THROMBOSIS RESEARCH Printed in the United
Vol. States
7, pp. 579-588, Pergamon Press,
STUDIES
OF FIBRINOLYTIC AND COAGULATION DURING OPEN HEART SURGERY PROBLEMS DURING OPEN I. FIBRINOLYTIC SURGERY WITH ECC +
1975 Inc.
FACTORS HEART
Popov-Cenic,W. BUttner,N. Miiller and H. Egli R.G. Kladetzky,Smilja Institute of Experimental Hematology and Blood Transfusion and Department of Anesthesiology, University of Bonn, GFR
(Received
30.5.1975; Accepted
by
in revised form Editor L. R6ka)
22.8.1975.
ABSTRACT 7 patients (group I) with congenital and 2 patients (group XI) with acquired cardial vitia were examined during open heart surgery with extracorporeal circulation (ECC) using the following fibrinolytic tests: paperfibrinolysis, fibrinogen, plasminogen, antiplasmins, FFD, reptilase time, SDPS-test. During ECC, similar changes of fibrinolytic and coagulation systems took place in both groups, but fibrinolysis as well as the increased turnover of coagulation factors were more of heparin with marked in group II. After neutralization protamine chloride the coagulation parameters in group I but in group II there was showed a tendency to normalize, a development of hemorrhage due to consumption coagulopathy. INTRODUCTION A number of investigations have suggested that bleeding complications following open heart surgery on ECC are caused by the so alone (2,3) or called heparin-rebound-effect (I), by fibrinolysis DIC with reactive fibrinolysis (4,5). Gans et al. (4) and Castaneda et al. (6,7) have pointed out that the development of DIC as well as the bleeding diathesis could be correlated to neutralization of But it is far from clear why many heparin by protamine chloride. patients suffered from life threatening hemorrhage after open heart surgery on ECC. We observed in 32 cases this massive hemorrhage together with pulmonary hyaline membranes following ECC (8). As our routine tests did not give any (platelet count, thrombin time, clot observation) explanation for these bleeding complications, we examined a large parameters. In the number of fibrinolytic as well as coagulation we shall look for fibrinolysis as an first part of our studies, important matter of coagulation dJsturbances. +Reported in part at September Jeruslaem,
the 3,
15th 1974
International 579
Congress
of Hematology,
580
HEART SURGERY 1:FIBRINOLYSIS
vo1.7,No.4
MATERIAL AND METHODS We examined 7 patients with congenital cardial vitia without postoperative bleeding complications (group I) and 2 patients with acquired heart vitia (group II), who showed signs of DIC before the beginning of surgery and suffered from serious postoperative bleeding after heparin neutralization with protamine chloride. All operations were carried out with the same bubble-oxygenator. Details of date concerning the patients, perfusion duration, machine filling, anticoagulation doses during ECC, neutralization, substitution etc. are shown in tables I and II. TABLE I Date about patients, diagnoses and treatment. 1-7 = group I; x l-2 - group II. xx = additional treatment with antifibrinolytica, application of fraction I Cohn, PPSB-concentrate. septum defect, (ASD = atria1 septum defect, VSD - ventricular ST = stenosis).
TABLE II Data about length of perfusion, machine filling and substitution with whole blood. x - anticoagulant 3 ooo IU heparin were added to every unit of blood.
At the end of the bypass, heparin neutralization was achifjved with protamine chloride l:l.l and a single dose of TRASYLOL between 400 ooo and 800 ooo KIU. In contrast to group I, the patient8 of group II who developed a hemorrhagic diathesis received additional doses of protamine chloride, TRASYLOLR, synthetic antifibrinolytica (AMCHA), fraction I according to COHN and PPSB-concentrate (table I).
HEART
SURGERY
TABLE Overview 1 -
BEFORE
2 - AT
THE
of blood
samples
III
taken
THE
BEGINNING
OF
END
OF BYPASS
TIME
for
AFTER
NEUTRALIZATION
OF
4 - 45 MIN.
AFTER
A CONSECUTIVE
DOSE
5-
3 HRS.
6 - AT
THE
the
various
tests.
