F. De Cock

Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions☆

Rapid inhibition of tissue-type plasminogen activator (t-PA) in human plasma was measured by addition of 5 IU (50 ng) of purified t-PA per ml plasma and measurement of residual t-PA in the euglobulin precipitate after 5 min incubation at 37 degrees C. The recovery of both t-PA activity and t-PA related antigen in pooled plasma from healthy individuals was approximately 90 percent, indicating that one ml of pooled normal plasma inhibits less than 1 IU or 10 ng of t-PA within 5 min. Of 20 control subjects 13 had less than 1 IU inhibitor activity; 5 subjects inhibited between 1 and 3 IU of t-PA and 2 subjects inhibited around 4.5 IU. The inhibitor titer in the latter two had however decreased to 1.8 and 2.7 IU after two days. Markedly increased rapid inhibition of t-PA (greater than 4 IU per ml) was found in plasma of patients with severe liver disease (3 of 8), pancreatitis (4 of 8), malignancy (5 of 26), but only very occasionally and transiently in that of patients with myocardial infarction (5 of 28) or deep vein thrombosis (2 of 9). Increased inhibition was observed on the first day following coronary bypass (22 of 42) or open heart (16 of 27) surgery but this had disappeared in 15 of 16 patients on the fifth postoperative day. Titration of inhibitor levels revealed maximal amounts of 30 to 50 IU per ml plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of antithrombin III (heparin cofactor) in man: effects of venous thrombosis and of heparin administration

Abstract. The metabolism of human antithrombin III (heparin cofactor) was studied in four control subjects, in four subjects with peripheral obliterative arterial disease, in six patients with recent venous thrombosis and in one patient with clinically severe haemophilia A. The labelled antithrombin III had a high specific activity (5.75 units/mg) and displayed a single band on SDS‐polyacrylamide gel electrophoresis. On Sephadex G‐100 gel filtration the labelled material eluted in the same position as the antithrombin III activity in plasma. Crossed immunoelectrophoresis of a mixture of fresh plasma and labelled antithrombin III against a specific antiserum, revealed a single precipitin line in which radioactivity was concentrated. The changes in electrophoretic mobility of both the plasma antithrombin III and the labelled material following the addition of heparin to the mixture or following coagulation were identical. The purified antithrombin III behaved as a homogeneous protein in the turnover experiments. The plasma radioactivity data were approximated by a sum of two exponential terms and the metabolism of antithrombin III represented by a two compartment mammillary model.

Analysis of coagulation and fibrinolysis during intravenous infusion of recombinant human tissue-type plasminogen activator in patients with acute myocardial infarction.

Coagulation and fibrinolysis were studied in patients with acute myocardial infarction during intravenous infusion of recombinant human tissue-type plasminogen activator (rt-PA) (0.75 mg/kg over 90 min, n = 101), streptokinase (1,500,000 IU over 60 min, n = 61), or placebo (n = 40). In the rt-PA group, the plasma level of rt-PA antigen was 1.2 +/- 0.6 micrograms/ml (mean +/- SD) and the euglobulin fibrinolytic activity (EFA) was 910 +/- 735 IU t-PA/ml. In the streptokinase group, the EFA was equivalent to 430 +/- 435 IU t-PA/ml. At the end of the infusion, the plasma fibrinogen level measured with a coagulation rate assay was decreased to 57 +/- 33% of the preinfusion value in the rt-PA group, to 7 +/- 10% in the streptokinase group, and remained unchanged in the placebo group. Fibrinogen-fibrin degradation products increased to 0.75 +/- 0.54 mg/ml in the streptokinase group but to only 0.10 +/- 0.13 mg/ml in the rt-PA group. The plasma levels of alpha 2-antiplasmin, plasminogen, and factor V decreased to between 30% and 45% in the rt-PA group but significantly more in the streptokinase group (to between 15% and 25%). Thus rt-PA induced much less systemic fibrinolytic activation than streptokinase. In the patients who received rt-PA, a weak correlation (r = .21, n = 89, .1 greater than p greater than .05) was found between the extent of fibrinogen breakdown at 90 min and the plasma rt-PA concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma fibrinopeptide A levels in patients with acute myocardial infarction treated with alteplase. Correlation with concomitant heparin, coronary artery patency, and recurrent ischemia. The European Cooperative Study Group.

