Biomedical subjects

J M Stassen

Publications and source records attributed to J M Stassen.

At least 55 records · Page 3Linked to original sources

PubMed1993

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

BACKGROUND: Streptokinase 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 RESULTS: The inhibitory capacity of baboon plasma (in a human plasma-based clot lysis assay) was 0.39 +/- 0.25 microgram streptokinase and 0.04 +/- 0.05 microgram recombinant staphylokinase per milliliter of plasma (mean +/- SD, n = 9). Intravenous infusion over 1 hour of 0.300 mg/kg of streptokinase at 0, 1, 2, 3, and 5 weeks in five baboons given heparin and the antiplatelet agent ridogrel increased the streptokinase-neutralizing titer from 0.22 +/- 0.18 microgram/mL plasma at baseline to 3.8 +/- 4.4 micrograms/mL after 2 weeks (p = 0.043 versus baseline by Wilcoxon signed rank test) and to 4.4 +/- 4.6 micrograms/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 micrograms/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% 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-fibrin-labeled 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 x hr-1) and aspirin (10 mg/kg) did not produce a significant prolongation of the median template bleeding time. CONCLUSIONS: Recombinant 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.

Animals↗

PubMed1993

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.

Analysis of Variance↗

PubMed1992

Synergistic antithrombotic properties of G4120, a RGD-containing synthetic peptide, and argatroban, a synthetic thrombin inhibitor, in a hamster femoral vein platelet-rich thrombosis model.

The synergistic antithrombotic properties of G4120, a synthetic Arg-Gly-Asp (RGD) containing peptide which strongly inhibits platelet aggregation, and of Argatroban, a synthetic thrombin inhibitor, were examined in a reproducible quantitative hamster femoral vein platelet-rich mural thrombosis model. Bolus injections of G4120 and Argatroban inhibit thrombus formation in a dose-dependent way; 50% inhibition (ID50) is obtained with 11 micrograms/kg G4120 and with 2 mg/kg Argatroban. Combined bolus injections of 3 micrograms/kg G4120 with 0.5, 0.75 or 1 mg/kg Argatroban and of 1 mg/kg Argatroban with 1.5 or 3 micrograms/kg G4120 caused linear dose-dependent inhibition of thrombus formation, whereas 3 micrograms/kg G4120 or 1 mg/kg Argatroban alone had very little effect (less than 20% inhibition). ID50 was obtained with the combination of 3 micrograms/kg G4120 and 0.5 mg/kg Argatroban, corresponding to an equi-effective fractional combination of 0.62 with a 95% confidence interval of 0.50 to 0.74. Alternatively the ID50 was obtained with the combination of 1 mg/kg Argatroban and 1.3 micrograms/kg G4120, corresponding to an equi-effective fractional combination of 0.52 with a 95% confidence interval of 0.18 to 0.86. In both instances these results are indicative of a significant synergistic interaction. Bolus injection of 10 mg/kg aspirin, 100 U/kg heparin or the combination did not inhibit thrombus formation. The synergistic effect of the combination of platelet inhibiting RGD-peptides and synthetic thrombin inhibitors could be useful in the prevention of arterial occlusion with platelet-rich thrombus in patients with ischemic heart disease following thrombolytic therapy or angioplasty, although this combination is not expected to reverse platelet thrombus formation.

Animals↗

PubMed1992

Antithrombotic properties of L-cysteine, N-(mercaptoacetyl)-D-Tyr-Arg-Gly-Asp-sulfoxide (G4120) in a hamster platelet-rich femoral vein thrombosis model.

