THROMBOSIS RESEARCH 61; 369-373,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
FIBRIN-DEPENDENT FIBRINOLYTICACI’IVITY DURING
Ricardo Giuliani, Eduardo Szwarcer,Eleno MartinezAquino, GracielaPalumbo. Departamento de Trombosis, Instituto de Cardiologia, HospitalEspanolde Buenos Aires. Argentina. (Received
in revised form 15.11 .1990 by Editor H. Kwaan)
Editorial Offfice 19.12.1990)
By-Pass (CPB) Surgerymay at times induce a haemostatic defect, at presentnot too well understood, causing severe bleeding from the operativesite and chest tube drain. We present here some data on antigen increasein tissue PlasminogenActivator (tPA) and D 2 Dimer (D2D) detectedduring CPB and apparentlynot compensated by enhancedPlasminogen Activator Inhibitor type 1 (PA1 1) activity. tPA concentration (antigenic) ranged around 6.15 ng/ml (SD 5.6) before thoracotomyand 5.8 g/ml (SD 4.74) 5-10 minutes after a heparin 250 IU/Eg bolus injection.During CPB, tPA increased to 20.34 ng/ml (SD 9.17) before protamineinfusion, and 16.93 ng/ml (SD 8.13) after heparin neutralization. As the D2D went up to 2000-4000ng/ml (before/after protamine)and it was not correlated by fibrinogenconsumption or FDP production,we find these observations suggestive of fibrin-dependent fibrinolytic activity, as an acquired haemostaticdefect developedduring CPB.
INTRGDUcPION Bleeding after Cardiopulmonary By-Pass (CPB) in open heart surgery, may be heavy enough to force the surgeonto reexplorethe chest in some 3 to 5 percent of patients (1,2) sometimesfindingan open vessel where the haemorrhage can be efficientlycontrolled,but occasionally extracorporeal circulation (the pump oxygenatorin particular) causes a severehaemostaticdefect,at present not too well characterizedand difficultto restore (3,4). There are not many studieson haemostasisduring and after CPB, based on a tight correlation between test tube data and physiopharmacological changesduring the procedure, such as hypothermiaand haemodilutionlevels, effect of drugs and anaesthetics,type of oxygenator,blood transfusionproducts,etc.(l) In at Key words:
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least one study (4) it was fairly clear that clotting factors and inhibitors, plasminogen, alfa 2 antiplasmin and platelets usually drop related to haemodilutionlevela. There is Bornefurtherdrop in platelet8of about 10 percent and in factorV to about 20-25 percent. However theae haemostatic changes alone do not explain the, at times, severebleeding associated with CPB. In the above mentionedstudy Mammen et al could demonstrate a decrease in platelet responseto riatocetin,after heparin neutralization,in CPB surgery (4). There is at least one communicationon aix patientashowingan increasein tiaaue Plaaminogen Activator (tPA) during CPD, measuredby a fibrinplate technique(5). Plaamin digestion of glycoproteinIB aeema to hinder ita reactivitywith VW factor (6), and in a recent prospective, randomized and double blind aprotinin trial, a significantbleeding time shorteningwas demonstratedin treatedpatientaafter CPB surgery (7). In thia paper, we offer further informationon fibrinolyticchanges induced by CPB surgery,at differentatage8, in a atandarizedprotocol.
MATHRIALANDMETHODS A group of 29 patientsranging in age between 40 and 66 were inveatigated. 7 had valve replacementaand 22 coronaryby-paaa grafting. Standard surgical protocol and deviceswere uaed: a roller pump, a bubble oxygenator and a priming aolutioncomposedof 3 unite of plasma, 500 cc 5% dextroseand about 400 cc aaline. The mean CPB time averaged95 minutea. In this aet of patients,blood aampleswere drawn at the followingsurgical atagea: 1) prethoracotomy,2) prepump (5 to 10 minutes after 250 IU/Kg of a heparin bolua infuaion), 3) preprotamine (immidiatelybefore heparin neutralization)and 4) poatprotamine (after protamine infusion). The aaaaya were done on Platelet Poor Plaama (PPP) obtainedby centrifuging titrated whole blood (9 parts blood plus 1 part 0.13 mol/L sodium The PPP was atored in sealed polypropylenetubes at minua 20-O citrate). Centigrade until analyzed. Hematocritwas registeredin each blood sample to reflect in this way the haemodilutionlevel at each stage. tPA determinations were performed by an enzyme immunoaorbent assay (ELISA) with commercial reagenta (Tint ELISA, Biopool,Umea,Sweden),and data expreaaedin rug/mlactPA (singlechain tPA) protein (6,9). activity was measured by a two-step procedure, according to PA1 Chmielewska et al (lo), alao using a commercialreagentkit kindly provided PA1 1 was by Dr.Mata Ranby (Spectrolyee, Biopool,Umea, Sweden).Functional defined in actPA unita remainingafter interaction(15-20minutes)with the PA1 1 sample (initialactPA concentration= 40 IU/ml). D 2 Dimer antigen concentration was assayed by an agglutination aemiquantitativeteat based on monoclonalantibodiesantiD 2 Dimer linked to latex beads (11). FDP was measuredby a Staphylococcal ClumpingTest, as described(12). Fibrinogen was determined as total coagulable protein by a standard procedure(13).
