Jm Stassen

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.

Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation.

BACKGROUND RGD-containing peptides are able to prevent binding of ligands to certain integrins such as alpha IIb beta 3 (glycoprotein IIb/IIIa) and alpha v beta 3 and as such are inhibitors for platelet aggregation and smooth muscle cell migration, both of which are involved in neointima formation. METHODS AND RESULTS Hamster carotid arteries were damaged, and neointima formation was determined at different time points. G4120, a cyclic RGD-containing peptide, was administered continuously intravenously by an implanted osmotic pump. Neointima formation was inhibited dose dependently. The inhibition was strongest when treatment was started before the vascular injury and continued for the full observation period. Treatment started after the damage and maintained until neointima assessment or started before and stopped earlier was less effective. CONCLUSIONS Inhibition of integrin function by an RGD-containing peptide results in reduction of the development of a neointima. This effect is due both to an early event, which could be due to inhibition of secretion of PDGF by the platelets with blocked alpha IIb beta 3, and to a late event, possibly by interference with smooth muscle cell alpha v beta 3.

Thrombolysis with Human Extrinsic (Tissue-type) Plasminogen Activator in Dogs with Femoral Vein Thrombosis

Extrinsic (tissue-type) plasminogen activator (plasminogen activator) was isolated either as a single-chain or as a two-chain molecule from the culture medium of a human melanoma cell line. The thrombolytic activity of both molecular forms of activator was investigated in beagle dogs with an experimental femoral vein thrombosis and compared with that of urokinase. The 125I-fibrinogen-labeled thrombus was formed in an isolated 4-cm segment of the vein, aged for 30 min, and the thrombolytic substances were infused over a 4-h period. The degree of thrombolysis was measured 2 h later as the difference between the injected and recovered 125I. In six control animals with a saline infusion the extent of thrombolysis was 16.3 +/- 3.8% (mean +/- SEM), in five dogs receiving 100,000 IU urokinase, 17.4 +/- 3.7% (P less than 0.4) and in four dogs with 1,000,000 IU urokinase 40.6 +/- 4.8% (P less than 0.001). Infusion of 100.000 IU single-chain plasminogen activator in five dogs resulted in 3.5 +/- 7.8% lysis (P less than 0.05) and of 100,000 IU two-chain plasminogen activator in five dogs in 60.1 +/- 10.8% (P less than 0.001). Infusion of 300,000 IU one-chain plasminogen activator yielded 57.5% lysis and of the same amount of two-chain plasminogen activator 72.9%. Significant activation of plasminogen, consumption of alpha 2-antiplasmin, and fibrinogen breakdown in plasma was only observed in animals receiving the high doses of urokinase but not in the saline, plasminogen activator, or the low-dose urokinase groups. It is thus concluded that in this thrombosis model human extrinsic plasminogen activator has a higher specific thrombolytic effect that urokinase. Plasminogen activator also appears to induce thrombolysis without systemic fibrinolytic activation and fibrinogen breakdown.

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.

Thrombolytic and pharmacokinetic properties of chimeric tissue-type and urokinase-type plasminogen activators.

BackgroundChimeric molecules comprising the A-chain of tissue-type plasminogen activator (t-PA) and the catalytic domain of urokinase-type plasminogen activator (u-PA) have intact enzymatic characteristics of u-PA, partial fibrin-binding properties of t-PA, and thrombolytic properties in animal models comparable with but not superior to those of single-chain u-PA (scu-PA). Deletion of the finger and growth factor domains (t-PA-AFE/scu-PA-e) in such chimeras further reduces their affinity for fibrin. Methods and ResultsA detailed investigation of the thrombolytic potency and the pharmacokinetics of t-PA and u-PA chimeras was performed in quantitative animal models for thrombolysis. In hamsters with pulmonary embolism, in rabbits with jugular vein thrombosis, and in baboons with femoral vein thrombosis, the thrombolytic potency (percent lysis per milligram of compound administered per kilogram of body weight) of t-PA-AFE/scu-PA-e was significantly higher than that of recombinant scu-PA (rscu-PA, Saruplase) as shown by a maximal rate of 720 ± 170% versus 45 ± 5% lysis per milligram of compound per kilogram of body weight (mean ± SEM, p < 0.01) in hamsters, 210 ± 18% versus 49 ± 3% lysis per milligram of compound per kilogram of body weight (mean ± SEM, p < 0.01) in rabbits, and 310 ± 73% versus 90 ± 0.3% lysis per milligram of compound per kilogram of body weight (p < 0.01) in baboons. However, the specific thrombolytic activity (percent lysis per microgram per milliliter steadystate plasma antigen level) of t-PA-AFE/scu-PA-e was not significantly different from that of rscu-PA in hamsters (210 ± 57% versus 160 ± 27% lysis per microgram per milliliter antigen level) and was lower than that of rscu-PA in rabbits (37 ± 4% versus 130 ± 5% lysis per microgram per milliliter antigen level; p < 0.01). In dogs with a combined femoral vein blood clot and a platelet-rich femoral arterial eversion graft thrombosis, 0.25 mg/kg body wt bolus injections of t-PA-AFE/scu-PA-e produced significantly more venous clot lysis (90 + 5%, n = 10) than 0.25 mg/kg rscu-PA (26 + 3%, n = 10) (p < 0.001) and, at the arterial side, more frequent (10 of 10 dogs versus three of 10 dogs) and more persistent (six of 10 dogs versus none of 10 dogs) recanalization (p = 0.002). After bolus injection in hamsters, rabbits, or baboons, t-PA-AFE/scu-PA-e had a fourfold to sixfold longer initial half-life than rscu-PA and a slower plasma clearance of sixfold in hamsters, 10-fold in rabbits, and more than 10-fold in baboons. ConclusionsThese results indicate that t-PA-AFE/scu-PA-e has a markedly enhanced thrombolytic potency toward venous and arterial thrombi caused by a delayed in vivo clearance with relatively maintained specific thrombolytic activity. These properties suggest that the chimera may be clinically useful for thrombolytic therapy by bolus administration in patients with thromboembolic disease.