WITH
PROTAMINE
SURGERY
15 MIN.
3-
581
1:FIBRINOLYSIS
AFTER
A CONSECUTIVE
DOSE
FIRST
POSTOPERATIVE
DAY
HEPARIN
CHLORIDE
OF TRASYLOLR OF TRASYLOLR
The blood was taken via polyaethylencytheter with the silicon techniwue according to Egli (9). All blood samples were decalcified in a ratio I:5 as well as I:10 with trisodium citrate 3,8X, which was in the syringe. Besides, the native blood was taken with and without the addition of TRASYLOLR. The citrate blood was centrifuged at 3 ooo revolutions per min. at +4'Celsius for 15 min. and than frozen separately for each test until the samples were needed. The native blood at -30' Celsius samples were left in a water bath for 4 hrs. at +37' Celsius and than centrifuged. While preparing the serum of sample 2, which was taken at the end of ECC during high heparinization, no blood clotting was observed until after neutralization of heparin with protamine chloride. For detecting of FFD all samples were divided in two parts. Part one was left in a water bath for 4 hrs. at +37' Celsius and after part two was prepared by application of 100 clotting centrifuged, NIB U thrombin/ml and thaa prepared in the same way as above. The following tests were carried out: paperfibrinolysis according to Goossens (10); fibrinogen in the F.-P.-T.-test according determinations of fibrinogen, FFD to Vermylen (II); immunologic alpha-I-antitrypsin (slow acting antiplasmin) in serum, plasminogen, and alpha-2-macroglobulin (fast acting antiplasmin) in serum by radial immunodiffusion according to Mancini (12) with "Behring immunoelectrophoresis of FFD in 1% agarose acPartigenplatten"; time according to Latallo cording to Scheidegger (13); reptilase et al. (14) with test reagence "Boehringer Mannheim"; SDPS-test according to Niewiarowsky et al. (15) modified by Kidder (16) - with protamine sulphgte 1% diluted I:5 and I:10 by tris buffer (ph 6.5) Celsius after.15 minutes and at room temperature recorded at +37 after 2 hrs. and 24 hrs.. STATISTICS were tested In group I, all parameters the 2 patients of group to significance; statistic calculation.
byx2-test II did not
with regard allow any
RESULTS In the paperfibrinolytic test the fibrinolytic activity at the end of the bypass time is greater in group I than in group II. potential in group I After detaching the ECC, the fibrinolysis drops, while the values increase in group II and reach their maxi(Fig. 1). mum 3 hrs. after surgery
HEART SURGERY 1:FIBRINOLYSIS
582
Vo1.7,No.4
Fibrinogen shows similar reactions in the F.P.T.-test and in the but the values of the F.P.T.-test are lower radial immunodiffusion, than immunological ones. In comparing the reaction of fibrinogen in both groups, in group end of the bypass time and an increase I we noticed a drop at the In group II the initial values were after the end of the surgery. 2 there was a more pronounced drop which rein sample elevated, and an increase in sample 6 mained after the end of surgery,
(Fig.
1).
w.7. 30
In*)
PAPER - FIBRINOLVSIS II”.1
I”.,
PLASMINOGEN ,,“.I
I
,,{
: _ :
,,A*
0 &,- ANTITRYPSIN F3 *I-MACROGLGWLIN !,“.I
Fig. 1 Upper part: changes of paperfibrinolysis;lower part: changes of fibrinogen (pale pillars = F.P.T.test,stripped pillars = immunologic determinations). Group I: left side, group II: right side.
Fig. 2 Upper part: changes of plasminogen;lower part: changes of alphaI-antitrypsin (stripped pillars), alpha-2-macroglobulin (pale pillars). Group f:left side, group II: right side.
The reaction of plasminogen and antiplasmins (alpha-I-antitrypsin and alpha-2-macroglobulin) corresponds to their fibrinogen levels. In group II the initial values of plasminogen and antiplasmins are reduced (Fig. 2). In contrast to group I, we already found FFD in the serum of group II before surgery started. At the end of the bypass time, all patients have very high values, up to 470 mgX FFD, but only in serum which has been obtained by neutralization of heparin with protamine chloride. After in vitro addition of 100 NIH U thrombin only the vestiges of split products remained to a maximum of 28 mg%. After the end of the observation time, in group 11, however, no FFD are detectable in samples 4 and 5 (Fig. 3).
