation of spiraling chain of protamine interacting with DNA of sperm. The positively charged arginine residues bind with the negatively charged phosphate groups in the DNA grooves. From Horrow (1), reproduced with permission of the International Anesthesia Research Society.
with less frequent insulin injections. Isophane (NPH) insulin contains 0.3-0.5 mg protamine/100 U of inslflin, and protaminezinc iusulin (PZI) contains 1-1.7 mg protamine/100 U of insulin. The second major medicinal use ofprotamine, that of reversing heparin anticoagulation, was first reported by Chargaff and Olson (4). Heparin, a polyanionic mucopolysaccharide, induces anticoagulation by activating antithrombin III. The polycationic protamlne combines ionically with the polyanionic heparin to form a stable complex that is devoid of anticoagulant activity. Each milllgram ofprotamine neutralizes approx 90 USP U ofheparin activity derived from lung tissue or about 115 USP U of heparin activity derived from intestinal mucosa. Because heparin disappears rapidly from the circulation, the dose of protamine required also
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decreases rapidly with the time elapsed following iv injection of heparin. Therefore, it is useful to follow coagulation studies, such as the activated dotting time (ACT), to help determine the zmount of prot~mine required to reverse heparin anticoagulation (5). Heparin anticoagulation is frequently used during vascular surgery, cardiac catheterization, dialysis, and phoresis. The use of iv prot~mine to reverse heparin anticoagulation has increased in the last decade with the evolution of coronary artery bypass surgery and cardiac catheterization as common therapeutic and diagnostic modalities.
ADVERSEPROTAMINEREACTIONS With its increased use came increased reports of adverse reactions associated with iv protamine administration (6-17). Acute prot~mine reactions include mild reactions, such as rash, urticaria, and transient mild elevations in pnlmonary artery pressure. Other reactions are more severe, and may include bronchospasm, hypotension, and at times, cardiovascular collapse and death (6). Adverse reactions to protamine may include any or all of the abovementioned clinical and hemodynamic manifestations, Because systematic, prospective studies are now just being undertaken, the exact incidence of adverse reaction to iv protamine is presently unclear. The task is made more difficult because multiple immune and nonimmune mechanisms may be involved in adverse prot_~mine reactions (16,18) (see below). Where data exist, incidence rates vary widely. One retrospective study consisting of 2996 consecutive coronary artery bypass patients receiving iv prot~mine between 1981-1986 found that 2.9% of diabetic patients who had been on protarnlne-insuliu injections developed hypotension following protamine administration, compared with 0.076% ofnondiabetics (12). A second retrospective study of 651 consecutive cardiac catheterization patients between 19801982 found that 26.6% of diabetic patients who had been receiving protsmine-insulin injections developed significant hypotension requiring treatment, following iv protamine administration, compared with 0.4% of patients with no prior exposure to protamine-insulin injections (11). A prospective study looking only at
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Weiss and Adkinson Table 1 Groups at Putative Increased Risk for Protamine Reactions
Diabetics receiving injections ofinsullns containing protamine Isophane (NPH)---0.3-0.5 rag protamine/100 U insulin Protamine-zinc--l-l.7 mg protamine/100 U insulin Fish allergic individuals Vasectomized men Prior exposure to iv protamine given for reversal of heparin anticoagulation
pulmonary vasoconstriction and not hypotension found that 1.2% of 904 consecutive cardiopulmonary bypass patients developed pulmonary artery pressure elevation following iv protamine administration (10).
HIGH-RISK GROUPSFOR PROTAMINEREACTIONS Certain patient populations are at higher risk for developing adverse reactions to iv protamine (Table 1). As noted above, diabetic patients receiving daily subcutaneous injections of protamineinsulin preparations have a 40- to 50-fold increased risk of adverse reactions when given protamine intravenously (11,12). These studies, along with mlmerous reports of protamine reactions in protamine-insulin dependent diabetic patients (6-9,14,17,18), suggest that prior immunologic sensitization by subcutaneous injections ofproto_mine are, in some part, responsible for subsequent reactions when 100- to 1000-fold larger doses of protamine are administered intravenously. We published a case-control study showing that, in diabetic patients who had received prot~mlue-insulin injections, the presence ofsez~m antiprotamine IgE antibodies was a significant risk factor for acute protamine reactions (relative risk = 95), as was antiprot~mlne IgG antibodies (relative risk = 38) (16). Another group at theoretical risk for protamine reactions are fish-allergic individvals. Since prot~mine is produced from the sperm or matured testes of sulmon or related species of fish, it has been
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suggested that individuals allergic to fish may have semlm antibodies directed against protamine, or conversely, that commercial prot~mlne preparations may be contaminated with other fish antigens that fish-allergic patients react to. To date, studies supporting the increased risk of prot~miue reactions in fish-allergic patients is lacking and Hm~ted to case reports (19,20). Vasectomized men are another group at putative high risk for proWmine reactions. Beginning at puberty, a"blood-testes" barrier sequesters developing sperm from the rest of the body, providing immunotolerance to the developing sperm. With occlusion of ejaculatory paths at vasectomy, sperm are absorbed systemically. Samuel showed that, with disruption of the blood-testes barrier following vasectomy, about 65% of men developed hemagglutinating autoantibodies against sperm and 22-30% developed autoantibodies against human proteins similar to protamine (21,22). Sera from some vasectomlzed men contained an antibody that crossreacted with s~lmon pro~mlne as measured by a microcomplement fixation test (23). We have recently found that 35% of vasectomized men have antiprotamine IgE antibodies in their serum compared to 0% in age matched controls (24). Finally, prior exposure to iv protemine given for reversal of heparin anticoagulation may theoretically increase the risk of a reaction on subsequent protamine administration. Sharath described a patient who suffered a fatal reaction to iv protamine 1 mo after an uneventful initial exposure to iv protamine (6). Considering the large number of patients who receive protamine during cardiac catheterization and then are rechaUenged at the conclusion of cardiopulmonary bypass during their coronary artery bypass surgery, it seems unlikely that a single iv exposure to protamine is likely to stimulate significant antiprotamine antibodies. However, Lakin et al. (13) found that none of 22 hemaphoresis donors experienced reactions on their first exposure to iv protamine, whereas 4 of 11 donors experienced urticaria, bronchospasm, and/ or hypotension on their second exposure to protamine following hemaphoresis. We are presently evaluating the development of antipro~mine antibodies in patients following the administration of a large iv dose of protamine.