Thrombolytic and Pharmacokinetic Properties of Conjugates of Urokinase-type Plasminogen Activator with a Monoclonal Antibody Specific for Crosslinked Fibrin

Abstract A primarily 1:1 stoichiometric conjugate between a murine monoclonal antibody directed against fragment D-dimer of crosslinked human fibrin (MA-15C5) and recombinant single chain urokinase-type plasminogen activator (rscu-PA) was produced by chemical crosslinking with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Dewerchin et al, in press). The thrombolytic and pharmacokinetic properties of this conjugate (rscu-PA/MA-15C5) and of its two chain derivatives obtained by digestion with thrombin (rtcu-PA/MA-15C5/T) or plasmin (rtcu-PA/MA-15C5/P) were compared with those of their unconjugated counterparts. Continuous intravenous infusion of these compounds over 4 h in rabbits with a 0.2 ml t 2 s1-fibrin labelled human plasma clot in the jugular vein resulted in linear dose-response curves of clot lysis. The thrombolytic potency of rscu-PA/MA-15C5 was 8 to 9-fold higher (50% lysis with 0.3 mg/kg) than that of rscu-PA (50% lysis with 2.6 mg/kg), while that of rtcu-PA/MA-15C5/P (25% lysis with 0.3 mg/kg) was 4-fold higher than that of rtcu-PA/P 25% lysis with 1.2 mg/kg). Both rtcu-PA/MA-15C5/T and rtcu-PA/T, which are inactive towards purified plasminogen, had significant thrombolytic potency in vivo, causing 15–26% lysis at doses of 1.0–2.0 mg/kg. Systemic fibrinogen degradation during infusion was only observed with rtcu-PA/P but not with rtcu-PA/MA-15C5/P, which is suggestive of an increased fibrin specificity of the conjugate. The enhanced thrombolytic potency of the conjugates as compared to their unconjugated counterparts was not observed when jugular vein clots were produced with rabbit plasma or when u-PA was conjugated with an irrelevant antibody. This suggests that the increased potency is due to specific antibody targetting of u-PA to human fibrin. After intravenous bolus injection in rabbits, u-PA related antigen disappeared from plasma in a biphasic manner with t 1 2 of 2.0 and 7.0 min for rscu-PA and 12 and 90 min for rscu-PA/MA-15C5. Plasma clearance rates were 14 ± 1.1 ml/min for rscu-PA and 3.4 ± 0.56 ml/min for rscu-PA/MA-15C5. In conclusion, conjugation of rscu-PA with MA-15C5 increases its in vivo specific thrombolytic potency about 8-fold and decreases its plasma clearance rate about 4-fold.