Vo1.7,No.4
BEART SURGERY 1:FIBRINOLYSIS
583
FFD 0
WlTHOUT
Ed
WITH
THROMBIN THROMBIN lln.2 .L69
1
2
3
4
5
Fig. Reaction cation
123456
6
3
of FFD; pale pillars before, of 100 NIH U thrombin.(Group
I:
stripped left,
pillars group-II:
after right
appliside)
In immunoelectrophoresis FFD were pointed out corresponding well In the upper part of figure 4 is to quantitative immunodiffusion. shown a band in the attachment area which reacted with anti-fibrinogen-serum, and a second one which moved to anode. We found this immunoelectrophoretic picture in each sample of both groups, in sample 5 of group I only the immunoelectrophoresis - seen in the bands (above with 10 pl serum, lower part of figure 4 - shows three below with 1 pl serum against 50 /ul antifibrinogen-serum).
Fig. Immunoelectrophoresis;
4 details
see
text.
584
BEART
SURGERY
1:FIBRINOLYSIS
Vo1.7,No.4
The preoperative values of reptilase time are normal in group I, whereas they are longer in group II. In both groups reptilase time is prolonged during perfusion, more in group II than in group I. After detaching the apparatus, reptilase time stays prolonged in group I; in group II it goes back to normal (Fig. 5).
REPTILASE
SDPS
Fig.
- TIME Il”Z2
- TEST I,“:?
5
reaction of Upper half of the figure: reaction of SDPS-test. of the figure: right side)
reptilase time, (Group I: left,
lower group
half II:
In group I the SDPS-test is negative in sample 1. During perfusion SDPS-test becomes slightly positive. After neutralization of heparin with protamine chloride, the test is more strongly positive, and after application of antifibrinolytica, the intensity of the test increases even further. In group II, this test is already positive before surgery. The further development of SDPStest in group II corresponds to that of group I, but the intensity of the reaction is more marked (Fig. 5). DISCUSSION Looking for signs of fibrinolysis at the end of ECC, we have seen a much greater loss of fibrinogen as well as plasminogen in group II than in group I. This differenting decrease was also to be seen in the fast acting antiplasmin which is a useful indicator of the amount of plasmin neutralized (4). These drops of fibrinogen, plasminogen and alpha-2-macroglobulin suggested that fibrinolysis was active (17) much more in group II than in group I, despite a low level of activator (18) which was pointed out by paperfibrinolysis. A more pronounced fibrinolysis in group II was also suggested by a larger amount of FFD and a more prolonged reptilase time (14,191.
Vo1.7,No.4
HEART
SURGERY
1:FIBRINOLYSIS
585
This drop in fibrinogen and plasminogen might not only be produced by fibrinolysis but could also be caused by DIC (20,21). That would be pointed out by the increase of FM in the SDPS-test The immunoelectrophoresis showed FR-antigen material (5,15). which could be complexes of FM with FDP/fdp and which were unclottable (21,22). Therefore we thought that during the ECC an intravascular coagulation had developed in spite of high doses of heparin (4,23). After neutralization of heparin with protamine chloride we observed a different development of these coagulation parameters between group I and II. Whilst in group I fibrinogen, plasminogen and antiplasmin showed a tendency to increase during an increased turnover of coagulation factors, there was a further decrease of these parameters in group II. The bleeding which had now developed after application of protamine chloride could be correlated to the increasing of FM and a simultaneous drop of fibrinogen and plasminogen. But at this time there was a small amount of FFD only in sample 3 or none in samples 4 and 5 which might be able to disturb the coagulation system and produce fibrinolytic bleeding (24,25). Therefore it could not be a fibrinolytic bleeding, but it had to be the consequence of increased DIC which lead to a consumption coagulopathy (27). The question now arose why after giving protamine chloride at the end of the bypass time in sample 3 the amount of FFD diminished and FFD disappeared after application of antifibrinolytica and substitution of fraction I according to Cohn in group II. We thought this disappearance of FFD could be called a paracoagulation phenomenon in vivo which is triggered by protamine chloride (28). Our observations could be correlated to the results of Castaneda (6) and RLidegran (29) who have observed a drop in the count of red triggered by injection of procells (6) and of platelets (29), tamine. A further question was to be answered, why group II had de- as we have already said veloped such a severe hemorrhage while had taken place in both groups qualitatively similar changes The reason for the development in group II could be during ECC. explained by the preoperative values of group II. This had shown signs of chronic DIC with reactive fibrinolysis, e.g. FM, high low antithrombin-III-activity (see level of fibrinogen and FFD, high activity of factors VIII and IX (see part II), part II), low level of plasminogen and antiplasmins (25,27,30,31). This chronic DIC could be responsible for a reduced clearance of RES This disturbed clearance lead to a reduced elimination (4,30,32). (4,27,33) which was set activators of FM, FFD and prothrombin an other reason for the developfree from parts of cells and was ment of DIC (4) after neutralizing of heparin and could be seen as the cause of a consumption coagulopathy (27). the neutralization of heparin From-the above we may follow, with protamine at the end of the bypass time is contraindicated (4,7). We thank technical
Miss H. Schreiber assistance.