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Weiss and Adkinson MECHANISMSOF PROTAMINEREACTIONS
The exact mechanism(s) by which proWmine produces adverse reactions are incompletely understood at present. A ~ m a l studies initiallysuggested that prot~mine could cause direct,nonimmunologic release of histamine from rat and hamster peritoneal mast cellsin vitro (25). Subsequent studies using hvman basophils and mast cellshave been unable to demonstrate hisWmlne releasewith protsmine at concentrationsup to 30-100 Hg/mI, (17,26,27). Differences a m o n g species, such as those described above, m a k e extrapolating from anlmal experiments to humans dlmcult. Tobin et al. (28) found that proWmine, although not being able to cause nonimmunologic basophil histamine releasein humans, m a y potentiate IgE-mediated release of histAmlne from human basophils in vitro through a polycationin-recognitionsite. Also, protamineheparin complexes can activatecomplement by the classicpathway both in vitroand in vivo leading to the generation of anaphylatoxins C3a, C4a, and C5a (29-32). Lakin et al. (13) provided evidence in a patient who developed urticaria,bronchospasm, and life-threateninghypotension following pro~mlne administrationthat complement-fixing antiprot~mlne IgG antibodies were involved in the acute reaction. Since then, others have also noted an associationbetween adverse protam~ne reactions and the presence of antipro~mine IgG antibodies (1416). W e conducted a case-controlstudy and found that, in diabetic patients who had received protamine-insulin injections,the presence of serum antiprot~m~ne IgG antibody was a significantrisk factor for acute prot~m~ne reactions (relativerisk = 38). Similarly, in patients without previous exposure to pro~m~ne-insulin injections, the presence of antiprotamine IgG antibody was also a significant risk factor for protamine reactions (relativerisk = 25) (16) (Table 2). Using the technique of heterologous interpolation (33,34),we quantitated the amount of antiprotam~ne IgG antibody in the sera of these patients.Protamlne-insulin-dependent diabetic patients who had acute reactions to intravenously adm~rtistered protamine had significantlyhigher levels of antiprotamine IgG antibodies than other patients (median = 36.4 ~tg/mL,p < 0.0002). In fact,antiprotamine IgG levelsin some of these patients were as high as 200 ~tg/mL, levels comparable to hyperimmunized beeClinical Reviews in Allergy
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Table 2 Association of Protamine Reactions and the Presence of Serum IgE and IgG Antibodies to Protamine Protamine-specific antibody
Cases, reaction
Controls, no reaction
Relative risk
Lower 95% C.L.
p Value
With prior exposure to subcutaneous protamine-insulin preparations IgE present
9
0
IgE absent
4
22
IgG present
11
2
IgG absent
2
95
6.6
1.0 x 10-5
38
5
1.2 x 10-5
20
No prior exposure to subcutaneous protamine-insulin preparations IgE present
0
0
IgE absent
14
21
IgG present
5
0
NA
NA
0.99
25 1.6 0.006 IgG absent 9 21 C. L. = confidence limit, NA = not applicable. From Weiss (16), reproduced with permission of the Massachusetts Medical Society.
keepers (16). Using a double-antibody, radioimmunoprecipitation assay, Kurtz et al. (35) found that 38-91% ofprotamine-insulindependent diabetic patients had serum antiprotamine IgG antibodies. Using an ELISA, Nell and Thomas (36) found that 38% of 319 NPH insulin-treated diabetic patients and only 2.5% of 202 normal control subjects had serum antiprotamine IgG antibodies. Neither of these two studies quantitated the amount ofantiprotamine IgG antibodies. Preliminary data suggest that antiprotamine IgG antibodies are predominantly of the complement-fixing IgG 1subclass (15) (personal observation). Thus, the ability of protamine to activate complement, either through protamine-heparin complexes or through protamineantiprotamine IgG antibody interaction, with the subsequent generation of anaphylatoxins, has been implicated in causing some protamine reactions, particularly those associated with pulmonary Clinical Reviews in Allergy
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vasoconstriction (see below). Once generated, anaphylatexins can induce histamine release from human mast cells and basophils, cause neutrophil migration and release ofpolymorphonuclear cell enzymes, initiate platelet aggregation, cause smooth muscle contraction, and stimulate prostaglandin and thromboxane production from macrophages (37,38). Recent data suggested that protsmlue could inhibit plasma Carboxypeptidase N's ability to convert the anaphylatoxins and bradyldnins to the less active des arg metabolites (39), potentially allowing these vasoactive compounds previously generated to produce more significant hemodynRmic manifestations. Lowenstein and colleagues (18,40) found that pulmonary artery pressure elevation following rapid protamlne injection in three patients was associated with elevations of thromboxane B~ along with C5a. Plasma histamine levels were unchanged in these patients. In contrast, a patient who developed a decrease in systemic vascular resistance and profound systemic hypotension without pulmonary artery hypertension had a tenfold elevation in plasma his~mine and no change in thromboxane B~ or C5a levels. Examples of these two different hemodynsmic reactions are seen in Fig. 2. McIntyre et al. (42) also noted that an elevation in thromboxane B~ and 6-keto-PGFla, a stable metabolite of PGI 2, in a prot~mlrm-insuliu-dependent diabetic aider iv protAmlne administration resulted in pulmonary artery pressure elevation. Animal studies have shown that the elevation of pulmonary artery pressure and thromboxane following prot~mlne admiuistration could be blocked by inhibitors ofcycloxygenase or thromboxane synthetase (40,43-45). A study is presently planned to evaluate whether a thromboxane receptor blocker will be able to diminish the incidence of pulmonary artery pressure elevation following pro~mine administration in hnmAn_S. We developed an agarose-based radioallergosorbent test (RAST) to measure antiprotAmine IgE antibodies. We tested our ability to insolubflize the polycationic protRmine to the agarose beads by first immunizing rabbits to protamlne. Using a solid-phase prot~mlneagarose assay, there was a 25-fold increase in immunoreactive antibody in rabbits after proWmine immunization (coNfirmed by soluble inhibition assays), thus validating the assay. We next showed that, in diabetic patients who had received protsmine-insulin injections, the presence of serum antiproWmine IgE antibody was a significant Clinical Reviews in Allergy
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Fig. 2. Two hemodynamic patterns of acute protamine reactions. A. Acute protamine reaction showing pulmonary vasoconstriction with elevation of pulmonary artery pressure, and right atrial pressure followed by decline of left atrial pressure and systemic arterial pressure. B. Systemic hypotensionfollowing protamine administration without any change in pulmonary artery pressure. From Lowenstein (40) and Levy (41), reproduced with permission of the Journal of Cardiothorasic Anesthesia and Butterworth Publishers.