BackgroundFibrin generation during and after therapy with alteplase may depend on the level of concomitant anticoagulation. The hypothesis that fibrinopeptide A (FPA) levels, as markers of ongoing in vivo fibrin formation, correlate with the angiographic and clinical outcome of thrombolysis is tested. Methds and ResultsSerial plasma FPA levels were determined in 334 patients of the randomized European Cooperative Study Group trial comparing heparin versus placebo plus alteplase and aspirin in patients with acute myocardial infarction. Median FPA levels (with the 10th to 90th percentiles) were 21 ng/ml (2–390 ng/ml) before treatment in placebo-allocated patients (n = 166) and increased to 49 (15–580), 34 (4–320), 27 (2–240), 29 (2–430), and 30 (3–390) ng/ml after 0.75, 3, 12, 24, and 36 hours, respectively. In heparin-allocated patients (n = 168), median baseline FPA values were 18 ng/ml (2–210 ng/ml) and decreased to 6 (1–110), 5 (1–75), 5 (1–60), 7 (1–100), and 10 (1–170) ng/ml at corresponding time points (p<0.0001 for the difference at each time point). Adequate anticoagulation, defined as no activated partial thromboplastin time value below twice the pretreatment value at 3, 12, 24, and 36 hours after initiation of treatment, was obtained in 48 patients assigned to heparin. It was associated with normal median FPA levels (≤4 ng/ml) at all time points compared with 12 (2–80), 16 (2–240), and 15 (2–240) nglml at 12, 24, and 36 hours, respectively, in heparin-assigned but inadequately anticoagulated patients (n = 102, p<0.001 for each time point). In the heparin-treated group, median FPA values tended to be lower at all time points in patients with patent vessels than in patients with occluded arteries, but the difference was significant only at 24 hours (p = 0.04). FPA levels did not correlate with clinically apparent recurrent ischemia or with left ventricular thrombosis on two-dimensional echocardiography. ConclusionsDuring and after thrombolytic therapy with alteplase, the enhanced fibrin generation is suppressed by sustained concomitant anticoagulation with intravenous heparin. Adequate anticoagula-tion warrants individual titration of the heparin dose. High plasma FPA levels 24 hours after alteplase therapy are specific but insensitive markers of vessel occlusion in anticoagulated patients. They do not correlate with clinical outcome.

Comparative immunogenicity and thrombolytic properties toward arterial and venous thrombi of streptokinase and recombinant staphylokinase in baboons.

BackgroundStreptokinase is a routinely used thrombolytic agent that is immunogenic and relatively inefficient toward platelet-rich thrombus, whereas staphylokinase is a poorly studied fibrinolytic agent. Here, the comparative immunogenicity and thrombolytic properties toward arterial platelet-rich thrombus and venous whole blood clots of streptokinase and recombinant staphylokinase were studied in baboons. Methods and ResultsThe inhibitory capacity of baboon plasma (in a human plasma-based clot lysis assay) was 039±0.25 μg streptokinase and 0.04±0.05 jag recombinant staphylokinase per milliliter of plasma (mean±+SD, n=9). Intravenous infusion over 1 hour of0300 mg/kg of streptokinase at 0, 1, 2,3, and 5 weeks in five baboons given heparin and the antiplatelet agent ridogrel increased the streptokinaseneutralizing titer from 0.22±0.18 μg/mL plasma at baseline to 3.8±4.4 μg/mL after 2 weeks (p=0.043 versus baseline by Wilcoxon signed rank test) and to 4.4±4.6 μg/mL after 5 weeks, whereas the thrombolytic potency toward a 125I-fibrin-labeled plasma clot inserted into an extracorporeal arteriovenous loop was reduced from 84±7% at baseline to 45±8% after 2 weeks and to 36±8% after 5 weeks (p<0.01 versus baseline). Administration over 1 hour of 0.065 mg/kg recombinant staphylokinase at 0, 1, 2, 3, and 5 weeks in four baboons did not induce measurable staphylokinase-neutralizing activity in three of the four animals after 5 weeks. In the fourth baboon, a staphylokinase-neutralizing activity of 0.8 and 1.5, ug/mL was found at 3 and 5 weeks, respectively. Repeated staphylokinase administration was not associated with inhibition of clot lysis (43+4% lysis at baseline, 52+9%o at 3 weeks, and 61+14% at 5 weeks; p=NS versus baseline). Repeated administration of streptokinase but not of staphylokinase caused a marked (>50%) decrease in blood pressure, requiring administration of steroids and intravenous fluids, and a marked increase in leukocyte count and hemoglobin concentration. Intravenous infusion of streptokinase or recombinant staphylokinase over 1 hour in doses ranging between 0 and 1.0 mg/kg in three groups of four baboons each induced dose-dependent lysis of a 125I-fibrinlabeled autologous jugular vein blood clot (50% lysis requiring 0.140 mg/kg streptokinase and 0.058 mg/kg recombinant staphylokinase, representing equimolar amounts of 3.25 nmol/kg) without systemic fibrinogen depletion. The thrombolytic potency toward platelet-rich arterial thrombus of streptokinase and recombinant staphylokinase were studied in a femoral arterial eversion graft model. Arterial recanalization with recombinant staphylokinase was more frequent and more persistent than with streptokinase (all p<0.05). Intravenous infusion of 1.0 mg/kg streptokinase or 0.25 mg/kg recombinant staphylokinase in two groups of four baboons each given intravenous heparin (200-unit bolus and 50 units. kg-1. hr-1) and aspirin (10 mg/kg) did not produce a significant prolongation of the median template bleeding time. ConclusionsRecombinant staphylokinase has a thrombolytic potency toward jugular vein blood clots in baboons comparable to that of streptokinase, but it is less immunogenic and less allergenic and it does not induce resistance to lysis upon repeated administration; it is significantly more efficient than streptokinase for the dissolution of platelet-rich arterial eversion graft thrombi. Recombinant staphylokinase, which can be easily obtained in active form by expression in Escherichia coli, may constitute a potentially useful alternative to streptokinase for the treatment of acute myocardial infarction.