ACKNOWLEDGEMENTS Gerhard and Miss I.
for
their
valuable
586
HEART
SURGERY
1:FIBRINOLYSIS
Vo1.7,No.4
REFERENCES KOLFF, A.J., EFFLER, D.B., GROVES, L.K., PEERBOOM, G. and MORACA, membrane oxygenator (heart-lung machine) and its P.P.: Disposable use in experimental surgeray. Clev. Clin. Quart. 23, 69, 1965. EKERT, H., MONTGOMERY, D. and ABERDEEN, E.: Fibrinolysis extracorporeal circulation. Circul. Res. 28, 512, 1971. GANS, heart Surg.
during
H. and LILLEHEI, C.W.: Problems in hemostasis during surgery: I. On the release of plasminogen activator. 154, 915, 1961.
openAnn.
GANS, H., SUBRAMANIAN, V., JOHN, S., CASTANEDA, A.R. and LILLEHEI, and practical (clinical) considerations conC.W.: Theoretical cerning proteolytic enzymes and their inhibitors with particular reference to changes in the plasminogen-plasmin system observed during assisted circulation in man. Ann. N.Y. Acad. Sci. 146, 721, 1968, 5
PALESTER-CHLEBOWCZYK, M., STRYEWSKA, E., SITKOWSKI, W., OLENDER, Z. and LATALLO, Z.S.: Preliminary results of A ., WEGRZYNOWICZ, the protamine sulphate test in plasma of various patients. Stand. J. Haemat. Suppl. 13, 183, 1971. CASTANEDA, A.R., GANS, H., WEBER, K.C. and FOX, I.J.: Heparin Experimental and clinical studies. Surgery 62, neutralization: 686, 1967. CASTANEDA, A.R.: heart operations?
Must heparin be neutralized following openJ. Thorac. Cardiovasc. Surg. 52, 716, 1966. c CREMER, H., SCHULTE AM ESCH, J. and POPOV-CENIC, S.: Zum Problem der pulmonalen hyalinen Membranen beim Erwachsenen. Anaesthesist 23, 241, 1974. H.: Zur Differenzierung EGLI, H. and BUSCHA, hangigkeit von Kontaktwirkungen benetzbarer EinfluR auf die Blutthrombokinase-Bildung. Haemorrh. 3, 604, 1959. ..
und Kalzium-AbOberflHchen. Ihr Thromb. Diath.
IO
GOOSSENS, 21, 1816,
11
VERMYLEN, C., DE VRECKER, F.A. and VERSTRAETE, M.: A rapid enzymatic method for assay of fibrinogen fibrin polymerization time (F.P.T.-test). Clin. Chim. Acta 8, 418, 1963.
12
MANCINI, G., CARBONARA, A.O. and quantition of antigens by single chemistry 2, 235, 1965.
13
SCHEIDEGGER, J.J.: Int. Arch. Allergy
14
LATALLO, Z.S. and TEISSEYRE, E.: Evaluation of reptilase and thrombin clotting time in the presence of fibrinogen degradation products and heparin. Stand. J. Haemat. Suppl. 13, 261, 1971.
15
NIEWIAROWSKI, intravascular
N.: Standartisierte'Fibrinolysemethoden. 1970.
Med.
Welt
HERMANNS, J.F.: Immunchemical radial immundiffusion. Immuno-
Une micro-mfthode de l'immunoelectrophorgse. Appl. Immunol. 7, 103, 1955.
S. and GUREWICH, V.: coagulation. J. Lab.
Laboratory Clfn. Med.
identification 77, 665, 1971.
of
Vo1.7,No.4
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SURGERY
1:FIBRINOLYSIS
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16
KIDDER, W.R., LOGAN, L.J., RAPOPORT, S.J. and PATCH, M.J.: plasma protamine paracoagulation test. Am. J. Pathol. 58, 675, 1972.
17
MERSKEY, C., JOHNSON, A.J., KLEINER, defibrination syndrome: Clinical and 13, 528, 1967. Br. J. Haemat.
C.J. and WOHL, H.: The laboratory diagnosis.