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risk factor for acute protam~ne reactions (relative risk = 95) (16). The presence of antiprotamine IgE was associated with reactions to prot~mlne that were of longer duration and that were more frequently severe in nature than reactions in patients without antiprot~m~ne IgE antibodies. In fact, nine of nine patients with antiprotAm~ne IgE antibodies experienced severe protamlne reactions involving hypotension and/or bronchospasm t h a t were potentially life-threatening. In general, these patients also tended to have very high levels of antiprotamine IgG antibodies (>10 pg/ mL) (16). In that study, we did not find antiprotamine IgE antibodies in any patient without prior exposure to protamineinsulin injections. Subsequently, we have observed one patient who had an acute protsmine reaction and antiprotamine IgE antibodies without having received previous subcutaneous protamine-insulin injections. We serially measured sen]m antiprotamine IgE and IgG antibody levels in an NPH-insulin-dependent diabetic who had a severe, protracted anaphylactic reaction to protamine. At the time of his pro~mlne reaction, his sez~m contained 8.5 ng/mI, of antiprotamlne IgE and 1.3 pg/ml, of antipro~mine IgG antibody. One month following the reaction, antiprotamlne IgE and IgG increased to 16 ng/mL (twofold rise) and 90.5 pg/mT,(70-fold rise), respectively. With time, both antipro~mine IgE and IgG antibody declined (17) (Fig. 3). Serial intradermal skin tests with native protamine did not discriminate between this profound prot_~mine reactor and nine norreal control subjects who had no prior exposure or any demonstrable serum IgE antibody to pro~mlne. Intradermal injections of protamine between 100-1000 pg/ml, induced irritative skin responses in normal control subjects. We were able to show that the protam~ne reactor's skin mast cells were able to respond to IgE-dependent stimuli by obtaining a positive wheel and flare skin response to an intradermal injection of rabbit F (ab')~ ant[human IgE antibody. Although reports ofprot~mlne reactors with positive prot~mine skin tests exist, two of these reports (7,14) do not provide information on the concentrations of protum~ne used for skin tests, and a third report (19) employed concentrations ofprotamine that we have shown caused false-positive, irritative reactions in the skin. Lakin et al. (13) reported a prot,mine reactor with a positive skin test to
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20
1O0
_15
80
,E=
60
40
9
5
"~
~
20
0 0
I 1
I 2
i 3
0 9
Months from reaction
Fig. 3. Serial antiprotamine IgE- and IgG-antibody levels in the serum of a protamine reactor. The patient had been receiving NPH insulin injections for 10 yr prior to receiving iv protamine at cardiac catheterization. He developed acute rash, bronchospasm, and life-threatening hypotension. At the time of the reaction, the patient's serum contained 8.5 ng/mL of antiprotamine IgE and 1.3 ~g/mL of antiprotamine IgG antibody. From Weiss (17), reproduced with permission of Blackwell Scientific Publications.
protamine at 0.001 ~ / m L , which should be a nonirritative concentration. A recent study found that results ofprotamine skin testing were not useful in predicting subsequent acute protamine reactions (46). In our patient, in vitro basophil histamine release to protomine was also inconclusive in discriminating between normal control subjects and the protamine reactor (17). Based on the above information, we postulate that protamine may be an incomplete or univalent antigen, which first must combine with a tissue macromolecule or possibly heparin to become a complete, multivalent antigen capable ofeliciting IgE antibody-dependent mediator release from mast cells and basophils. This testable hypothesis is presently being studied. In summary, protamine may elicit both immune and nonimmune adverse reactions through multiple mechanism.q dependent on its charge and antigenicity. Figure 4 shows the most likely mechanisms causing acute protamine reactions in humans.
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Weiss and Adkinson O + hepann ' ~
/ PROTAMINE
Protamine-heparin r
Complement activation (classic
e.,
+ Protamine-specific IgG Ab
,-ca e. e.,
(D Q
~
+ Protamine-speci fllc IgEAbboundto
|
+
Carboxypeptidase N
:#
C3, 4 ,5a des arg ~ ~
Mast cell / basophil " d ' ' ~ Mediator release
C3a, C4 a, C5a r
? cell source platelets PMNs TX / PG generation mast cells AIv. macro ? VEC ul vasoconstriction
PAP S[I R
Bronchoconstriction R heart failure (~afterload)
SBP q
~ L sided filling pressure
Fig. 4. Possible mechanism for acute protamine reactions. 1. Protamine combines with heparin to activate complement via the classic pathway. The generation of anaphylatoxins may cause mast cell or basophil mediator release, or may stimulate thromboxane and prostaglandin generation leading to pulmonary vasoconstriction. If severe, this could lead to acute right-sided heart failure and systemic hypotension. 2. Protamine combines with antiprotamine IgG antibody, leading to complement activation with the same cascade as noted in number 1.3. Protamine may inhibit the ability of carboxypeptidase N to convert the anaphylatoxins to their less active des arg metabolites. 4. Protamine crosslinks cell surface antiprotamine IgE antibodies on mast cells and basophils, leading to mediator release and systemic vascular dilatation and hypotension and/or bronchoconstriction. 5. Protamine may combine with a polycationic activation site on mast cells and basophils, and potentiate IgE-mediated histamine release. TX = thromboxane, PG = prostaglandins, PAP = pulmonary artery pressure, SVR = systemic vascular resistance, SBP = systemic blood pressure, VEC = vascular endothelial cells, AIv. macro = alveolar macrophage.
PROTAMINEALTERNATIVES Bemuse patientswho have had previous acute prot~miue reactions may, in the future,require reversalofheparin anticoagulation and because certainpatient populations may be at high risk for protamine reactions (i.e., protamine-insulin-dependent diabetics), alternatives to pro~mine for reversal of heparin anticoagulation Clinical Reviews in Allergy
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100
80
o
Protamine from Company
60
J3 .m r'r
--
0
A
9
B
[]
C
9
D
40
20
i
i
.001
.01
i
.1
i
1
S o l u b l e Protamine (mg/ml)
Fig. 5. Increasing concentrations of soluble protamine from four commercially available manufacturers (A-D) are plotted on the X axis. Antiprotamine antibody in sera from seven patients is inhibited from binding to the protamine solid phase in a similar fashion.
are needed. Premedication with glucocortcosteroids and antihistamines, which have been successfully employed to reduce the risk of recurrent reactions to radiocontrast media (47,48), has not been successful in preventing IgE-antibody-mediated reactions, such as those induced by penicillin (49,50). At present, no studies have been done to evaluate whether premedication regimens prevent or reduce the severity of acute prot~mine reactions. Since there are both intra- and interspecies differences in protsmines (2), we studied four commercially available protRmlrm preparations from different manufacturers to determine ff their abilities to bind to human antiprot~mine antibody differed. Ifa particular commercially available protamine is less immunoreactive with h u m a n antiprotsm~ne antibody, then it may be expected to cause less frequent and/or less severe reactions and, therefore, would be more advantageous to use. Seven protsmine-ins11Hn-dependent diabetics who had experienced life-threatening reactions to iv pro~mine and whose sera contained a mean of 67.4 ~g/mL (range of 16-200 roT,) ofantiprot~m~ne IgG antibody were evaluated. Inhibition studies, where each sen~m was preincubated with buffer and increasing concentrations of the four prot~mine preparations before addition to the prot~mine solid phase were done. As seen in Fig. 5, no signifiClinical Reviews in Allergy
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cant differences were found in the immunoreactivity of the four different prot~mlne preparations. Therefore, no benefit might be expected from using one commercial source of protamine over another (51). Allowing heparin anticoagulation to reverse spontaneously has been successfully accomplished in patients who had experienced previous acute protsmlne reactions, but this alternative is not without potential for significant hemorrhage and cannot be generally recommended. Hexadimethrine (Polybrene), a quartenary 3mmoni!lm polycationic salt, was used to reverse hepatin anticoagulation in the early days of cardiac surgeryin the United States. Reports of renal toxicity, in combination with animal studies demonstrating lesions of the kidney, adrenal cortex, and interior pituitary, led to the voluntary removal of hexadimethrine from the United States market. Hexadimethrine is available through the Food and Drug Administration (as a compassionate-use drug). We have successfully used it in two patients who had prior life-threatening reactions to protum~ne, both of whom had antiprotamine IgE antibodies (personal observation).