Platelet aggregation plays an important role in the pathogenesis in arterial thrombotic disorders. The binding of fibrinogen via the Arg-Gly-Asp (RGD) recognition sequence to the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is an essential step of platelet aggregation induced by various physiologic agonists, and RGD-containing peptides that bind to the GPIIb/IIIa receptor inhibit thrombus formation in vivo. L-cysteine, N-(mercaptoacetyl)D-tyrosyl-L-arginylglycyl-L alpha-aspartyl-cyclic (1----5)-sulfide, 5-oxide (G4120), a cyclic RGD-containing synthetic pentapeptide, inhibits adenosine diphosphate (ADP)-induced platelet aggregation with 50% inhibition (IC50) at a concentration of 0.05 microgram/mL in human plasma, 0.12 microgram/mL in hamster plasma, and 11 micrograms/mL in rat plasma. Corresponding values for the linear tetrapeptide Arg-Gly-Asp-Phe (RGDF) were 7 and 100 micrograms/mL in human and hamster plasma. The antithrombotic effects of G4120 and RGDF were evaluated in a hamster model consisting of a mural platelet-rich femoral vein thrombus induced by standardized endothelial cell damage. Bolus intravenous injection of G4120 was followed by a biphasic disappearance of G4120 from plasma with t1/2 alpha of 3.7 minutes and t1/2 beta of 63 minutes, corresponding to a plasma clearance of 5.2 +/- 0.68 mL/min. Bolus intravenous injection of G4120 inhibited ex vivo platelet aggregation with 0.5 mumol/L ADP and in vivo thrombus formation in a dose-dependent manner, with ID50 of 11 and 11 micrograms/kg, respectively. Bolus injection of RGDF inhibited in vivo thrombus formation; 43% inhibition was obtained at a dose of 30 mg/kg. Thus, this hamster platelet-rich femoral vein thrombosis model may be useful for the investigation of the antithrombotic properties of platelet GPIIb/IIIa antagonistic peptides. The cyclic synthetic peptide G4120 appears to have a very potent antithrombotic activity in vivo.

Amino Acid Sequence↗

PubMed1992

Thrombolytic and pharmacokinetic properties of a recombinant chimeric plasminogen activator consisting of a fibrin fragment D-dimer specific humanized monoclonal antibody and a truncated single-chain urokinase.

A recombinant chimeric plasminogen activator consisting of a humanized monoclonal antibody specific for cross-linked human fibrin (MA-15C5Hu) and a 32 kDa single-chain urokinase-type plasminogen activator (scu-PA-32k) comprising amino acids Leu144-Leu411, MA-15C5Hu/scu-PA-32k, was previously found to have a 12-fold higher fibrinolytic potency than recombinant scu-PA-32k towards a human plasma clot in a human plasma milieu in vitro (Vandamme et al., Eur J Biochem 1992; 205: 139-46). Therefore, the thrombolytic and pharmacokinetic properties of MA-15C5Hu/scu-PA-32k were compared with those of recombinant single-chain urokinase-type plasminogen activator (scu-PA) in 3 different venous thrombosis models in vivo. In hamsters with a pulmonary embolus consisting of a human plasma clot, the thrombolytic potency (% lysis per dose in mg/kg administered) of MA-15C5Hu/scu-PA-32k was 23-fold higher than that of scu-PA (p less than 0.0005). In rabbits with a jugular vein clot prepared from human plasma, the thrombolytic potency of MA-15C5Hu/scu-PA-32k was 11-fold higher than that of scu-PA (p = 0.012). In baboons with an autologous whole blood clot in the femoral vein, the chimera had a 5-fold higher thrombolytic potency than scu-PA. In all three animal species, the clearance of the chimera was 10- to 27-fold reduced as compared to scu-PA. The specific thrombolytic activity (% lysis per micrograms/ml steady-state plasma u-PA antigen) was increased up to 7-fold with MA-15C5Hu/scu-PA-32k as compared with scu-PA, which is indicative of targeting of the chimera to the clot. No fibrinogen breakdown or alpha 2-antiplasmin depletion was observed during thrombolysis with the chimera. Thus, MA-15C5Hu/scu-PA-32k constitutes a recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency in 3 different animal models of venous thrombosis.

Animals↗

PubMed1992

Thrombolytic and pharmacokinetic properties of an immunoconjugate of single-chain urokinase-type plasminogen activator (u-PA) and a bispecific monoclonal antibody against fibrin and against u-PA in baboons.