18
FLUTE, P.T.: An overview.
and fibrinolytic Suppl. 54, 203,
19
VERMYLEN, J.: Fibrinogen derivates. royale de medicine de Belgique 34,
20
LACKNER, H. and JAVID, plasminogen level. Am.
21
DEYKIN, D.: The lar coagulation.
22
MARDER, V.J. and BUDZYNSKI, A.Z.: Fibrinogen and its derivates, hereditary and acquired abnormalities. Schweiz. med. Wschr. 104, 1338, 1974.
23
MtiLLER-BERGHAUS, G., RGKA, L. and LASCH, glomerular microclot formation by fibrin Thromb. Diath. Haemorrh. 29, 375, 1973.
24
MARDER, V.J., SHULMAN, N.R. and CAROLL, W.R.: The of intermediate degradation products of fibrinogen lytic hemorrhage. Trans Assoc. Am. Physicians 80,
25
POPOV-CENIe, S., KALINKE, H., ETZEL, F., BAYMANN, E. and EGLI, changes during and after liver transplantations H . : Coagulation problems in transplanted organs. in man. In: Coagulation (Ed.). Springfield, Thomas-Publisher, 1972. K.N. von KAULLA
26
MARDER, V.J., SHULMAN, N.R. and CAROLL, W.R.: High molecular weight derivates of human fibrinogen produced by plasmin. I. Physiochemical and immunological characterization. J. Biol. Chem. 244, 2119, 1969.
27
LASCH, H.G., HEENE, D.L., HUTH, K. and SANDRITTER, W.: Pathoclinical manifestations and therapy of consumption physiology, coagulopathy ("Verbrauchskoagulopathie"). Am. J. Cardiol. 20, 1381, 1967.
28
LIPINSKI, B., WEGRZYNOWICZ, Z., BUDZYNSKI, A.Z., KOPEC, M., LATALLO, Z.S. and KOWALSKI, E.: Soluble unclottable complex formed in the presence of fibrinogen degradation products (FDP) during the fibrinogen-fibrin conversion and their potential significance in pathology. Thromb. Diath. Haemorrh. 17, 657, 1967.
29
RRDEGRAN, Kt, BERGENTZ, S.-E., LEWIS, D.H., LJUNGQUIST, U. and effects of induced platelet aggregation. OLSSON, P.: Pulmonary Intravascular obstruction or vasoconstriction? Stand. J. Clin. Lab. Invest. 28, 423, 1971.
30
BOWIE, E.J.W., COOPER, H., FUSTER, V., KAZMIER, F. and OWEN, of intravascular coagulation. Thromb. Diath. C.A.: The diagnosis Haemorrh. Suppl. 56, 137, 1973.
31
STRAUB, P.: generalized?
Coupling of coagulation Thromb. Diath. Haemorrh.
S. M&moires 125, 1972.
de
systems: 1973.
1'Academi
J.P.: The clinical significance J. Clin. Pathol. 60, 175, 1973.
clinical challenge N. Engl. J. Med.
of disseminated 283, 636, 1970.
The
of
the
intravascu-
H.G.: Induction of monomer infusion. importance in fibrino156, 1967.
Chronic intravascular coagulation: Localized Thromb. Diath. Haemorrh. 56, I, 1973.
or
588
HEART SURGERY
1:FIBRINOLYSIS
Vo1.7,No.4
32
MULLER-BERGHAUS, G.: Pathophysiology of DIC. In: Disseminated intravascular coagulation. E.F. MAMMEN, G.F. ANDERSON and M.I. BARNHART (Eds.). Stuttgart, Schattauer, 1969.
33
FLETCHER, A.P., BIEDERHANN, O., MOORE, D., ALKJAERSIG, N. and system activity SHERRY, S.: Abnormal plasminogen-plasmin (fibrinolysis) in patients with hepatic cirrhosis. J. Clin. Invest. 43, 681, 1964.
Vol. States
7, pp. 579-588, Pergamon Press,
STUDIES
OF FIBRINOLYTIC AND COAGULATION DURING OPEN HEART SURGERY PROBLEMS DURING OPEN I. FIBRINOLYTIC SURGERY WITH ECC +
1975 Inc.
FACTORS HEART
Popov-Cenic,W. BUttner,N. Miiller and H. Egli R.G. Kladetzky,Smilja Institute of Experimental Hematology and Blood Transfusion and Department of Anesthesiology, University of Bonn, GFR
(Received
30.5.1975; Accepted
by
in revised form Editor L. R6ka)
22.8.1975.