SUMMARY Recent advances in medicine, such as cardiac catheterization, phoresis, dialysis, and cardiopulmonary bypass technology, have increased the need for heparin anticoagulation. To antagonize heparin's effect and prevent hemorrhagic complications a f a r the procedure, protamine has likewise been used more frequently. With its increased use have come increased reports of adverse proWmlne reactions consisting of rash, urticaria, elevation of pulmonary artery pressure, systemic hypotension, and, at times, death. The elevation of pulmonary artery pressure, which appears to be a rather common occurrence in animals, may be an isolated finding without clinical consequences in humans. However, this pulmonary vasoconstriction may, when severe, lead to acute right-sided heart failure and systemic hypotension. Other protamine reactions involve a decrease in systemic vascular resistance and systemic hypotension without changes in pulmonary artery pressure. Causes of acute protsmine reactions may involve the generation of anaphyatoxins and prostanoids either from protsmine-heparin complexes or compleClinical Reviews in Allergy
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ment-fixing antiprotsmine IgG antibodies, from inhibition of plasmA Carboxypeptidase N, from crosslinldng of cell-surface antiprotsmine IgE on mast cells and basophils with subsequent mediator release, or from potentiation of IgE-mediated release of histamine through a polycationin-recognition site. Although we have come a long way in understanding the mechanisms by which protamine can cause its ill effects in humans, more work is clearly needed to define, in prospective studies, the incidence of and risk factors for protamine reactions in various patient groups, and to delineate more clearly which mechanisms are involved in each clinical type of acute protamine reaction. Hopefully, this will lead to strategies and prot~mine alternatives that will prevent or dimimsh, in frequency or severity, adverse protamine reactions. Alternatively, a dearer picture of the risk factors important for protamine reactions and the predictive value of diagnostic tests (e.g., protamine IgE antibody) can also minimize the clinical impact of this increasingly common adverse event.
ACKNOWLEDGMENTS This work was supported in part by grant (AI 20136) from the National Institutes of Health and by the United States Pharmacopeial Convention.
REFERENCES 1. Horrow, J. C. (1985), Anesth. Analg. 64, 348-361. 2. Delange R. J. and Smith, E. L. (1979), Neurath, H. and Hill, R. L. (eds.), The Proteins, Academic, New York, pp. 119-243. 3. Hagedorn, H. C., Jensen, B. N., Kraup, N. B., and Woodstrup, I. (1936), JAMA 1069 177-180. 4. Chargaff, E. and Olson, IL B. (1937), J. Biol. Chem. 122, 153-165. 5. Bull, B. S., Huse, W. M., Braver, F. S., and Korpman, R. A. (1975), J. Thorac. Cardiovasc. Surg. 69, 685-689. 6. Sharath, M. D. Metzger, W. J., Richerson, H. B., Scupham, R. IL, Meng, R. L., Ginsberg, B. H., and Weiler, J. M. (1985), J. Thorac. Cardiovasc. Surg. 90, 86-90. 7. Doolan, L., McKenzie, I., Krafchek, J., Parson, B., and Buxton, B. (1981), Anaesth. Intens. Care 9, 147.
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8. Chung, F. and Miles, J. (1984), Can. Anaesth. Soc. J. 31, 314-318. 9. Vontz, F. I~, Puestow, E. C., and Cahill, D. J., Jr. (1982), The American Surgeon 48, 549-551. 10. Lowenstein, E., Lynch, I~, Robinson, D. R., Fitzgibbon, C. M., and Zapol, W. (1988), Am. Rev. Resp. Dis. 137(abst.), 245. 11. Stewart, W. J., McSweeney, S. M., Kellett, M. A., Faxon, D. P., and Ryan, T. J (1984), Circulation 70, 788-792. 12. Gottschlich, G. M., Gravlee, G. P., and Georgitis, J. W. (1988),Ann. Allergy 61, 277-281. 13. Lakin, J. D., Blocker, T. J., Strong, D. M., and Yocum, M. W. (1977), J. Allergy Clin. Immunol. 61, 102--107. 14. Grant, J. A., Cooper, J. R., Albyn, I~ C., Bulmer, D., Farnam, J., and Yunginger, J. W. (1984), J. Allergy Clin. Immunol. 73(abst.), 180. 15. Gottschlich, G. M. and Georgitis, J. W. (1988), J. Allergy Clin. Immunol. 81 (abstract), 238. 16. Weiss, M. E., Nyhan, D., Zhikang, P., Harrow, J. C., Lowenstein, E., Hirshman, C., and Adkinson, N. F., Jr. (1989), N. Engl. J. Med. 320, 886-892. 17. Weiss, M. E., Chatham, F., Kngey-Sobotka, A., and Adkinson, N. F., Jr. (1990), Clinical Exper. Allergy, 20, 713-720. 18. Morel, D. R., Zapol, W. M., Thomas, S. J., Kitain, E. M., Robinson, D. R., Moss, J., Chenoweth, D. E., and Lowenstein, E. (1987), Anesthesiol. 66, 597-604. 19. Knape, J. T. A., Schuller, J. L., De Haan, P., De Jong, A. P., and Bovill, J. G. (1981), Anesthesiol. 55, 324,325. 20. Caplan, S. N. and Berkman, E. M. (1976), N. Engl. J. Med. 295, 172. 21. Samuel, T., Kolk, A. H. J., Rumke, P., and Van Lis, J. M. J. (1975), Clin. Exp. Immunol. 21, 65. 22. Samuel, T. and Linnet, L. (1978), Clin. Exp. Immunol. 33, 261. 23. Samuel, T. (1977), Clin. Exp. Immunol. 30, 181. 24. Adourian, U., Fischer, E., Adkinson, N. F. Jr., Hirshman, C. A. (1990) Anesthesiology, 73, A1257 (Abst.) 25. Keller, R. (1968), Int. Arch Allergy 34, 139-144. 26. Foreman, J. C. and Lichtenstein, L. M. (1980), Biochim. Biophys. Acta 629, 587-603. 27. Sauder, R. A. and Hirshman, C. & (1989), American Rev. Resp. Dis. 139, A485. 28 Tobin, M. C., Kams, B. K., Anse]mino, L. M., and Thomas, L. L. (1986), Molecular Immunology 23, 245-253. 29. Rent, R., Ertel, N., Eisenstein, R.,and Gewurz, H. (1975), J. Immunol. 114, 120-124. 30. Best, N., Teisner, B., Grudzinskas, J. G., and Fisher, M. M. (1983), Br. J. Anaesth. 55, 1149. 31. Best, N., Sinosich, M. J., Teisner, B., Grudzinskas, J. G., and Fisher, M. M. (1984), Br. J. Anaesth. 56, 339.
