E. Demarsin

Recombinant human microplasmin: production and potential therapeutic properties

Summary.  The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen (µPlg), which lacks the five ‘kringle’ domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin (µPli) and equilibrated with 5 mmol L−1 citrate, pH 3.1, yielding a stable preparation. In mice with middle cerebral artery (MCA) ligation, an intravenous (i.v.) bolus of 5.0 mg kg−1µPli reduced infarct size at 24 h from 27 (26–30) to 25 (21–28) mm3 (median and range, n= 16 each, P= 0.0001), whereas 4.0 mg kg−1 rt‐PA and 40 mg kg−1µPlg had no effect. Infarct reduction was observed with administration at 4 h after occlusion. In mice with MCA, infarct size at 24 h was reduced from 20 (14–30) to 9.1 (3.1–25) mm3 with 5.0 mg kg−1µPli (n = 15 each, P < 0.002) and to 11 (5.2–27) mm3 with 4.0 mg kg−1 rt‐PA (n = 6; P= 0.02). Infarct reduction was still observed at 10 h after occlusion with µPli but not with t‐PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg−1µPli over 2 h, induced 51 ± 15% lysis (mean ± SD, n= 11) vs. a control value of 23 ± 5.5%. µPli did not prolong template bleeding times, whereas equipotent doses of rt‐PA were associated with extensive rebleeding. The potency of µPli in both models was similar to that of intact plasmin. These findings indicate that recombinant µPli may be useful for treatment of ischemic stroke and arterial thrombosis.

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.

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.

Characterisation of rt-PA I276G, a recombinant human tissue-type plasminogen activator mutant with altered plasmin cleavage site

Abstract rt-PA I276G, a mutant of recombinant human tissue-type plasminogen activator (rt-PA) with altered plasmin cleavage site, was obtained by site-directed mutagenesis of Ile 276 to Gly. rt-PA I276G, purified to homogeneity from the conditioned medium of transfected Chinese hamster ovary cells, was obtained as a single chain molecule, which was quantitatively converted to a two chain moiety by cleavage of the Lys 277 -Gly 278 peptide bond with plasmin. The specific activities on fibrin films of the single chain and two chain forms of rt-PA I276G were 8900 and 15000 IU/mg respectively, as compared to 210000 and 130000 IU/mg respectively for the single chain and two chain forms of wild-type rt-PA obtained in the same expression system. The amidolytic activity of the rt-PA I276G moieties was 3- to 5-fold lower and their catalytic efficiency for plasminogen activation 20- to 50-fold lower than those of the wild-type rt-PA moieties. Both single chain and two chain rt-PA I276G induced concentration-dependent lysis of a 125 I-fibrin labelled plasma clot submersed in human plasma; equi-effective concentrations (causing 50% clot lysis in 2 h) were 0.55 and 1.40 μg/ml respectively, as compared to 0.36 and 0.60 μg/ml for single chain or two chain wild-type rt-PA respectively. Continuous infusion over 60 min of single chain rt-PA 1276G or wild-type rt-PA in hamsters with a pulmonary embolus, revealed an approximately 2-fold lower thrombolytic potency (clot lysis versus dose) for the mutant, but a comparable specific thrombolytic activity (clot lysis versus steady state plasma antigen level). It is concluded that replacement of Ile 276 by Gly in single chain rt-PA significantly reduces the intrinsic enzymatic activity in purified systems. In a plasma milieu in the presence of fibrin the fibrinolytic potential of the mutant is, however, only 2-fold lower than that of wild-type rt-PA obtained in the same expression system.