ABSTRACT 7 patients (group I) with congenital and 2 patients (group XI) with acquired cardial vitia were examined during open heart surgery with extracorporeal circulation (ECC) using the following fibrinolytic tests: paperfibrinolysis, fibrinogen, plasminogen, antiplasmins, FFD, reptilase time, SDPS-test. During ECC, similar changes of fibrinolytic and coagulation systems took place in both groups, but fibrinolysis as well as the increased turnover of coagulation factors were more of heparin with marked in group II. After neutralization protamine chloride the coagulation parameters in group I but in group II there was showed a tendency to normalize, a development of hemorrhage due to consumption coagulopathy. INTRODUCTION A number of investigations have suggested that bleeding complications following open heart surgery on ECC are caused by the so alone (2,3) or called heparin-rebound-effect (I), by fibrinolysis DIC with reactive fibrinolysis (4,5). Gans et al. (4) and Castaneda et al. (6,7) have pointed out that the development of DIC as well as the bleeding diathesis could be correlated to neutralization of But it is far from clear why many heparin by protamine chloride. patients suffered from life threatening hemorrhage after open heart surgery on ECC. We observed in 32 cases this massive hemorrhage together with pulmonary hyaline membranes following ECC (8). As our routine tests did not give any (platelet count, thrombin time, clot observation) explanation for these bleeding complications, we examined a large parameters. In the number of fibrinolytic as well as coagulation we shall look for fibrinolysis as an first part of our studies, important matter of coagulation dJsturbances. +Reported in part at September Jeruslaem,
the 3,
15th 1974
International 579
Congress
of Hematology,
580
HEART SURGERY 1:FIBRINOLYSIS
vo1.7,No.4
MATERIAL AND METHODS We examined 7 patients with congenital cardial vitia without postoperative bleeding complications (group I) and 2 patients with acquired heart vitia (group II), who showed signs of DIC before the beginning of surgery and suffered from serious postoperative bleeding after heparin neutralization with protamine chloride. All operations were carried out with the same bubble-oxygenator. Details of date concerning the patients, perfusion duration, machine filling, anticoagulation doses during ECC, neutralization, substitution etc. are shown in tables I and II. TABLE I Date about patients, diagnoses and treatment. 1-7 = group I; x l-2 - group II. xx = additional treatment with antifibrinolytica, application of fraction I Cohn, PPSB-concentrate. septum defect, (ASD = atria1 septum defect, VSD - ventricular ST = stenosis).
TABLE II Data about length of perfusion, machine filling and substitution with whole blood. x - anticoagulant 3 ooo IU heparin were added to every unit of blood.
At the end of the bypass, heparin neutralization was achifjved with protamine chloride l:l.l and a single dose of TRASYLOL between 400 ooo and 800 ooo KIU. In contrast to group I, the patient8 of group II who developed a hemorrhagic diathesis received additional doses of protamine chloride, TRASYLOLR, synthetic antifibrinolytica (AMCHA), fraction I according to COHN and PPSB-concentrate (table I).
HEART
SURGERY
TABLE Overview 1 -
BEFORE
2 - AT
THE
of blood
samples
III
taken
THE
BEGINNING
OF
END
OF BYPASS
TIME
for
AFTER
NEUTRALIZATION
OF
4 - 45 MIN.
AFTER
A CONSECUTIVE
DOSE
5-
3 HRS.
6 - AT
THE
the
various
tests.
