THROMBOSIS RESEARCH 57; 783-794,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.

HYPERCOAGULABLE STATE AFTER THROMBOLYTIC THERAPY IN PATIENTS WITH MYOCARDIAL INFARCTION (AMI) TREATED WITH STREPTOKINASE.

ACUTE

Virtudes Vilaa, Edelmiro Regaiihnz,Justo Aznari, Victoria Lacuevac, Miguel Ruano , BegofiaLaiz a

Research Center,

b

Department of Clinical Pathology, 'Intensive Care Unit. La Fe Hospital, Valencia. Spain.

(Received 1.8.1989; accepted in revised form 5.1.1990 by Editor F. Haverkate)

ABSTRACT Fibrinogen activity was studied in 70 patients with AM1 who were treated with an intravenous infusion of SK (800,000 U/30 min or 1.5 miil U/60 min). Patients received a continuous infusion of heparin after thrombolytic therapy was completed. 800 000 U and 1.5 mill U SK recanalized infarct-related arteries at's rate of 7S%. Early re-infarction occurred in 6% in each group. Upon admission to the hospital patien'isshowed a hypercoagulable state that may be related to an elevated level of fibrinogen and HMW fibrinogen (70.522 vs 6522 % in patient and normal plasmas, respectively) that changed to a transitory hypocoagulable state indicated by decreased fibrinogen levels after SK treatment. Forty-eight hours after SK, a new fibrinogen hyperfunction, related to an increase in fibrinogen level and especially HMW synthesized fibrinogen (82+1 or 81+1 %, 800,000 and 1.5 mill U SK, respectively) was observed, which was neutralized by heparin therapy (1,660 U/h with continuous infusion). The elevated levels of fibrinogen (363221 vs 24028 mg/dl in patient and normal plasmas, respectively) and HMW fibrinogen (70+3 % with both SK doses) observed seven days after SK may -be related to a hypercoagulable state that is not neutralized by the heparin dose used (5,000 U/4 h bolus). Patients who showed non-recanalization were compared with those whose arteries recanaiized. The former group had a higher concentration of fibrinogen (197+31 vs 147+18 mg/dl), HMW fibrinogen (7820.5 vs 7420.3 %, respectiv&y), and 'PA (130+3 vs 624 pmol/ml) and more extensive fibrin gel formatian kinetics (gelation rate 3.321.4 vs 1.120.2 OD/s x 10 , respectively) than the second group. The hypercoagulable state found in patients with acute myocardial infarction undergoing thrombolytic therapy may be related mainly to the progression of HMW fibrinogen and fibrinogen levels. Key words: Thrombolysis, fibrinogen, streptokinase 783

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INTRODUCTION Thrombolytic agents are used mainly for treatment of acute myocardial infarction (l-5), peripheral artery occlusion (6-7), deep-vein thrombosis (8-9) and pulmonary embolism (10). However, there are still many unanswered questions about the complications observed after successful recanalization of the coronary artery. One of the many factors that determine non-recanalization and early reocclusion may be associated with fibrinogen activity, which may produce a hypercoagulable state in patients after thrombolytic therapy (ll-13), and with the patient response to heparin therapy initiated after thrombolytic treatment (12,14-15). Studies of patients with acute myocardial infarction have shown elevated levels of fibrinopeptide A in plasma (16-18), which is a useful index of fibrin formation "in viva" (19-21). Heparin usually reduces the elevated fibrinopeptide A level in plasma, but in many patients, neither subcutaneous nor intravenous heparin is sufficient at conventional doses to bring the elevated FPA level back to normal (16-17,22). The cause of this insufficient response to heparin therapy remains unclear. The hypercoagulability indicated by the high levels of fibrinopeptide A after thrombolytic therapy is begun and during heparin therapy may bear a relationship to the early reocclusions detected in some of these patients (21). It is an established fact that normal human plasma fibrinogen contains two major fractions, one of high (HMW, 340 Kd) and the other of low (LMW, 320 Kd) molecular weight (23-25). HMW fibrinogen is more sensitive to thrombin than LMW fibrinogen (26-27) and has a higher gelation rate (28). The 340 Kd fibrinogen is the most sensitive to plasmin degradation (28). While it is clear that patients suffering from extensive acute myocardial infarction display a short-term increase in fibrinogen, few studies have dealt with the question of whether this increase is conditioned by the increase in high molecular weight fibrinogen (24). After thrombolytic therapy it is not clear which fibrinogen species is responsible for the concentration increase resulting from the new synthesis of fibrinogen. In the present study we examined changes in the activity of fibrinogen, fibrinopeptide A, fibrin(ogen) and cross-linked fibrin degradation products before and after administration of streptokinase (800,000 U/30 min, 1.5 mill U/60 min) and heparin. The subjects of the study were acute myocardial infarction patients undergoing thrombolytic therapy. We compared patients in whom thrombolysis was successful with those in whom it was unsuccessful. The progression of the lesion and, consequently, the symptoms and signs of ischaemia in these patients after thrombolytic therapy may, therefore, be related to a hypercoagulable state. PATIENTS AND METHODS Patients: The study group comprised 70 patients (64 males, 6 females), aged 51~8 (range: 40-67). Criteria used for initial diagnosis of acute myocardial infarction (AMI) were: typical chest pain, at least 30 min in duration, and electrocardiogram ST-segment elevation of more than 0.2 mV in two electrocardiographic leads, even after sublingual administration of nitroglycerin. AM1 was confirmed in all patients by a rise in the creatine kinase level. Patients were eligible for treatment with intravenous streptokinase (SK) if they were evaluated within 4 hours of the onset of pain. Patients were excluded for any of the following reasons: more than 70 years of age, known bleeding diathesis, recent cerebrovascular accident, transient ischemic attack, diabetes mellitus, chronic renal or hepatic failure, recent treatment with streptokinase, streptococcal infection, previous cardiac surgery, oral anticoagulant therapy, prolonged cardiopulmonary resuscitation. Thirty-five patients received 800,000 U SK for 30 min and 35 patients