Targeting of plasminogen activators to the fibrin component of a thrombus with the use of monoclonal antibodies (MA) directed against human fibrin may enhance their thrombolytic potency and fibrin-specificity. The thrombolytic and pharmacokinetic properties of rscu-PA/MA-FU1-74, an immunoconjugate of recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and a bispecific MA directed against u-PA and against the beta-chain of human fibrin (MA-FU1-74), were investigated in baboons with a [125I]fibrin-labeled autologous blood clot in the femoral vein. Continuous intravenous infusion of rscu-PA/MA-FU1-74 (1:1.2 molar ratio) over 2 hours showed a fivefold increased thrombolytic potency (lysis per unit dose) over that of unconjugated rscu-PA, as evidenced both by a higher maximal rate of lysis (380% +/- 68% v 78% +/- 25% lysis per mg u-PA equivalent of compound administered per kg body weight, P less than .001), and by a lower dose at which the maximal rate of lysis occurs (0.19 +/- 0.03 v 0.82 +/- 0.10 mg compound per kg body weight, P less than .001). The specific thrombolytic activity (percent lysis per unit steady-state plasma u-PA antigen level) was lower for rscu-PA/MA-FU1-74 than for rscu-PA, as shown by both a lower maximal rate of lysis (60% +/- 13% v 220% +/- 22% lysis per microgram/mL u-PA antigen level in plasma, P less than .001) and a higher plasma antigen level at which maximal lysis is achieved (1.2 +/- 0.17 v 0.20 +/- 0.01 microgram/mL, P less than .001). The thrombolytic potency of rscu-PA/MA-UK1-3, an immunoconjugate of rscu-PA with the parental anti-u-PA antibody was similar to that of unconjugated rscu-PA. Clot lysis was achieved without systemic fibrinogen or alpha 2-antiplasmin consumption, and with a minor transient prolongation of the bleeding time. After the end of the infusions, u-PA-related antigen disappeared from plasma in a biphasic manner, with an initial half-life of 3.3 +/- 0.4 minutes for rscu-PA, 13 +/- 1 minutes for rscu-PA/MA-FU1-74, and 13 +/- 1 minutes for rscu-PA/MA-UK1-3, with corresponding plasma clearances of 340 +/- 28, 10 +/- 1, and 37 +/- 4 mL/min, respectively (mean +/- SEM). rscu-PA/MA-FU1-74 has a fivefold higher thrombolytic potency than unconjugated rscu-PA, as a result both of fibrin targeting by the specific idiotype of the antibody and of a slower plasma clearance.

Animals↗

PubMed1992

Biochemical and biological properties of a recombinant tissue-type plasminogen activator derived from the rat JMI-229 cell line.

Recombinant tissue-type plasminogen activator (rt-PA), produced by expression of the genomic t-PA DNA from the JMI-229 cell line, which is of rat origin, in the host cell line, was purified to homogeneity. JMI-229 rt-PA was obtained essentially as a single chain molecule which was quantitatively converted to a two-chain moiety by treatment with plasmin. The plasminogen activating potential of single chain JMI-229 rt-PA was 5-fold lower than that of commercially available human rt-PA (Actilyse) in the absence of fibrin, but comparable in the presence of fibrin; it showed a concentration-dependent binding to fibrin, with a significantly more pronounced binding than Actilyse at low fibrin concentration (85 +/- 8% versus 20 +/- 7% at 0.025 mg/ml fibrin; p = 0.004). In human plasma in the absence of fibrin, the concentrations of both single chain and two-chain JMI-229 rt-PA required to induce 50% fibrinogen degradation in 2 h, were about 15-fold higher than those of Actilyse. Both single chain and two-chain forms of JMI-229 rt-PA and of Actilyse induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma, in the absence of significant systemic fibrinolytic activation. Equally effective concentrations (causing 50% clot lysis in 2 h) were 0.11 or 0.10 micrograms/ml for single chain or two-chain JMI-229 rt-PA, as compared to 0.11 or 0.15 micrograms/ml for single chain or two-chain Actilyse.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗

PubMed1992

Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1.