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RESULTS mean 6.15 ng/ml (SD 5.6), in tPA leve1 ranged between 3.5-18.5 &ml, mean 5.8 ng/ml (SD 4.74), in Stage 1 (prethoracotomy)and 3.5-18 &ml, Stage 2 (prepump);but increasedto values spread between 3.1-37 r&ml, mean and 3.5-35 ng/ml, mean 20.34 ng/ml (SD 9.17), in Stage 3 (Preprotamine) 16.93 ng/ml (SD 8.31) in Stage IV (postprotamine). PA1 1 stayed within the normal range: 4.86 IU/ml (SD 2.48) in Stage 1; 4.91 IU/ml (SD 3.17) in Stage 2; 4.52 N/ml (SD 2.49) in Stage 3 and 5.26 IU/ml (SD 3.97) in Stage 4.
6.15 + 5.6
4.91 f 3,17
5.80 2 4.74
4.52 + 2.49
20.34 f 9.17
5.26 f 3.97
16.93 + 8.31
1 V8 2
P < 095
p < 0.7
2 VB 3
p < 0.6
p < 0.001
3 VB 4
p < 0.6
p < 0.001
D2D antigen concentration increased remarkably to levels of 2000-4000 ng/ml. = at
- OOQ na/ml).
Stane I Stapea
o* 1% * Numberof patients.
FDP were detected in the normal range below 5 ug/ml. Fibrinogen concentration followed the haemodilution changes accordingto haematocrit(363 + 76 mg/dl to 259 _+_ 47 mg/dl).
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It should be noted that haematocrit must be taken into account, as release of PA1 1, s&PA and D 2 D might be even higher, by about 30 percent, because of haemodilution during CPB. DISCUSSION The normal concentration of sctPA in PPP, in resting healthy adults, has already been determined by other authors using different methods. Results were 4 ng/ml and 6.6 &ml, obtained by IRMA (immunoradiometric assay) (14) In our CPB observations, in stage 1, mean . and ELISA (B) respectively value was 4.86 ng/ml. 5-10 minutes after an infusion of 250 IU/Kg Sodium Heparin bolus, sctPA antigen did not increase as we would have expected, because of heparin effect (15). CPB procedure boosted sctPA antigen about 2.5 times in stages 3 and 4, to values obtained after strenous exercise or venous stasis (8). similar This increase in tPA seems to remain steady, throughout and even after according to some (incomplete) results not extracorporeal circulation, shown. PA1 1 activity does not seem to compensate tPA hike, as it kept its normal values during CPB. D 2 D antigen increment may be explained as an expression of a fibrin-dependant fibrinolytic activity. As fibrinogen decrease followed the haematocrit fall (haemodilution effect) and FDP values were below upper normal (5 ug/ml), fibrinogen lysis may be . In fact, Mammen et al (4) also demonstrated no consumption of excluded plasminogen and alfa 2 antiplasmin, providing CPB procedure is fibrinogen, properly managed with steady plasma heparin levels of about 2-2.5 IU /ml. Quite often the only clear abnormality detected in patients with haemorrhage after CPB surgery is still a prolonged bleeding time (16). after heparin unresponsiveness to ristocetin A relative platelet neutralization by protamine was also noted (4). it was shown that platelets can be less reactive In some in vitro studies, to VW factor after glycoprotein Ib plasmin digestion (6), and in vivo, in a randomized trial, in cardiovascular surgery, it prospective, double blind, was demonstrated a shortening of bleeding time and a marked reduction in in patients treated with trasylol(7). blood transfusion requirements, Therefore more studies should be performed to see if these tPA increments are related to excessive mediastinal blood shed after heart surgery.
ACKNOWLEDGMENTS The authors
wish to thank Mr.Federico
Bleeding after HARKER L.A. 314 (22), 14451447, 1986.
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12. 13. 14.
BACHMAN F., McKENNA R, C!X)LE E.R., NAJAFI H. The hemostaticmechanism after open heart surgery.1. Studies on plasma coagulation factors and fibrinolysis in 512 patients after extracorac Car&ipvasc Surg 70:7, 6-85, poreal circulation. 1975. McKENNA R, BACHMANN F, WHITTAKER B, GILSON JR, and WEINBERGM-Jr. hemostatic mechanism after open heart surgery. J m &vaac Swg 70:298-308 1975 MAMMEN E. F, KOETS M:H, WASHINGTON B.C, WOLK L-W, BROWN J.M, BURDICK M, SELIK N.R. WILSON R.F. Hemostasischangesduring cardio 3, 281-292,1985. pulmonaryby pass surgery. Semin KLLJFT C, BROMMER E.J. GOMES M, de *JONGD-S, NAUTA J. Enhanced fibrinolytic activity during cardiopulmonaryby pass in open heart plasmiriogen surgery in man is caused by extrinsic (tissuety'pe) activator. H\arJ (=lin 14 (5), 375382, 1984.