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received 1.5 mill U SK infused over one hour. Immediately after thrombolytic therapy was completed, a regimen of heparin treatment was initiated. Patency of the recanalization was determined at 48 h by quantitative coronary angiography. Reinfarction and recurrent angina were detected by applying clinical, electrocardiographic and enzymatic criteria. Blood was tested every hour during the first 24 hrs for the cardiac enzyme CK and its myocardial band fraction (CK-MB). Blood pressure and electrocardiographic changes were monitored. Patient samples were classified in the following groups: pre-SK, prior to SK administration; post-SK, 5 min after intravenous SK infusion; 24 hr after SK, with 500 U/hour intravenous heparin; 48 hrs after SK, with 1,660 U/hr intravenous heparin; and 7 days after SK. In this last group the plasma samples were taken two hours after 5 000 U of heparin bolus every 4 hours. Control subjects Fifty healthy male volunteers, aged 4527 years. Blood samples were drawn, with minimal stasis, from patients and normal subjects into vacutainer tubes containing l/10 volume of 0.13 mol/l sodium citrate. 200 U of Trasylol/ml blood were added to the blood samples. The plasma was separated immediately following centrifugation at 3,000 rpm for 15 minutes. The level of anticoagulation was monitored with serial partial thromboplastin times (PTT). Heparin infusion dosages were titrated in an attempt to maintain the PTT between 90 and 150 seconds. Thrombin and Reptilase coagulable material was determined in plasma or purified fibrinogen following the fibrin gel formation kinetic method (29). Thrombin and Reptilase were added to a final concentration of 0.45 U/ml and the turbidity was measured (absorbance at 350 nm) in a double beam spectrophotometer. The gelation rates were obtained from the turbidity curve (29). The gelification rate varies according to the fibrinogen concentration and coagulation capacity. The standard curve was obtained by clotting plasmas at different known fibrinogen concentrations. The calculated gelification rate values corresponding to the fibrinogen levels of each patient and control plasmas were evaluated from the standard curve. Fibrinogen purification was performed by precipitation according to the polyethylene glycol 6,000 method as described previously (30). The precipitated fibrinogen was dissolved in 0.018 M phosphate buffer, pH 7.8. Aliquots were stored in polystyrene tubes at -2OeC. Fibrinogen concentration was measured spaces photometrically at 280 and 320 1.506 (31). Plasma fibrinogen nm, using the extinction coefficient, E ’ concentration was measured by heat precipitation (32) and by gelation methods (29). Fibrin monomers were obtained as previously described (33). Human thrombin was added to decalcified plasma, at a final concentration of 6.25 U/ml, and the clot was dissolved in 0.02 M acetic acid. Serum fibrin(ogen) degradation products(FDP) were measured by the immunohaemoagglutinationmethod (34). Plasma cross-linked fibrin degradation products (D-dimer) were measured by ELISA (Diagnostica Stago, Germany) with a monoclonal antibody for D-dimer that does not cross-react with fibrinogen or non cross-linked fibrinogen degradation products. Glycosidically bound sialic acid was estimated by the thiobarbituric acid method (35). Plasma fibrinopeptide A (FPA) was measured by the radioimmuno-assay method (IMCO Corp, Sweden). Plasmas were treated twice with 10% bentonite and analyzed by SDS-PAGE to confirme the absence of fibrinogen. Gel electrophoresis and immunoblotting. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the samples was performed at 4eC on 140 x 110 x 0.9 mm gels, with an acrylamide gradient of 35-50 g/l and 50-70 g/l for