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32. Kirklin,J. K., Chenoweth, D. E., Naftel,D. C., Blackstone, E. H., Kirklin, J. W., Bitran,D. D., Curd, J. G., Reves, J. G., and Samuelson, P. N. (1986), Ann. Thor. Surg. 41, 193-199. 33. Hamilton, R. G. and Kagey-Sobotka, A. (1983),J. Clin. Immunoassay 6, 160-171. 34. Hamilton, R. G. and Adkinson, N. F. Jr. (1988), Kemeny, D. M. and Challacombe, S. J., eds.)ELISA and Other Solid Phase Immunoassays., John Wiley, Chichestor,pp. 57-84. 35. Kurtz, A. B., Gray, R. S., Markanday, S., and Nabarro, J. D. N. (1983), Diabetologia 25, 322-324. 36. Nell, L. J. and Thomas, J. W. (1988), Diabetes 37, 172-176. 37. Yancey, I~ B., Hammer, C. H., Harvath, L., Renfer, L., Frank, M. M.,and Lawley, T. J. (1985), J. Clin. Invest. 75, 486-495. 38. Ghebrehiwet, B. (1985), Kaplan, A. P. (ed.),Allergy. Churchill Livingstone, New York, pp. 131-152. 39. Skidgel, R. A., Tan, F., Jackman, H., Zsigmond, E. K., and Erdos, E. G. (1988), Fed. Proc. 2(abst.), A1382. 40. Lowenstein, E. (1989), J. Cardio.Thor. Aesth. 3, 99-107. 41. Levy, J. H. (1986), Anaphylactic Reactions in Anesthesia and Intensive Care, Buttorworths, Boston. 42. McIntyre, R. W., Flezzani, P., Knopes, I~ D., Reves, J. G., and Watkins, W. D. (1986), Amer. J. Cardiol. 58, 857,858.Hobbhahn, J., Conzen, P. F., Zenker, B., Goetz, A. E., Peter, IL, and Brendel, W. (1988), Anesth. Analg. 67, 253-260. 43. Hobbhahn, J., Conzen, P. F., Zenker, B., Goetz, ~ E., Peter, K., and Brendel, W. (1988), Anesth. Analg. 67, 253-260. 44. Degges, R. D., Foster, M. E., Dang, A. Q., and Read, R. C. (1987),Amer. J. Surg. 154, 696-699. 45. Morel, D. R., Lowenstein, E., Nguyenduy, T., Robinson, D. R., Repine, J. E., Chenoweth, D. E., and Zapol, W. M. (1988), Circulation Res. 62, 905-915. 46. Vegh, & B., Gellhaus, M. &, Benson, P. M., Hottel, R. A., and Weiler, J. M. (1989), J. Allergy Clin. Immunol. 18(abst.), 256. 47. Kelly, J. F., Patterson, R., Lieberman, P., Mathison, D. A., and Stevenson, D. D. (1978), J. Allergy. Clin. Immunol. 62, 181-184. 48. Greenberger, P. & (1984), J. Allergy Clin. Immunol. 74, 600-605. 49. Mathews, I~ P., Hemphill, F. M., Lovell, R. G., Forsythe, W. E., and Sheldon, J. M. (1956), J. Allergy 27, 1-15. 50. Sciple, G.W.,Knox, J.M.,andMontgomery, C.H.(1959),N. Engl. J. Med. 261, 1123-1125. 51. Adourian, U., Adkinson, N. F., Jr., I-Iirshman, C. A., and Weiss, M. E. (1990), Anesthesiology 73, 328--331.
Clinical Reviews in Allergy
Volume 9, 1991
Allergy to Protamine Michael E. Weiss 1 and N. Franklin Adkinson, Jr.*,2 ~Assistant Clinical Professor of Medicine, University of Washington Hospital, Seattle, Washington; and2Professor of Medicine, Johns Hopkins University School of Medicine, Division of Clinical Immunology at the Johns Hopkins Asthma and Allergy Center, Baltimore, MD
INTRODUCTION
Protsm~nes are foreign proteins that, in nature, are bound to nuclear material in fish sperm heads (1). They were first discovered and named in 1868 by Friedrick Miescher during his investigations of the cell nucleus. Subsequent studies showed that protamines were simple proteins, and using prot~mines as prototypes, Miescher and his colleagues helped establish the fundamentals of protein chemistry (2). ProtAm~nes are polycationic, containing multiple positively charged arginine residues. In fact, nearly two-thirds of the amino acid composition of the 4500 dalton tool wt molecule is arginine (1). The multiple positive charges on prot~m~ne (arginine residues) associate with the negatively charged phosphate groups in sperm DNA to form the nucleoprotAmine complex (Fig. 1). At present, commercially available prot~mine is prepared from salmon m~lt, which has been defatted, precipitated with alcohol and sodium chloride, and then depyrogenated. In the late 1930s, the two major medicinal uses of protsmine were established. Hagedorn et al. discovered that combining protamine with insulin retarded the absorption of insulin (3). This allowed insulin-dependent diabetic patients to achieve euglycemia *Authorto whom all correspondenceand reprint requestsshould be addressed.
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Fig. 1. Schematic representation of spiraling chain of protamine interacting with DNA of sperm. The positively charged arginine residues bind with the negatively charged phosphate groups in the DNA grooves. From Horrow (1), reproduced with permission of the International Anesthesia Research Society.
with less frequent insulin injections. Isophane (NPH) insulin contains 0.3-0.5 mg protamine/100 U of inslflin, and protaminezinc iusulin (PZI) contains 1-1.7 mg protamine/100 U of insulin. The second major medicinal use ofprotamine, that of reversing heparin anticoagulation, was first reported by Chargaff and Olson (4). Heparin, a polyanionic mucopolysaccharide, induces anticoagulation by activating antithrombin III. The polycationic protamlne combines ionically with the polyanionic heparin to form a stable complex that is devoid of anticoagulant activity. Each milllgram ofprotamine neutralizes approx 90 USP U ofheparin activity derived from lung tissue or about 115 USP U of heparin activity derived from intestinal mucosa. Because heparin disappears rapidly from the circulation, the dose of protamine required also
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decreases rapidly with the time elapsed following iv injection of heparin. Therefore, it is useful to follow coagulation studies, such as the activated dotting time (ACT), to help determine the zmount of prot~mine required to reverse heparin anticoagulation (5). Heparin anticoagulation is frequently used during vascular surgery, cardiac catheterization, dialysis, and phoresis. The use of iv prot~mine to reverse heparin anticoagulation has increased in the last decade with the evolution of coronary artery bypass surgery and cardiac catheterization as common therapeutic and diagnostic modalities.
ADVERSEPROTAMINEREACTIONS With its increased use came increased reports of adverse reactions associated with iv protamine administration (6-17). Acute prot~mine reactions include mild reactions, such as rash, urticaria, and transient mild elevations in pnlmonary artery pressure. Other reactions are more severe, and may include bronchospasm, hypotension, and at times, cardiovascular collapse and death (6). Adverse reactions to protamine may include any or all of the abovementioned clinical and hemodynamic manifestations, Because systematic, prospective studies are now just being undertaken, the exact incidence of adverse reaction to iv protamine is presently unclear. The task is made more difficult because multiple immune and nonimmune mechanisms may be involved in adverse prot_~mine reactions (16,18) (see below). Where data exist, incidence rates vary widely. One retrospective study consisting of 2996 consecutive coronary artery bypass patients receiving iv prot~mine between 1981-1986 found that 2.9% of diabetic patients who had been on protarnlne-insuliu injections developed hypotension following protamine administration, compared with 0.076% ofnondiabetics (12). A second retrospective study of 651 consecutive cardiac catheterization patients between 19801982 found that 26.6% of diabetic patients who had been receiving protsmine-insulin injections developed significant hypotension requiring treatment, following iv protamine administration, compared with 0.4% of patients with no prior exposure to protamine-insulin injections (11). A prospective study looking only at
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Weiss and Adkinson Table 1 Groups at Putative Increased Risk for Protamine Reactions
Diabetics receiving injections ofinsullns containing protamine Isophane (NPH)---0.3-0.5 rag protamine/100 U insulin Protamine-zinc--l-l.7 mg protamine/100 U insulin Fish allergic individuals Vasectomized men Prior exposure to iv protamine given for reversal of heparin anticoagulation
pulmonary vasoconstriction and not hypotension found that 1.2% of 904 consecutive cardiopulmonary bypass patients developed pulmonary artery pressure elevation following iv protamine administration (10).