A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296-G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296-G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt-PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1992

Comparative antithrombotic effects of heparin, recombinant hirudin and argatroban in a hamster femoral vein platelet-rich mural thrombosis model.

The antithrombotic properties of bolus i.v. injections of heparin, of recombinant hirudin (r-hirudin) or of the synthetic competitive thrombin inhibitor Argatroban were investigated in a quantitative hamster femoral vein platelet-rich mural thrombosis model. Heparin at a dose of 100 U/kg prolonged the activated partial thromboplastin time from 26 +/- 15 to 177 +/- 45 sec (P = .001), but did not significantly inhibit platelet-rich thrombus formation (7 +/- 44% inhibition, P = NS vs. placebo). However, 400 U/kg of heparin produced total inhibition of thrombus formation (101 +/- 14+, P less than .06 vs. control). R-hirudin and argatroban inhibited thrombus formation in a dose-dependent manner: 50% inhibition was obtained with 1.4 mg/kg for r-hirudin and with 2.0 mg/kg for Argatroban. A linear correlation was observed between the percentage of inhibition of thrombus formation vs. Activated partial thromboplastin time (r = 0.57, P = .003 for r-hirudin and r = 0.66, P = .002 for Argatroban). These results suggest that thrombin plays a pivotal role in platelet-rich mural thrombus formation, that this small animal model may be useful for investigation of the pharmacodynamics of synthetic thrombin inhibitors and that platelet-rich thrombus formation is inhibited effectively by heparin, r-hirudin and Argatroban. However, r-hirudin and Argatroban cause less profound changes in the coagulant function at doses that inhibit platelet-rich thrombus formation than heparin.

Animals↗

PubMed1991

Enhancement of clot lysis in vitro and in vivo with a bispecific monoclonal antibody directed against human fibrin and against urokinase-type plasminogen activator.

A hybrid hybridoma (FU1-74), secreting a bispecific monoclonal antibody (bs mAb), was obtained by fusion of a murine hybridoma secreting a monoclonal antibody (mAb) specific for human fibrin with a murine hybridoma secreting a mAb against urokinase-type plasminogen activator (u-PA). The bs mAb (MA-FU1-74), purified to homogeneity from mouse ascitic fluid, migrated as a single band with apparent Mr 150,000 on nonreduced SDS-PAGE and had an affinity for both human fibrin (Ka = 2 x 10(7) M-1) and for u-PA (Ka = 10(8) M-1) comparable to that of the mAbs obtained from the respective parental hybridomas. MA-FU1-74 did not influence the enzymatic activity of two-chain u-PA (tcu-PA) towards plasminogen or towards a chromogenic substrate. The complex of MA-FU1-74 with recombinant single chain u-PA (rscu-PA) or with tcu-PA (urokinase) enhanced the fibrinolytic potency of the plasminogen activator towards clotted human plasma 20-fold and 5-fold, respectively. In a hamster pulmonary embolism model, the rscu-PA/MA-FU1-74 complex had a 13- to 17-fold increased thrombolytic potency (percent lysis per mg/kg u-PA administered) relative to that of rscu-PA. The specific thrombolytic activity (percent lysis per microgram/ml steady state plasma level of u-PA antigen) of the complex was, however, not significantly different from that of rscu-PA. The complex of rscu-PA with the parental anti-u-PA mAb (MA-UK1-3) had only a 2-fold enhanced thrombolytic potency relative to that of rscu-PA and had a 5-fold decreased specific thrombolytic activity. The plasma clearance rates of the complexes of rscu-PA with both MA-FU1-74 and MA-UK1-3 were about 10-fold lower than that of rscu-PA. In a rabbit jugular vein thrombosis model, the rscu-PA/MA-FU1-74 complex had a 4-fold enhanced thrombolytic potency, an unchanged specific thrombolytic activity and 20-fold reduced plasma clearance. In both animal models, the rscu-PA/MA-FU1-74 complex did not cause more extensive systemic activation of the fibrinolytic system than rscu-PA. It is concluded that the bispecific anti-fibrin/anti-u-PA mAb MA-FU1-74 targets u-PA to the fibrin clot, resulting in a significantly enhanced thrombolytic potency of the plasminogen activator.