Comparative thrombolytic and immunogenic properties of staphylokinase and streptokinase

The comparative thrombolytic properties of natural (STAN) and recombinant (STAR) staphylokinase (STA) and of streptokinase were studied in hamsters with a pulmonary embolus consisting of a platelet-poor, platelet-rich (300000 platelets/μI), platelet-enriched (1500 000 platelets/ μl) or mechanically compressed human plasma clot. Intravenous infusion over 1 h in doses ranging between 0 and 0.50 mg/kg induced dose-dependent progressive clot lysis without systemic fibrinogen breakdown. The relative thrombolytic potencies, on a weight base, of the STA preparations versus streptokinase were comparable in the platelet-poor clot model (50% lysis requiring 13 μg/kg STA and 12 μg/kg streptokinase) and in the platelet-rich clot model (50% lysis with 28 μg/kg STA and with 32 μg/kg streptokinase), but 5-fold higher in the platelet-enriched clot model (50% lysis with 46 μg/kg STA and with 210 μ/kg streptokinase) and 2-fold higher in the mechanically compressed clot model (50% lysis with 36 μg/kg STA and with 71 μg/kg streptokinase). Dog plasma at baseline contained streptokinase-neutralising activity (neutralising 0.24±0.14 μg streptokinase per ml plasma in a human plasma-based clot lysis assay, mean ±SD) but apparently no STA-neutralising activity. Repeated administration of 40 μg/kg over 1 h of streptokinase at weekly intervals in 5 dogs, increased the streptokinase-neutralising titre to 1.1±0.98 μg/ml plasma after 2 weeks and to 2.9±2.5 μg/ml after 3 weeks (p=0.13 and 0.05 versus baseline, respectively), whereas the thrombolytic potency towards a 125I-fibrin-labelled plasma clot inserted into an extracorporeal arteriovenous loop was reduced from 58±4% at baseline to 10±3% after 2 weeks and 4±1% after 3 weeks (p<0.001 vs baseline). Repeated weekly administration of an equipotent dose of STA (4 μg/kg) did not induce measurable STA-neutralising activity after 2 weeks and was associated with persistent, although somewhat reduced clot lysis (58 ± 4% lysis at baseline and 33±7% at 2 weeks, p=0.02). After 3 weeks however, STA-neutralising activity became detectable in plasma (0.34±0.30 μg/ml, p=0.07 vs baseline) and resistance to clot lysis became obvious (6±2% versus a baseline value of 58±4%, p<0.01). Thus, in the hamster model, STA has a thrombolytic potency towards platelet-poor and platelet-rich clots comparable to that of streptokinase, but it is relatively more efficient than streptokinase for the dissolution of platelet-enriched or retracted clots. Furthermore, STA might be less immunogenic than streptokinase as evidenced by less rapid induction of antibody formation and resistance to clot lysis upon repeated administration. Thrombolytic therapy of patients with acute myocardial infarction with STA thus might constitute a potential alternative to treatment with streptokinase, with a better efficacy towards platelet-rich arterial clots and possibly with reduced allergic side effects.