HIGH-RISK GROUPSFOR PROTAMINEREACTIONS Certain patient populations are at higher risk for developing adverse reactions to iv protamine (Table 1). As noted above, diabetic patients receiving daily subcutaneous injections of protamineinsulin preparations have a 40- to 50-fold increased risk of adverse reactions when given protamine intravenously (11,12). These studies, along with mlmerous reports of protamine reactions in protamine-insulin dependent diabetic patients (6-9,14,17,18), suggest that prior immunologic sensitization by subcutaneous injections ofproto_mine are, in some part, responsible for subsequent reactions when 100- to 1000-fold larger doses of protamine are administered intravenously. We published a case-control study showing that, in diabetic patients who had received prot~mlue-insulin injections, the presence ofsez~m antiprotamine IgE antibodies was a significant risk factor for acute protamine reactions (relative risk = 95), as was antiprot~mlne IgG antibodies (relative risk = 38) (16). Another group at theoretical risk for protamine reactions are fish-allergic individvals. Since prot~mine is produced from the sperm or matured testes of sulmon or related species of fish, it has been
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suggested that individuals allergic to fish may have semlm antibodies directed against protamine, or conversely, that commercial prot~mlne preparations may be contaminated with other fish antigens that fish-allergic patients react to. To date, studies supporting the increased risk of prot~miue reactions in fish-allergic patients is lacking and Hm~ted to case reports (19,20). Vasectomized men are another group at putative high risk for proWmine reactions. Beginning at puberty, a"blood-testes" barrier sequesters developing sperm from the rest of the body, providing immunotolerance to the developing sperm. With occlusion of ejaculatory paths at vasectomy, sperm are absorbed systemically. Samuel showed that, with disruption of the blood-testes barrier following vasectomy, about 65% of men developed hemagglutinating autoantibodies against sperm and 22-30% developed autoantibodies against human proteins similar to protamine (21,22). Sera from some vasectomlzed men contained an antibody that crossreacted with s~lmon pro~mlne as measured by a microcomplement fixation test (23). We have recently found that 35% of vasectomized men have antiprotamine IgE antibodies in their serum compared to 0% in age matched controls (24). Finally, prior exposure to iv protemine given for reversal of heparin anticoagulation may theoretically increase the risk of a reaction on subsequent protamine administration. Sharath described a patient who suffered a fatal reaction to iv protamine 1 mo after an uneventful initial exposure to iv protamine (6). Considering the large number of patients who receive protamine during cardiac catheterization and then are rechaUenged at the conclusion of cardiopulmonary bypass during their coronary artery bypass surgery, it seems unlikely that a single iv exposure to protamine is likely to stimulate significant antiprotamine antibodies. However, Lakin et al. (13) found that none of 22 hemaphoresis donors experienced reactions on their first exposure to iv protamine, whereas 4 of 11 donors experienced urticaria, bronchospasm, and/ or hypotension on their second exposure to protamine following hemaphoresis. We are presently evaluating the development of antipro~mine antibodies in patients following the administration of a large iv dose of protamine.
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Weiss and Adkinson MECHANISMSOF PROTAMINEREACTIONS
The exact mechanism(s) by which proWmine produces adverse reactions are incompletely understood at present. A ~ m a l studies initiallysuggested that prot~mine could cause direct,nonimmunologic release of histamine from rat and hamster peritoneal mast cellsin vitro (25). Subsequent studies using hvman basophils and mast cellshave been unable to demonstrate hisWmlne releasewith protsmine at concentrationsup to 30-100 Hg/mI, (17,26,27). Differences a m o n g species, such as those described above, m a k e extrapolating from anlmal experiments to humans dlmcult. Tobin et al. (28) found that proWmine, although not being able to cause nonimmunologic basophil histamine releasein humans, m a y potentiate IgE-mediated release of histAmlne from human basophils in vitro through a polycationin-recognitionsite. Also, protamineheparin complexes can activatecomplement by the classicpathway both in vitroand in vivo leading to the generation of anaphylatoxins C3a, C4a, and C5a (29-32). Lakin et al. (13) provided evidence in a patient who developed urticaria,bronchospasm, and life-threateninghypotension following pro~mlne administrationthat complement-fixing antiprot~mlne IgG antibodies were involved in the acute reaction. Since then, others have also noted an associationbetween adverse protam~ne reactions and the presence of antipro~mine IgG antibodies (1416). W e conducted a case-controlstudy and found that, in diabetic patients who had received protamine-insulin injections,the presence of serum antiprot~m~ne IgG antibody was a significantrisk factor for acute prot~m~ne reactions (relativerisk = 38). Similarly, in patients without previous exposure to pro~m~ne-insulin injections, the presence of antiprotamine IgG antibody was also a significant risk factor for protamine reactions (relativerisk = 25) (16) (Table 2). Using the technique of heterologous interpolation (33,34),we quantitated the amount of antiprotam~ne IgG antibody in the sera of these patients.Protamlne-insulin-dependent diabetic patients who had acute reactions to intravenously adm~rtistered protamine had significantlyhigher levels of antiprotamine IgG antibodies than other patients (median = 36.4 ~tg/mL,p < 0.0002). In fact,antiprotamine IgG levelsin some of these patients were as high as 200 ~tg/mL, levels comparable to hyperimmunized beeClinical Reviews in Allergy
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Table 2 Association of Protamine Reactions and the Presence of Serum IgE and IgG Antibodies to Protamine Protamine-specific antibody
Cases, reaction
Controls, no reaction
Relative risk
Lower 95% C.L.
p Value
With prior exposure to subcutaneous protamine-insulin preparations IgE present
9
0
IgE absent
4
22
IgG present
11
2
IgG absent
2
95
6.6
1.0 x 10-5
38
5
1.2 x 10-5
20
No prior exposure to subcutaneous protamine-insulin preparations IgE present
0
0
IgE absent
14
21
IgG present
5
0
NA
NA
0.99
25 1.6 0.006 IgG absent 9 21 C. L. = confidence limit, NA = not applicable. From Weiss (16), reproduced with permission of the Massachusetts Medical Society.