Amino Acid Sequence↗

PubMed1991

Comparative fibrinolytic properties of staphylokinase and streptokinase in animal models of venous thrombosis.

The thrombolytic and pharmacokinetic properties of staphylokinase were compared with those of streptokinase in hamsters with a pulmonary embolus produced from human plasma or from hamster plasma, and in rabbits with a jugular vein blood clot produced from rabbit blood. In both models, a continuous intravenous infusion of staphylokinase and streptokinase over 60 min in hamsters or over 4 h in rabbits, induced dose-dependent progressive clot lysis in the absence of significant systemic activation of the fibrinolytic system. The results of thrombolytic potency (clot lysis at 30 min after the end of the infusion, in percent, versus dose administered, in mg/kg) were fitted with an exponentially transformed sigmoidal function (formula: see text) and the maximal percent clot lysis (c), the maximal rate of lysis (z = 1/4 ac.eb) and the dose at which the maximal rate of lysis is achieved (b) were determined. In hamsters with a pulmonary embolus produced from human plasma, streptokinase had a somewhat higher thrombolytic potency than staphylokinase, as revealed by a higher z value (2,100 +/- 1,100% lysis per mg/kg streptokinase administered versus 1,100 +/- 330% lysis per mg/kg for staphylokinase). In hamsters with a pulmonary embolus produced from hamster plasma, staphylokinase had a somewhat higher thrombolytic potency than streptokinase (z = 1,600 +/- 440 versus 1,200 +/- 370% lysis per mg/kg). Staphylokinase had a higher thrombolytic potency than streptokinase in rabbits,as revealed by a higher z-value (950 +/- 350% lysis per mg/kg staphylokinase administered versus 330 +/- 39% lysis per mg/kg for streptokinase) and a lower b-value (0.035 +/- 0.010 mg/kg staphylokinase versus 0.091 +/- 0.008 mg/kg for streptokinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal antibody specific for human fibrin fragment D-dimer.

A recombinant single-chain molecule, scFv-K12G0, containing the variable domains of the monoclonal antibody MA-15C5, specific for fragment D-dimer of human cross-linked fibrin, was constructed and expressed in Spodoptera frugiperda, Sf9, insect cells. The Arg108 carboxyl-terminal amino acid of the variable domain of the light-chain of the antibody was connected through a synthetic Ala-Gly-Gln-Gly-Ser-Ser-Val peptide linker with the Gln1 amino-terminal amino acid of the variable domain of its heavy chain. scFv-K12G0 was secreted by the infected Sf9 cells at a rate of 10 micrograms/10(6) cells within 48 h, resulting in conditioned medium with a maximal concentration of 15 mg of scFv-K12G0/liter. The molecule, purified to homogeneity by ion exchange chromatography and gel filtration, migrated as a single Mr band on reduced sodium dodecyl sulfate-gel electrophoresis. It bound to immobilized fragment D-dimer with an affinity constant of 4.0 x 10(9) M-1 (2.0 x 10(10) M-1 for intact MA-15C5). Clearing of scFv-K12G0 from the circulation in rabbits occurred with an initial half-life (t1/2 alpha) of 10 min and a clearance of 5.1 ml min-1, as compared to 90 min and 210 ml min-1 for intact MA-15C5. Nephrectomy resulted in a prolongation of t1/2 alpha to 110 min, suggesting that the rapid clearance of scFv-K12G0 occurs primarily via the kidney, presumably by glomerular filtration. The results indicate that the single-chain recombinant molecule scFv-K12G0 is secreted in functionally intact form and suggest that it may be useful for targeting of radioisotopes or plasminogen activators to blood clots in vivo.