WITH
PROTAMINE
SURGERY
15 MIN.
3-
581
1:FIBRINOLYSIS
AFTER
A CONSECUTIVE
DOSE
FIRST
POSTOPERATIVE
DAY
HEPARIN
CHLORIDE
OF TRASYLOLR OF TRASYLOLR
The blood was taken via polyaethylencytheter with the silicon techniwue according to Egli (9). All blood samples were decalcified in a ratio I:5 as well as I:10 with trisodium citrate 3,8X, which was in the syringe. Besides, the native blood was taken with and without the addition of TRASYLOLR. The citrate blood was centrifuged at 3 ooo revolutions per min. at +4'Celsius for 15 min. and than frozen separately for each test until the samples were needed. The native blood at -30' Celsius samples were left in a water bath for 4 hrs. at +37' Celsius and than centrifuged. While preparing the serum of sample 2, which was taken at the end of ECC during high heparinization, no blood clotting was observed until after neutralization of heparin with protamine chloride. For detecting of FFD all samples were divided in two parts. Part one was left in a water bath for 4 hrs. at +37' Celsius and after part two was prepared by application of 100 clotting centrifuged, NIB U thrombin/ml and thaa prepared in the same way as above. The following tests were carried out: paperfibrinolysis according to Goossens (10); fibrinogen in the F.-P.-T.-test according determinations of fibrinogen, FFD to Vermylen (II); immunologic alpha-I-antitrypsin (slow acting antiplasmin) in serum, plasminogen, and alpha-2-macroglobulin (fast acting antiplasmin) in serum by radial immunodiffusion according to Mancini (12) with "Behring immunoelectrophoresis of FFD in 1% agarose acPartigenplatten"; time according to Latallo cording to Scheidegger (13); reptilase et al. (14) with test reagence "Boehringer Mannheim"; SDPS-test according to Niewiarowsky et al. (15) modified by Kidder (16) - with protamine sulphgte 1% diluted I:5 and I:10 by tris buffer (ph 6.5) Celsius after.15 minutes and at room temperature recorded at +37 after 2 hrs. and 24 hrs.. STATISTICS were tested In group I, all parameters the 2 patients of group to significance; statistic calculation.
byx2-test II did not
with regard allow any
RESULTS In the paperfibrinolytic test the fibrinolytic activity at the end of the bypass time is greater in group I than in group II. potential in group I After detaching the ECC, the fibrinolysis drops, while the values increase in group II and reach their maxi(Fig. 1). mum 3 hrs. after surgery
HEART SURGERY 1:FIBRINOLYSIS
582
Vo1.7,No.4
Fibrinogen shows similar reactions in the F.P.T.-test and in the but the values of the F.P.T.-test are lower radial immunodiffusion, than immunological ones. In comparing the reaction of fibrinogen in both groups, in group end of the bypass time and an increase I we noticed a drop at the In group II the initial values were after the end of the surgery. 2 there was a more pronounced drop which rein sample elevated, and an increase in sample 6 mained after the end of surgery,
(Fig.
1).
w.7. 30
In*)
PAPER - FIBRINOLVSIS II”.1
I”.,
PLASMINOGEN ,,“.I
I
,,{
: _ :
,,A*
0 &,- ANTITRYPSIN F3 *I-MACROGLGWLIN !,“.I
Fig. 1 Upper part: changes of paperfibrinolysis;lower part: changes of fibrinogen (pale pillars = F.P.T.test,stripped pillars = immunologic determinations). Group I: left side, group II: right side.
Fig. 2 Upper part: changes of plasminogen;lower part: changes of alphaI-antitrypsin (stripped pillars), alpha-2-macroglobulin (pale pillars). Group f:left side, group II: right side.
The reaction of plasminogen and antiplasmins (alpha-I-antitrypsin and alpha-2-macroglobulin) corresponds to their fibrinogen levels. In group II the initial values of plasminogen and antiplasmins are reduced (Fig. 2). In contrast to group I, we already found FFD in the serum of group II before surgery started. At the end of the bypass time, all patients have very high values, up to 470 mgX FFD, but only in serum which has been obtained by neutralization of heparin with protamine chloride. After in vitro addition of 100 NIH U thrombin only the vestiges of split products remained to a maximum of 28 mg%. After the end of the observation time, in group 11, however, no FFD are detectable in samples 4 and 5 (Fig. 3).
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FFD 0
WlTHOUT
Ed
WITH
THROMBIN THROMBIN lln.2 .L69
1
2
3
4
5
Fig. Reaction cation
123456
6
3
of FFD; pale pillars before, of 100 NIH U thrombin.(Group
I:
stripped left,
pillars group-II:
after right
appliside)
In immunoelectrophoresis FFD were pointed out corresponding well In the upper part of figure 4 is to quantitative immunodiffusion. shown a band in the attachment area which reacted with anti-fibrinogen-serum, and a second one which moved to anode. We found this immunoelectrophoretic picture in each sample of both groups, in sample 5 of group I only the immunoelectrophoresis - seen in the bands (above with 10 pl serum, lower part of figure 4 - shows three below with 1 pl serum against 50 /ul antifibrinogen-serum).
Fig. Immunoelectrophoresis;
4 details
see
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The preoperative values of reptilase time are normal in group I, whereas they are longer in group II. In both groups reptilase time is prolonged during perfusion, more in group II than in group I. After detaching the apparatus, reptilase time stays prolonged in group I; in group II it goes back to normal (Fig. 5).