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non-reduced and reduced samples, respectively. Electrophoresis was done in a Tris-gly buffer continuous medium at 25 mA/slab for 2 hrs (36-37). After electrophoresis the gels were scanned in a Shimadzu (Japan) chromatogram scanner. It was ascertained that the area of peaks obtained by scanning the gels with purified proteins was a linear function of the amount of protein loaded. Molecular weights were estimated on a calibration curve obtained under identical conditions with proteins of known molecular weights. Electroblot transfer of separated proteins to nitrocellulose sheets was adapted from the method of Towbin et al (38). The transfer was done at 20 V/cm for 18 hours. 25 mmol/l Tris, 192 mmol/l glycine and 0.2 l/l methanol were used as the electrode buffer. Immediately after transfer, the nitrocellulose sheet was immersed in Tris-saline buffer (20 mmol/l Tris-HCl, 0.15 mol/l NaCl pH 7.4) containing 30 g/l albumin and incubated for 60 min. The sheet was then transferred to Tris-saline buffer containing 0.5 g/l Tween-20 and rabbit-antihuman fibrinogen diluted 1:200 and incubated for 2 hours at 37aC. The sheet was rinsed in Tris-saline-buffer containing 0.5 g/l Tween for 30 min, incubated with horseradish peroxidase conjugated goat anti rabbit IgG (diluted 1:2000) for 60 min at 37gC!,and rinsed with Tris-saline-Tween buffer and stained for peroxidase. Initial experiments were performed to examine the specificity of the antibody. The rabbit-antihuman fibrinogen IgG, when tested by immunoblotting, reacted only with fibrinogen and related proteins. This antibody did not react with any other plasma proteins (fibronectin or von Willebrand or IgG). Statistical analysis. All numerical results are expressed as mean 2 SEM. Tests for mean differences were performed with the unpaired Student t-test for analytical data. A p value of 0.05 was considered statistically significant. RESULTS Table I shows the data recorded for the groups of AM1 patients receiving 800,000 U or 1.5 mill U intravenous SK and for the healthy control group. The fibrinogen concentration is significantly increased in AM1 patients before thrombolytic therapy. When the high molecular weight fibrinogen concentration of groups in therapy was compared with the control group values, a significant increase was observed in the pre-SK group. In the post-SK group the concentration dropped to less than 20 % of the values in samples before administration of SK. Later the concentration of high molecular weight fibrinogen rose continously and 48 hours and 7 days after SK reached values that were significantly higher than in the controls. The functional activity of plasma fibrinogen (gelation rate) was estimated by fibrin gel formation from the patients and control plasmas (experimental values). The gelation rate values were also obtained from fibrinogen concentration on the standard curve (Table I). Because of the increase in fibrinogen concentration, the experimental and calculated gelation rate values with thrombin and Reptilase are higher in the pre-SK group than in the controls. The experimental values, however, are higher than those calculated theoretically from plasma fibrinogen concentration, which shows that fibrinogen hyperfunction is related to the increase in the high molecular weight species. Gelation rate decreased to nil after each dosage regimen of SK, then increased steadily until day 7, when it reached the same level as that of the pre-SK group. At 24 hours the gelation rates had decreased significantly with respect to the control group. When the experimental values are compared with those calculated from the fibrinogen concentration, an important discrepancy in the values obtained with thrombin is observed, whereas those obtained with Reptilase are only slightly reduced in both dosage regimens. Moreover, the PTT values are significantly higher. This is due to the presence of heparin and fibrinogen degradation products. At 48 hours the