keepers (16). Using a double-antibody, radioimmunoprecipitation assay, Kurtz et al. (35) found that 38-91% ofprotamine-insulindependent diabetic patients had serum antiprotamine IgG antibodies. Using an ELISA, Nell and Thomas (36) found that 38% of 319 NPH insulin-treated diabetic patients and only 2.5% of 202 normal control subjects had serum antiprotamine IgG antibodies. Neither of these two studies quantitated the amount ofantiprotamine IgG antibodies. Preliminary data suggest that antiprotamine IgG antibodies are predominantly of the complement-fixing IgG 1subclass (15) (personal observation). Thus, the ability of protamine to activate complement, either through protamine-heparin complexes or through protamineantiprotamine IgG antibody interaction, with the subsequent generation of anaphylatoxins, has been implicated in causing some protamine reactions, particularly those associated with pulmonary Clinical Reviews in Allergy
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vasoconstriction (see below). Once generated, anaphylatexins can induce histamine release from human mast cells and basophils, cause neutrophil migration and release ofpolymorphonuclear cell enzymes, initiate platelet aggregation, cause smooth muscle contraction, and stimulate prostaglandin and thromboxane production from macrophages (37,38). Recent data suggested that protsmlue could inhibit plasma Carboxypeptidase N's ability to convert the anaphylatoxins and bradyldnins to the less active des arg metabolites (39), potentially allowing these vasoactive compounds previously generated to produce more significant hemodynRmic manifestations. Lowenstein and colleagues (18,40) found that pulmonary artery pressure elevation following rapid protamlne injection in three patients was associated with elevations of thromboxane B~ along with C5a. Plasma histamine levels were unchanged in these patients. In contrast, a patient who developed a decrease in systemic vascular resistance and profound systemic hypotension without pulmonary artery hypertension had a tenfold elevation in plasma his~mine and no change in thromboxane B~ or C5a levels. Examples of these two different hemodynsmic reactions are seen in Fig. 2. McIntyre et al. (42) also noted that an elevation in thromboxane B~ and 6-keto-PGFla, a stable metabolite of PGI 2, in a prot~mlrm-insuliu-dependent diabetic aider iv protAmlne administration resulted in pulmonary artery pressure elevation. Animal studies have shown that the elevation of pulmonary artery pressure and thromboxane following prot~mlne admiuistration could be blocked by inhibitors ofcycloxygenase or thromboxane synthetase (40,43-45). A study is presently planned to evaluate whether a thromboxane receptor blocker will be able to diminish the incidence of pulmonary artery pressure elevation following pro~mine administration in hnmAn_S. We developed an agarose-based radioallergosorbent test (RAST) to measure antiprotAmine IgE antibodies. We tested our ability to insolubflize the polycationic protRmine to the agarose beads by first immunizing rabbits to protamlne. Using a solid-phase prot~mlneagarose assay, there was a 25-fold increase in immunoreactive antibody in rabbits after proWmine immunization (coNfirmed by soluble inhibition assays), thus validating the assay. We next showed that, in diabetic patients who had received protsmine-insulin injections, the presence of serum antiproWmine IgE antibody was a significant Clinical Reviews in Allergy
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Fig. 2. Two hemodynamic patterns of acute protamine reactions. A. Acute protamine reaction showing pulmonary vasoconstriction with elevation of pulmonary artery pressure, and right atrial pressure followed by decline of left atrial pressure and systemic arterial pressure. B. Systemic hypotensionfollowing protamine administration without any change in pulmonary artery pressure. From Lowenstein (40) and Levy (41), reproduced with permission of the Journal of Cardiothorasic Anesthesia and Butterworth Publishers.
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risk factor for acute protam~ne reactions (relative risk = 95) (16). The presence of antiprotamine IgE was associated with reactions to prot~mlne that were of longer duration and that were more frequently severe in nature than reactions in patients without antiprot~m~ne IgE antibodies. In fact, nine of nine patients with antiprotAm~ne IgE antibodies experienced severe protamlne reactions involving hypotension and/or bronchospasm t h a t were potentially life-threatening. In general, these patients also tended to have very high levels of antiprotamine IgG antibodies (>10 pg/ mL) (16). In that study, we did not find antiprotamine IgE antibodies in any patient without prior exposure to protamineinsulin injections. Subsequently, we have observed one patient who had an acute protsmine reaction and antiprotamine IgE antibodies without having received previous subcutaneous protamine-insulin injections. We serially measured sen]m antiprotamine IgE and IgG antibody levels in an NPH-insulin-dependent diabetic who had a severe, protracted anaphylactic reaction to protamine. At the time of his pro~mlne reaction, his sez~m contained 8.5 ng/mI, of antiprotamlne IgE and 1.3 pg/ml, of antipro~mine IgG antibody. One month following the reaction, antiprotamlne IgE and IgG increased to 16 ng/mL (twofold rise) and 90.5 pg/mT,(70-fold rise), respectively. With time, both antipro~mine IgE and IgG antibody declined (17) (Fig. 3). Serial intradermal skin tests with native protamine did not discriminate between this profound prot_~mine reactor and nine norreal control subjects who had no prior exposure or any demonstrable serum IgE antibody to pro~mlne. Intradermal injections of protamine between 100-1000 pg/ml, induced irritative skin responses in normal control subjects. We were able to show that the protam~ne reactor's skin mast cells were able to respond to IgE-dependent stimuli by obtaining a positive wheel and flare skin response to an intradermal injection of rabbit F (ab')~ ant[human IgE antibody. Although reports ofprot~mlne reactors with positive prot~mine skin tests exist, two of these reports (7,14) do not provide information on the concentrations of protum~ne used for skin tests, and a third report (19) employed concentrations ofprotamine that we have shown caused false-positive, irritative reactions in the skin. Lakin et al. (13) reported a prot,mine reactor with a positive skin test to
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20
1O0
_15
80
,E=
60
40
9
5
"~
~
20
0 0
I 1
I 2
i 3
0 9
Months from reaction
Fig. 3. Serial antiprotamine IgE- and IgG-antibody levels in the serum of a protamine reactor. The patient had been receiving NPH insulin injections for 10 yr prior to receiving iv protamine at cardiac catheterization. He developed acute rash, bronchospasm, and life-threatening hypotension. At the time of the reaction, the patient's serum contained 8.5 ng/mL of antiprotamine IgE and 1.3 ~g/mL of antiprotamine IgG antibody. From Weiss (17), reproduced with permission of Blackwell Scientific Publications.
protamine at 0.001 ~ / m L , which should be a nonirritative concentration. A recent study found that results ofprotamine skin testing were not useful in predicting subsequent acute protamine reactions (46). In our patient, in vitro basophil histamine release to protomine was also inconclusive in discriminating between normal control subjects and the protamine reactor (17). Based on the above information, we postulate that protamine may be an incomplete or univalent antigen, which first must combine with a tissue macromolecule or possibly heparin to become a complete, multivalent antigen capable ofeliciting IgE antibody-dependent mediator release from mast cells and basophils. This testable hypothesis is presently being studied. In summary, protamine may elicit both immune and nonimmune adverse reactions through multiple mechanism.q dependent on its charge and antigenicity. Figure 4 shows the most likely mechanisms causing acute protamine reactions in humans.