Kinetic properties of tripeptide lysyl chloromethyl ketone and lysyl p-nitroanilide derivatives towards trypsin-like serine proteinases

The steady-state kinetic parameters of the tripeptides D-Val-Leu-Lys-, Ala-Phe-Lys-, and < Glu-Phe-Lys- in which the free carboxyl group was substituted with p-nitroaniline (substrate) or chloromethane (inhibitor), towards the serine proteinases plasmin (EC 3.4.21.7), thrombin (EC 3.4.21.5), urokinase, factor Xa, and trypsin (EC 3.4.21.4) were investigated. The p-nitroanilide derives were found to be very good substrates for plasmin, 2.5--40-times less efficient towards trypsin and very poor (100--10 000-times less efficient) substrates for thrombin, factor Xa and urokinase. The chloromethyl ketone derivatives were comparably efficient inhibitors of plasmin and trypsin and in general very poor (100--10 000-times weaker) inhibitors of thrombin, factor Xa and urokinase. D-Val-Leu-Lys-pNA however was a very poor substrate but D-Val-Leu-Lys-CH2Cl a very efficient inhibitor for thrombin. The variability in susceptibility of the substrates towards the enzymes was due to differences in their Michaelis constant, in their deacylation rate constant or both. the variable efficiency of the inhibitors was mostly due to differences in their dissociation constant and much less to differences in their alkylation rate constant. Only a poor correlation (r = 0.25) was found between the efficiency of the p-nitroanilides as substrate and their homologous chloromethyl ketones as inhibitor. The most notable discrepancy was observed with the D-Val-Leu-Lys derivatives towards thrombin.

Comparative fibrinolytic and fibrinogenolytic properties of staphylokinase and streptokinase in plasma of different species in vitro

Abstract The comparative fibrinolytic and fibrinogenolytic properties of staphylokinase and streptokinase were studied in plasma of the human, baboon, rabbit, hamster, rat and dog species in vitro. Equipotent concentrations of staphylokinase and streptokinase were determined as the concentrations that caused 50% lysis in 2 h (C 50 ) of 60 μl 125 I-fibrin labelled autologous or human plasma clots submersed in 250 μl citrated plasma. In addition, residual fibrinogen levels at C 50 were determined. In human plasma, staphylokinase was 4-fold more potent than streptokinase (C 50 of 17 and 68 nM respectively) and was more fibrin-specific (residual fibrinogen level 95 and 50 of 32 nM, 27 nM and 36 nM vs. 370 nM, 270 nM and >450 nM respectively) or human plasma clots submersed in autologous plasma (C 50 of 24nM, 24nM and 17nM vs. 100nM, 87nM and 10nM respectively). Residual fibrinogen levels at C 50 in the autologous systems were ⩾80% of baseline. The autologous rat system was very resistant to lysis with both staphylokinase and streptokinase (less than 5% clot lysis at concentrations of 500 nM), whereas in the dog system staphylokinase appeared to be 60-fold more potent than streptokinase (C 50 of 7 nM and 430 nM respectively). These results indicate that the plasma fibrinolytic systems of baboons, rabbits and hamsters react comparably to the human system in terms of their fibrinolytic and fibrinogenolytic response to staphylokinase. The rat system appears to be more resistant and the dog system more sensitive.

Turnover of human histidine-rich glycoprotein in healthy subjects and during thrombolytic therapy

Abstract Human histidine-rich glycoprotein, a protein with a potential antifibrinolytic effect, was purified to homogeneity and labeled with 125I. This material was injected intravenously and its turnover followed in 4 control subjects and in 3 patients undergoing thrombolytic therapy with streptokinase. The purified labeled histidine-rich glycoprotein behaved as a homogeneous protein in the turnover experiments and the plasma radioactivity disappearance rate could adequately be described by a sum of two exponential terms. The main metabolic parameters in the controls are : plasma radioactivity half-life 2.93 ± 0.36 days, fractional catabolic rate constant 0.51 ± 0.06 of the plasma pool per day, and synthetic rate 1.59 ± 0.35 mg7kg/day. During thrombolytic therapy with streptokinase the labeled histidine-rich glycoprotein disappeared with a half-life of only 1.43 ± 0.10 days due to an increased fractional catabolic rate constant (1.21 ± 0.13 of the plasma pool per day). At the same time the synthetic rate increased 2–3-fold (3.39 ± 2.35 mg/kg/day) resulting in only a slight decrease of the plasma level of histidine-rich glycoprotein.

A latex agglutination test for rapid quantitative estimation of the plasmin–antiplasmin complex in human plasma

Abstract. An antiserum was raised in rabbits against human plasmin‐antiplasmin complex and rendered specific for neoantigens of this complex by absorption with purified plasminogen and plasma. Polystyrene particles were coated with the specific antibodies and used in an agglutination test for the determination of plasmin‐antiplasmin complex in the plasma from various patients. Purified plasmin‐antiplasmin complex at a concentration of 0.1–0.2 mg/l was found to cause a clear agglutination of the particles.