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gelation rate values with Reptilase are higher than those of the control group and those calculated from their plasma fibrinogen concentration. This demonstrates that there is an increase in fibrinogen concentration and an increase in the highest molecular weight species (Fig l), which is the species with the greatest coagulation activity. The gelation rate values obtained with thrombin show a significant decrease with respect to the calculated values from plasma fibrinogen concentration in the 48-hour group. The prolongation of PTT in this group indicates that this decrease is due to the antithrombin action of heparin. On day 7 the calculated and experimental gelation rate values are similar, and both are significantly higher than those of the control group. Changes in the percent and in the concentration of HMW fibrinogen during therapy are correlated with gelation rate. Analysis of PAGE-SDS in plasmas (Fig 1) shows that after SK, the fibrinogen was virtually zero. Twenty-four hours after SK the fibrinogen present is mainly HMW fibrinogen which is degraded to LMW fibrinogen. Table II shows tne average levels of FPA, FDP and D-dlmer. When the values for the two groups are compared, no significant differences are found at any of the sampling times. When the FPA, FDP and D-dimer of the two groups are compared with control values, significant differences are observed at all the times studied. FPA values decreased from the pre-SK group to the 48 hours after-SK group and then increased until seven days after SK. However, the FPA values always showed an increase when compared with the control values. This coincided with the change in the heparin regimen from 1,660 U/h (continuous infusion) to 5,000 U/4 hrs (bolus). FDP and D-dimer rose to a maximum after SK infusion and then gradually decreased without reaching the normal values. TABLE I Changes in Coagulation Variables in a Group of AM1 Treated with 800,000 U/30 min (n=35) or 1.5 mill U/60 min (n=35) of SK.

SK (Ux106)

Fg

HMW

(mg/dl) (mg/dl)

GR(Thrombin) Found Calcu ated -$ (%) (OD/sxlO )

GR(Reptilase) PTT Found Calcu ated J (OD/sxlO- ) (s)

70+3 7171

5.1+1.5a 4.2 4.750.5a 4.0

Pre

0.8 1.5

Post

0.8 1.5

80+10; 99+8

24 h

0.8 1.5

155+14c 163514'

118+18 122511

76+4 75+3

1.3~0.2' 2.8 1.7+0.5' 2.3

1.5+0.6 l.lT0.3

1.4 1.5

b 79+9 b 64;8

48 h

0.8 1.5

288+12' 279514c

236+13; 226+6

8221 8121

4.9+0.6 3.550.8

7.6 7.2

4.9+0.4a 4.1T0.5a

3.8 3.6

126213' 141+12'

7d

0.8 1.5

10.2+1.3a 10.4 9.5T1.5a 10.4

5.420.4' 5.820.6'

5.2 5.2

2.7+0.2

2.7

Control -

305_e31C 219+30a 284+16' 20053 a 31+12' 3354 '

39+3 40+2

7023 70+3 240+8

158+8

65+2 _

10.022 9.5+1

;

8.4 8.0

-

5.6+0.3

3121 3421 9726 ; 8827

5.5

33+2

Data expressed as mean + SEM. SK: streptokinase; Fg: fibrinogen; HMW: high molecular weight fibrinzgen; GR: gelation rate. GR calculated: values obtained from the standard curve. a: p < 0.05 ; b: p< 0.005 ; c: p < 0.0005

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Kd

FIGURE 1 Immunoblotting of fibrinfibrin and crosslinked fibrin degradation products in AM1 patients treated with 800,000 U/30 min or 1.5 mill U/60 min of SK. NP, normal plasma. The fibrinogen species (HMW and LMW),fibrinogen degradation products (X, Y and D) and the molecular weight in K daltons of electrophoretic bands are indicated.

w=n ,

PRE

POST 24 h 48 h

7 ci

NP

AFTER SK

SK

TABLE II FPA, FDP and D-dimer Levels in AM1 Patients Treated with 800,000 U/30 min (n=35) or 1.5 mill U/60 min (n=35) of SK.

FPA (nddl)

Pre

FDP t&d11

D-dimer

25+7 2254

530260 560274

(ng/mli

0.8 1.5

18+12 20;11

Post

0.8 1.5

14+4 19t5

9432205°C 993294

24 h

0.8 1.5

12+4 1152

98215 b c 192245

48 h

0.8 1.5

7+2 VT2

34+6 31z8

7d

0.8 1.5

12+5 15;5

15+4 6T12

Control

2~0.3

;

b b

420.5

b b

9971+1800' 10462T1478' 4400+990 c 4719T1090C 1900+320 c 22207548 ' b 1020+350 b 14*712380 10628

Data expressed as mean + SEM SK: streptokinase; FPA: fibrinopeptide A; FDP: fibrin(ogen) degradation products; D-dimer: cross-linked fibrin degradation products. a: p