Thrombolytic and pharmacokinetic properties of a conjugate of recombinant single-chain urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin in a baboon venous thrombosis model

Chemical conjugates between recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody directed against fragment D-dimer of cross-linked human fibrin (MA-15C5), rscu-PA/MA-15C5, and between rscu-PA and a control monoclonal antibody (MA-1C8), rscu-PA/MA-1C8, were produced by cross-linking with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). In an in vitro system composed of a [125 I]fibrin-labeled baboon plasma clot immersed in autologous citrated plasma, dose- and time-dependent lysis was obtained with a ratio of the potencies of free and conjugated rscu-PA similar to that in human plasma: 50% lysis in 2 hours required 4.3 micrograms/ml rscu-PA, 1.0 microgram/ml urokinase-type plasminogen activator (u-PA) equivalent rscu-PA/MA-15C5, or 15 micrograms/ml u-PA equivalent rscu-PA/MA-1C8. The thrombolytic and pharmacokinetic properties of rscu-PA and of rscu-PA/MA-15C5 were compared in baboons with a 0.8-1.0 ml [125 I]fibrin-labeled autologous blood clot produced in a femoral vein. Continuous intravenous infusion of these compounds during a 2-hour period resulted in dose- and time-dependent lysis. The thrombolytic potency of rscu-PA/MA-15C5 was 3.0 +/- 0.5 times higher (50% lysis with 0.3 +/- 0.02 mg u-PA equivalent/kg body wt) than that of rscu-PA measured by ex vivo isotope recovery from the femoral vein segment (p less than 0.001) and was 2.7 +/- 0.5 times higher (50% lysis with 0.35 +/- 0.02 mg/kg rscu-PA/MA-15C5) by external radioisotope counting (p less than 0.001). A dose of 0.5 mg/kg of rscu-PA/MA-1C8 was much less active than rscu-PA. After the end of the infusion, u-PA-related antigen disappeared from plasma in a biphasic manner with an initial half-time of 2.7 +/- 0.5 for rscu-PA, 24 +/- 1.2 for rscu-PA/MA-15C5, and 21 +/- 0.5 minutes for rscu-PA/MA-1C8 with corresponding plasma clearances of 340 +/- 40, 20 +/- 3, and 24 +/- 2 ml/min, respectively. In conclusion, the increased thrombolytic potency of rscu-PA/MA-15C5 is the result of a reduction of the thrombolytic potency due to coupling of rscu-PA to the antibody molecule, which is counter-balanced by an enhancement of the thrombolytic potency due to fibrin targeting by the specific idiotype.

A technique to investigate mural thrombus formation in small arteries and veins: I. Comparative morphometric and histological analysis

Numerous clinically relevant animal models exist for thrombosis studies. Few of these are suitable for both arteries and veins. In this investigation, an established venous thrombosis model was adapted through minimal technical adjustments to allow also the study of arterial thrombosis. A standardized subintimal crush injury was performed to carotid arteries or femoral veins of hamsters. Thrombus volumes were then quantified by direct morphometric measurements from serial microscopic sections or by on-line image analysis of light intensity changes from transilluminated vessels. The platelet-rich mural thrombus, which was established within minutes of the trauma, disintegrated during the observation period. The life cycle of the thrombus was different in arteries and veins, but significant linear correlation (p<0.01) was found in both types of vessel between thrombus volumes measured by the two techniques. The model can consequently be used for comparative in vivo thrombosis studies in small (≈1-mm) arteries and veins. Stockmans F, Stassen JM, Vermylen J, Hoylaerts MF, Nyström A. A technique to investigate mural thrombus formation in small arteries and veins: I. Comparative morphometric and histological analysis. Ann Plast Surg 1997;38:56-62

Isolation and conditioning of recombinant staphylokinase for use in man

Abstract Staphylokinase (STA), a M m 18000 protein produced by Staphylococcus aureus is known to have profibrinolytic properties for more than 40 years (Lack CH, Nature 1948; 161: 559–560) but its potential for thrombolytic therapy has not been adequately investigated. Therefore we have elaborated procedures for the large scale production of recombinant STA (STAR) from the culture broth of E. coli cells transformed with the recombinant plasmid pUC19 which contains a 2.9 kb insert obtained by Hin dIII restriction enzyme digestion of genomic DNA obtained from a selected Staphylococcus aureus strain. STAR, purified from 12 litre batches by chromatography on SP-Sephadex with pH gradient elution, SP-Sephadex with NaCl gradient elution and Sephacryl S-300 superfine gel filtration, with a recovery of 19 ± 4mg and a yield of 35 ± 15 percent, contained a single band on SDS-polyacrylamide gel electrophoresis with NH 2 -terminal sequence Ser-Ser-Ser-Phe-Asp-Lys-Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala-. It was obtained at a concentration of approximately 1 mg/ml with a specific activity of 185 000 ± 35 000 units/mg with an endotoxin content of 10 ± 7 U/mg. After filtration on 0.22 μm Millipore filters, the preparations were sterile under aerobic and anaerobic bacterial culture conditions and virus free by routine screening for human pathogenic viruses. The material remained active after incubation at 37°C for several days. Bolus injection of STAR at a dose of 3mg/kg in mice did not produce weight loss within 8 days. Thus, these materials appear to be suitable for the investigation, on a pilot scale, of the pharmacokinetic and thrombolytic properties of STAR in patients with thromboembolic disease.