REPTILASE
SDPS
Fig.
- TIME Il”Z2
- TEST I,“:?
5
reaction of Upper half of the figure: reaction of SDPS-test. of the figure: right side)
reptilase time, (Group I: left,
lower group
half II:
In group I the SDPS-test is negative in sample 1. During perfusion SDPS-test becomes slightly positive. After neutralization of heparin with protamine chloride, the test is more strongly positive, and after application of antifibrinolytica, the intensity of the test increases even further. In group II, this test is already positive before surgery. The further development of SDPStest in group II corresponds to that of group I, but the intensity of the reaction is more marked (Fig. 5). DISCUSSION Looking for signs of fibrinolysis at the end of ECC, we have seen a much greater loss of fibrinogen as well as plasminogen in group II than in group I. This differenting decrease was also to be seen in the fast acting antiplasmin which is a useful indicator of the amount of plasmin neutralized (4). These drops of fibrinogen, plasminogen and alpha-2-macroglobulin suggested that fibrinolysis was active (17) much more in group II than in group I, despite a low level of activator (18) which was pointed out by paperfibrinolysis. A more pronounced fibrinolysis in group II was also suggested by a larger amount of FFD and a more prolonged reptilase time (14,191.
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This drop in fibrinogen and plasminogen might not only be produced by fibrinolysis but could also be caused by DIC (20,21). That would be pointed out by the increase of FM in the SDPS-test The immunoelectrophoresis showed FR-antigen material (5,15). which could be complexes of FM with FDP/fdp and which were unclottable (21,22). Therefore we thought that during the ECC an intravascular coagulation had developed in spite of high doses of heparin (4,23). After neutralization of heparin with protamine chloride we observed a different development of these coagulation parameters between group I and II. Whilst in group I fibrinogen, plasminogen and antiplasmin showed a tendency to increase during an increased turnover of coagulation factors, there was a further decrease of these parameters in group II. The bleeding which had now developed after application of protamine chloride could be correlated to the increasing of FM and a simultaneous drop of fibrinogen and plasminogen. But at this time there was a small amount of FFD only in sample 3 or none in samples 4 and 5 which might be able to disturb the coagulation system and produce fibrinolytic bleeding (24,25). Therefore it could not be a fibrinolytic bleeding, but it had to be the consequence of increased DIC which lead to a consumption coagulopathy (27). The question now arose why after giving protamine chloride at the end of the bypass time in sample 3 the amount of FFD diminished and FFD disappeared after application of antifibrinolytica and substitution of fraction I according to Cohn in group II. We thought this disappearance of FFD could be called a paracoagulation phenomenon in vivo which is triggered by protamine chloride (28). Our observations could be correlated to the results of Castaneda (6) and RLidegran (29) who have observed a drop in the count of red triggered by injection of procells (6) and of platelets (29), tamine. A further question was to be answered, why group II had de- as we have already said veloped such a severe hemorrhage while had taken place in both groups qualitatively similar changes The reason for the development in group II could be during ECC. explained by the preoperative values of group II. This had shown signs of chronic DIC with reactive fibrinolysis, e.g. FM, high low antithrombin-III-activity (see level of fibrinogen and FFD, high activity of factors VIII and IX (see part II), part II), low level of plasminogen and antiplasmins (25,27,30,31). This chronic DIC could be responsible for a reduced clearance of RES This disturbed clearance lead to a reduced elimination (4,30,32). (4,27,33) which was set activators of FM, FFD and prothrombin an other reason for the developfree from parts of cells and was ment of DIC (4) after neutralizing of heparin and could be seen as the cause of a consumption coagulopathy (27). the neutralization of heparin From-the above we may follow, with protamine at the end of the bypass time is contraindicated (4,7). We thank technical
Miss H. Schreiber assistance.
ACKNOWLEDGEMENTS Gerhard and Miss I.
for
their
valuable
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MARDER, V.J., SHULMAN, N.R. and CAROLL, W.R.: The of intermediate degradation products of fibrinogen lytic hemorrhage. Trans Assoc. Am. Physicians 80,
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LASCH, H.G., HEENE, D.L., HUTH, K. and SANDRITTER, W.: Pathoclinical manifestations and therapy of consumption physiology, coagulopathy ("Verbrauchskoagulopathie"). Am. J. Cardiol. 20, 1381, 1967.
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