Amino Acid Sequence↗

PubMed1991

Effect of chemical conjugation of recombinant single-chain urokinase-type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo.

The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u-PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin-labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu-PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).

Adenosine Diphosphate↗

PubMed1991

Comparative thrombolytic properties of bolus injections and continuous infusions of a chimeric (t-PA/u-PA) plasminogen activator in a hamster pulmonary embolism model.

The recombinant chimeric plasminogen activator, rt-PA-delta FE/scu-PA-e, consisting of amino acids 1 to 3 and 87 to 274 of tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of single-chain urokinase-type plasminogen activator (scu-PA), has a markedly increased thrombolytic potency following its continuous intravenous infusion in animal models of venous thrombosis (Collen et al, Circulation, in press). In the present study, the thrombolytic potencies of intravenous bolus injections of rt-PA-delta FE/scu-PA-e, of recombinant t-PA (rt-PA), and of recombinant scu-PA (rscu-PA), given alone or in combination, were compared with those of intravenous infusions in a hamster pulmonary embolism model. Dose-dependent clot lysis was obtained in the absence of systemic activation of the fibrinolytic system and fibrinogen breakdown. In bolus injection experiments, the maximal rate of clot lysis, expressed in percent clot lysis per milligrams per kilogram compound administered, was 120 +/- 10 for rt-PA, 54 +/- 8 for rscu-PA, and 2,100 +/- 500 for rt-PA-delta FE/scu-PA-e (P less than .01 v rt-PA or rscu-PA). Comparative results with continuous infusion over 1 hour were 270 +/- 64, 99 +/- 18, and 1,500 +/- 250 (P less than .01 v rt-PA or rscu-PA) percent lysis per mg/kg compound infused for rt-PA, rscu-PA, and rt-PA-delta FE/scu-PA-e, respectively. Thus, rt-PA and rscu-PA are more potent when administered as an infusion than as a bolus, whereas rt-PA-delta FE/scu-PA-e is at least as potent when administered as a bolus. Combined bolus injections of rt-PA and rscu-PA had a 2.2-fold synergistic effect on clot lysis, but no synergism was observed with combined bolus injections or with combined infusions of rt-PA and rt-PA-delta FE/scu-PA-e, or of rscu-PA and rt-PA-delta FE/scu-PA-e. The present study thus shows that rt-PA-delta FE/scu-PA-e is much more potent for clot lysis than rt-PA or rscu-PA when administered as a bolus injection, but no synergistic interaction is observed between the chimera and either rt-PA or rscu-PA.

Animals↗

PubMed1991

Thrombolytic and pharmacokinetic properties of human tissue-type plasminogen activator variants, obtained by deletion and/or duplication of structural/functional domains, in a hamster pulmonary embolism model.

A pulmonary embolism model in hamsters was used for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of variants of tissue-type plasminogen activator (t-PA). A 25 microliters 125I-fibrin labeled human plasma clot was made in vitro and injected into the jugular vein of heparinized hamsters. The extent of thrombolysis within 90 min was determined as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs. Recombinant t-PA (home-made rt-PA or Activase) infused intravenously over 60 min caused dose-dependent progressive thrombolysis. The results of thrombolytic potency (clot lysis in percent versus dose administered in mg/kg) and of specific thrombolytic activity (clot lysis in percent versus steady state plasma level in microgram/ml) were fitted with an exponentially transformed sigmoidal function y = 100 c/(1 + e-a(ax-eh] and the maximal percent lysis (c), the dose or plasma level at which maximal rate of lysis is achieved (b) and the maximal rate of lysis (z = 1/4 ac.eb) were determined. With rt-PA, these parameters were c = 72 +/- 6% (mean +/- SEM), b = 0.19 +/- 0.08 mg/kg, z = 68 +/- 25% lysis per mg/kg, with corresponding values of 87 +/- 5%, 0.07 +/- 0.03 mg/kg and 150 +/- 38% lysis per mg/kg for Activase (p = NS). Deletion of the finger and growth factor domains in rt-PA (rt-PA-delta FE) was not associated with marked alteration of the thrombolytic potency (c = 90 +/- 30%, b = 0.34 +/- 0.35 mg/kg, and z = 54 +/- 14% per mg/kg), but was associated with a significant reduction of the specific thrombolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

