M De Mol

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.

Isolation and characterisation of natural and recombinant staphylokinase

Abstract Staphylokinase (STA), a Mr 18000 protein produced by Staphylococcus aureus has profibrinolytic properties (Lack CH, Nature 161, 559–560, 1948), but its potential for thrombolytic therapy has not been thoroughly investigated. Therefore we have elaborated procedures for the large scale production of natural (STAN) and recombinant (STAR) staphylokinase. A strain of Staphylococcus aureus (strain no. 23), selected from 100 consecutive cultures at our Bacteriology Laboratory, was found to secrete up to 300 μg staphylokinase per litre culture broth within 18h. The material, purified by batch adsorption on SP-Sephadex and chromatography on insolubilised active site-blocked plasmin with a yield of approximately 40 μg per l, migrated as a single band on SDS gel electrophoresis with Mr ≈ 16 500 and had NHZ-terminal sequence Lys-Gly-Asp-AspAla-. Restriction enzyme digested genomic DNA from this strain was hybridised with a degenerate 14-mer deoxyoligonucleotide encoding the NH2-terminal sequence, hybridising fragments were isolated and ligated into pUC19, competent E. coli cells were transformed, recombinant clones isolated and culture medium assayed for STA activity. A clone (subclone 159-2) transformed with recombinant pUC19 containing a 2.9 kb insert obtained by HindIII restriction enzyme digestion was found to secrete STAR without further manipulation (up to 20 mg/l into the culture broth and 30 mg/l into the periplasmic space). The material, purified by chromatography on insolubilised active-site blocked plasmin with a yield of 50 and 25%, respectively, contained three STAR variants with NH2-terminal sequence Ser-Ser-Ser-Phe-Asp-Lys-Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-M), with Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-Δ6) and with Lys-Gly-Asp-Asp-Ala- (STA-Δ10). Chromatography on CM-Sephadex yielded two peaks, STA-CM-I containing STA-Δ10 and STA-CM-11 containing STA-M and STA-Δ6. The specific activities of STAN, of STA-Δ10 obtained from STAR and of mixtures of STA-M and STA-Δ6 from STAR were indistinguishable by clot lysis and plasminogen coupled chromogenic substrate assays. Thus, highly purified preparations of natural (STAN) and recombinant (STAR) staphylokinase can be obtained by simple and straightforward techniques in sufficiently large amounts to allow detailed investigation of their biochemical and thrombolytic properties.

Studies of recombinant plasminogen activator inhibitor-1 in rabbits. Pharmacokinetics and evidence for reactivation of latent plasminogen activator inhibitor-1 in vivo.

The pharmacokinetics of human recombinant plasminogen activator inhibitor-1 (rPAI-1) was studied in rabbits. Latent rPAI-1 (0-2 units of tissue-type plasminogen activator neutralizing activity per microgram protein); reactivated rPAI-1 (approximately 150 units/micrograms); and chloramine T-oxidized, nonreactivatable rPAI-1 (approximately 0.7 units/microgram) were studied. The pharmacokinetic parameters for the disposition of rPAI-1 antigen after an intravenous bolus injection of 1.0 or 2.5 mg/kg rPAI-1 were very similar for all three forms: the initial volume of distribution was approximately 60 ml/kg, the initial half-life in plasma was 6 minutes, and the plasma clearance was approximately 4 ml/kg/min. The disposition of PAI activity after injection of reactivated rPAI-1 was similar to that of rPAI-1 antigen. Injection of latent rPAI-1 was associated with a nearly threefold increase in the specific activity of circulating PAI-1 from 2 units/micrograms to 5.0 +/- 1.1 units/micrograms (p less than 0.01) within 1 minute, followed by a cumulative 25-fold increase in specific activity over 1 hour (p = 0.01). In contrast, the specific activity of oxidized or reactivated preparations of rPAI-1 did not increase in the first several minutes after injection. These findings support the existence of a fast-acting but low-capacity mechanism for the reactivation of rPAI-1 in vivo.

Recombinant plasminogen activator inhibitor-1 reverses the bleeding tendency associated with the combined administration of tissue-type plasminogen activator and aspirin in rabbits.

The major side effect of thrombolytic therapy is bleeding; however, the pathogenesis of this potential complication is not well understood. Accordingly, we examined the effects of aspirin and recombinant human tissue-type plasminogen activator (rt-PA) on serial template bleeding times and on hemostasis parameters in rabbits. The administration of intravenous aspirin (15 mg/kg) produced a slight prolongation in bleeding times, from 2.1 +/- 0.5 to 2.6 +/- 0.5 min (mean +/- SD, n = 26, P less than 0.01), whereas rt-PA (1 mg/kg per h for 2 h) lengthened the bleeding time from 2.4 +/- 0.3 to 3.2 +/- 0.6 min (n = 5, P = NS). Combination of aspirin with 0.5 mg/kg per h of rt-PA for 2 h prolonged the bleeding time from 2.5 +/- 0.4 to 6.2 +/- 0.9 min (n = 10, P less than 0.01), with an associated fibrinogen decrease of approximately 15%. The combination of aspirin with 1 mg/kg per h of rt-PA for 2 h prolonged the bleeding time from 3.0 +/- 0.3 to 8.3 +/- 1.4 min (n = 8, P less than 0.01) and simultaneously induced a decrease of plasma fibrinogen by approximately 40%. Virtually all animals treated with rt-PA and aspirin manifested a bleeding tendency, as evidenced by spontaneous rebleeding at sites of previously performed template bleeding times or oozing at the femoral venous catheterization site. Intravenous bolus injection of 1 mg/kg of guanidine hydrochloride-reactivated recombinant human plasminogen activator inhibitor-1 (rPAI-1) at the end of the rt-PA infusion resulted in complete reversal, within 5 min, of the prolongation of the bleeding time, and in a disappearance of the bleeding tendency. Nonreactivated rPAI-1 and tranexamic acid were significantly less potent in reversing the bleeding time prolongation. These findings indicate that aspirin and rt-PA given separately do not markedly affect the template bleeding time, but in combination induce a marked prolongation associated with a significant bleeding tendency. This bleeding time prolongation can be rapidly normalized by the administration of reactivated rPAI-1.

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.