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Weiss and Adkinson O + hepann ' ~
/ PROTAMINE
Protamine-heparin r
Complement activation (classic
e.,
+ Protamine-specific IgG Ab
,-ca e. e.,
(D Q
~
+ Protamine-speci fllc IgEAbboundto
|
+
Carboxypeptidase N
:#
C3, 4 ,5a des arg ~ ~
Mast cell / basophil " d ' ' ~ Mediator release
C3a, C4 a, C5a r
? cell source platelets PMNs TX / PG generation mast cells AIv. macro ? VEC ul vasoconstriction
PAP S[I R
Bronchoconstriction R heart failure (~afterload)
SBP q
~ L sided filling pressure
Fig. 4. Possible mechanism for acute protamine reactions. 1. Protamine combines with heparin to activate complement via the classic pathway. The generation of anaphylatoxins may cause mast cell or basophil mediator release, or may stimulate thromboxane and prostaglandin generation leading to pulmonary vasoconstriction. If severe, this could lead to acute right-sided heart failure and systemic hypotension. 2. Protamine combines with antiprotamine IgG antibody, leading to complement activation with the same cascade as noted in number 1.3. Protamine may inhibit the ability of carboxypeptidase N to convert the anaphylatoxins to their less active des arg metabolites. 4. Protamine crosslinks cell surface antiprotamine IgE antibodies on mast cells and basophils, leading to mediator release and systemic vascular dilatation and hypotension and/or bronchoconstriction. 5. Protamine may combine with a polycationic activation site on mast cells and basophils, and potentiate IgE-mediated histamine release. TX = thromboxane, PG = prostaglandins, PAP = pulmonary artery pressure, SVR = systemic vascular resistance, SBP = systemic blood pressure, VEC = vascular endothelial cells, AIv. macro = alveolar macrophage.
PROTAMINEALTERNATIVES Bemuse patientswho have had previous acute prot~miue reactions may, in the future,require reversalofheparin anticoagulation and because certainpatient populations may be at high risk for protamine reactions (i.e., protamine-insulin-dependent diabetics), alternatives to pro~mine for reversal of heparin anticoagulation Clinical Reviews in Allergy
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100
80
o
Protamine from Company
60
J3 .m r'r
--
0
A
9
B
[]
C
9
D
40
20
i
i
.001
.01
i
.1
i
1
S o l u b l e Protamine (mg/ml)
Fig. 5. Increasing concentrations of soluble protamine from four commercially available manufacturers (A-D) are plotted on the X axis. Antiprotamine antibody in sera from seven patients is inhibited from binding to the protamine solid phase in a similar fashion.
are needed. Premedication with glucocortcosteroids and antihistamines, which have been successfully employed to reduce the risk of recurrent reactions to radiocontrast media (47,48), has not been successful in preventing IgE-antibody-mediated reactions, such as those induced by penicillin (49,50). At present, no studies have been done to evaluate whether premedication regimens prevent or reduce the severity of acute prot~mine reactions. Since there are both intra- and interspecies differences in protsmines (2), we studied four commercially available protRmlrm preparations from different manufacturers to determine ff their abilities to bind to human antiprot~mine antibody differed. Ifa particular commercially available protamine is less immunoreactive with h u m a n antiprotsm~ne antibody, then it may be expected to cause less frequent and/or less severe reactions and, therefore, would be more advantageous to use. Seven protsmine-ins11Hn-dependent diabetics who had experienced life-threatening reactions to iv pro~mine and whose sera contained a mean of 67.4 ~g/mL (range of 16-200 roT,) ofantiprot~m~ne IgG antibody were evaluated. Inhibition studies, where each sen~m was preincubated with buffer and increasing concentrations of the four prot~mine preparations before addition to the prot~mine solid phase were done. As seen in Fig. 5, no signifiClinical Reviews in Allergy
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cant differences were found in the immunoreactivity of the four different prot~mlne preparations. Therefore, no benefit might be expected from using one commercial source of protamine over another (51). Allowing heparin anticoagulation to reverse spontaneously has been successfully accomplished in patients who had experienced previous acute protsmlne reactions, but this alternative is not without potential for significant hemorrhage and cannot be generally recommended. Hexadimethrine (Polybrene), a quartenary 3mmoni!lm polycationic salt, was used to reverse hepatin anticoagulation in the early days of cardiac surgeryin the United States. Reports of renal toxicity, in combination with animal studies demonstrating lesions of the kidney, adrenal cortex, and interior pituitary, led to the voluntary removal of hexadimethrine from the United States market. Hexadimethrine is available through the Food and Drug Administration (as a compassionate-use drug). We have successfully used it in two patients who had prior life-threatening reactions to protum~ne, both of whom had antiprotamine IgE antibodies (personal observation).
SUMMARY Recent advances in medicine, such as cardiac catheterization, phoresis, dialysis, and cardiopulmonary bypass technology, have increased the need for heparin anticoagulation. To antagonize heparin's effect and prevent hemorrhagic complications a f a r the procedure, protamine has likewise been used more frequently. With its increased use have come increased reports of adverse proWmlne reactions consisting of rash, urticaria, elevation of pulmonary artery pressure, systemic hypotension, and, at times, death. The elevation of pulmonary artery pressure, which appears to be a rather common occurrence in animals, may be an isolated finding without clinical consequences in humans. However, this pulmonary vasoconstriction may, when severe, lead to acute right-sided heart failure and systemic hypotension. Other protamine reactions involve a decrease in systemic vascular resistance and systemic hypotension without changes in pulmonary artery pressure. Causes of acute protsmine reactions may involve the generation of anaphyatoxins and prostanoids either from protsmine-heparin complexes or compleClinical Reviews in Allergy
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ment-fixing antiprotsmine IgG antibodies, from inhibition of plasmA Carboxypeptidase N, from crosslinldng of cell-surface antiprotsmine IgE on mast cells and basophils with subsequent mediator release, or from potentiation of IgE-mediated release of histamine through a polycationin-recognition site. Although we have come a long way in understanding the mechanisms by which protamine can cause its ill effects in humans, more work is clearly needed to define, in prospective studies, the incidence of and risk factors for protamine reactions in various patient groups, and to delineate more clearly which mechanisms are involved in each clinical type of acute protamine reaction. Hopefully, this will lead to strategies and prot~mine alternatives that will prevent or dimimsh, in frequency or severity, adverse protamine reactions. Alternatively, a dearer picture of the risk factors important for protamine reactions and the predictive value of diagnostic tests (e.g., protamine IgE antibody) can also minimize the clinical impact of this increasingly common adverse event.
ACKNOWLEDGMENTS This work was supported in part by grant (AI 20136) from the National Institutes of Health and by the United States Pharmacopeial Convention.
REFERENCES 1. Horrow, J. C. (1985), Anesth. Analg. 64, 348-361. 2. Delange R. J. and Smith, E. L. (1979), Neurath, H. and Hill, R. L. (eds.), The Proteins, Academic, New York, pp. 119-243. 3. Hagedorn, H. C., Jensen, B. N., Kraup, N. B., and Woodstrup, I. (1936), JAMA 1069 177-180. 4. Chargaff, E. and Olson, IL B. (1937), J. Biol. Chem. 122, 153-165. 5. Bull, B. S., Huse, W. M., Braver, F. S., and Korpman, R. A. (1975), J. Thorac. Cardiovasc. Surg. 69, 685-689. 6. Sharath, M. D. Metzger, W. J., Richerson, H. B., Scupham, R. IL, Meng, R. L., Ginsberg, B. H., and Weiler, J. M. (1985), J. Thorac. Cardiovasc. Surg. 90, 86-90. 7. Doolan, L., McKenzie, I., Krafchek, J., Parson, B., and Buxton, B. (1981), Anaesth. Intens. Care 9, 147.
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