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

BACKGROUND: Chimeric 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-delta FE/scu-PA-e) in such chimeras further reduces their affinity for fibrin. METHODS AND RESULTS: A 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-delta FE/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 less than 0.01) in hamsters, 210 +/- 18% versus 49 +/- 3% lysis per milligram of compound per kilogram of body weight (mean +/- SEM, p less than 0.01) in rabbits, and 310 +/- 73% versus 90 +/- 0.3% lysis per milligram of compound per kilogram of body weight (p less than 0.01) in baboons. However, the specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma antigen level) of t-PA-delta FE/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 less than 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-delta FE/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 less than 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-delta FE/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. CONCLUSIONS: These results indicate that t-PA-delta FE/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.

Animals↗

PubMed1991

Small animal thrombosis models for the evaluation of thrombolytic agents.

BACKGROUND: Two thrombosis models for the evaluation of thrombolytic agents in small animals (less than 100 g) were evaluated: an iodine-125 fibrin-labeled rat plasma clot in the inferior caval vein of 3-4-week-old rats and a pulmonary embolus in adult hamsters that had been obtained by injection of a 125I fibrin-labeled human plasma clot. The extent of thrombolysis was determined by continuous external monitoring of radioisotope over the thrombus region and by ex vivo recovery of residual clot. METHODS AND RESULTS: In the rat model, infusion of solvent for 60 minutes was associated with mean +/- SEM lysis within 90 minutes of 13 +/- 3% (n = 8) by external counting and 26 +/- 4% (n = 8) by radioisotope recovery. Intravenous infusion of recombinant tissue-type plasminogen activator (rt-PA) over 60 minutes caused dose-dependent progressive clot lysis; with 0.5 mg/kg, producing a plasma level of 0.14 +/- 0.04 microgram/ml, lysis was 64 +/- 9% (n = 4) by external gamma counting and 78 +/- 4% (n = 4) by residual isotope in the vein segment and was not associated with significant fibrinogen or alpha 2-antiplasmin breakdown. In the hamster model, infusion of solvent for 60 minutes was associated with lysis within 90 minutes of 19 +/- 4% (n = 11) by external gamma counting and 31 +/- 3% (n = 14) by residual radioisotope. Intravenous rt-PA during 60 minutes resulted in dose-dependent progressive thrombolysis; with 0.5 mg/kg, producing a plasma level of 0.14 +/- 0.01 micrograms/ml, lysis was 50 +/- 4% (n = 4) by external gamma counting and 78 +/- 5% (n = 4) by residual radioactivity, without an extensive decrease in fibrinogen or alpha 2-antiplasmin. Parallel experiments in the rabbit jugular vein thrombosis model with a rabbit blood clot with intravenous infusion over 4 hours produced 7 +/- 2% (n = 9) lysis with solvent and dose-dependent progressive lysis with rt-PA; with 1 mg/kg, producing a plasma level of 0.20 +/- 0.03 microgram/ml, lysis was 56 +/- 7% (n = 7) by external gamma counting and 61 +/- 7% (n = 7) by residual radioactivity, without extensive consumption of fibrinogen or alpha 2-antiplasmin. CONCLUSIONS: These two thrombosis models in small animals are as reproducible and quantitative as the extensively used rabbit jugular vein thrombosis model. The hamster pulmonary embolism model is superior because it is simpler and more straightforward and allows the performance of as many as 10 experiments by one investigator in 1 day.

Animals↗

PubMed1990

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.

Animals↗