The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro

The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA- Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu- plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi- effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu- PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu- PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu- PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA- Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20- fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA- Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.     

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.     

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.     

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).     

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)     

Comparative thrombolytic properties of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (u-PA) and K1K2Pu (a t-PA/u-PA chimera) in a combined arterial and venous thrombosis model in the dog.

The chimeric molecule K1K2Pu, comprising the two kringle domains (K1 and K2) of tissue-type plasminogen activator (t-PA) and the COOH-terminal region with the serine protease domain (Pu) of urokinase-type plasminogen activator (u-PA), was previously shown to have a 5- to 10-fold reduced clearance rate with maintained specific thrombolytic activity, resulting in an increased thrombolytic potency in animal models of venous and arterial thrombosis. To document the thrombolytic potential of K1K2Pu, the thrombolytic potency and fibrin specificity were studied in a combined platelet-rich arterial eversion graft thrombosis and venous whole blood clot model in heparinized dogs (100 U/kg bolus and 50 U/kg per h infusion). Dose-response effects of bolus injections of K1K2Pu (0.032 to 0.25 mg/kg) were compared with those of recombinant t-PA (rt-PA) and of recombinant single chain u-PA (rscu-PA) (0.25 to 1.0 mg/kg each) in groups of five or six dogs, each given heparin with or without the thromboxane synthase inhibitor/prostaglandin endoperoxide receptor antagonist ridogrel. Heparin and ridogrel in the absence of a thrombolytic agent did not produce arterial reflow or venous clot lysis in five dogs. Addition of K1K2Pu, rt-PA or rscu-PA resulted in a dose-dependent induction of arterial reflow and of venous clot lysis in the absence of systemic fibrinolytic activation and fibrinogen breakdown. Consistent arterial reflow required 0.063 mg/kg of K1K2Pu and 0.5 mg/kg of rt-PA or of rscu-PA. The thrombolytic potency for venous clot lysis, expressed as percent lysis per mg compound administered per kg body weight, was (mean +/- SEM) 750 +/- 160 for K1K2Pu, 68 +/- 17 for rscu-PA (p less than 0.001 vs. K1K2Pu) and 110 +/- 29 for rt-PA (p less than 0.001 vs. K1K2Pu). The plasma clearance rates were significantly lower for K1K2Pu than for rscu-PA and rt-PA. In the absence of ridogrel, arterial reflow was significantly slower and was followed by cyclic reocclusion and reflow; however, venous clot lysis was unaffected. Template bleeding times were not significantly altered in the absence but were markedly prolonged in the presence of ridogrel. These results confirm and establish that, when given as a bolus injection, K1K2Pu has an approximately 10-fold higher thrombolytic potency for arterial and venous thrombolysis than does rt-PA or rscu-PA. Thrombolysis with K1K2Pu is obtained in the absence of systemic fibrinolytic activation and fibrinogen breakdown. These properties suggest that K1K2Pu offers potential for thrombolytic therapy by bolus administration in patients with thromboembolic disease.     

A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion

One (MA-1C8) of 36 monoclonal antibodies obtained by fusion of P3X63- Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified human tissue-type plasminogen activator (t-PA) blocked the activity of t-PA on fibrin plates but not on chromogenic substrates. MA- 1C8 at a concentration of 200 micrograms/mL inhibited plasma clot lysis and binding of t-PA to the clot. MA-1C8 had no influence on the activation of plasminogen by t-PA, which obeys Michaelis-Menten kinetics with Km = 105 mumol/L and kcat = 0.05 s-1; however, it abolished the influence of CNBr-digested fibrinogen on Km. These findings confirm that the stimulatory effect of fibrin on the activation of plasminogen by t-PA is mediated by binding of t-PA to fibrin and provide additional support for the kinetic model. Addition of t-PA to pooled fresh human plasma to a concentration of 5 micrograms/mL resulted in extensive fibrinogen breakdown after incubation for one hour at 37 degrees C or during storage at -20 degrees C for one day. In both instances, fibrinogen degradation was completely prevented by addition of MA-1C8 to a concentration of 200 micrograms/mL of plasma. MA-1C8 also effectively prevented in vitro fibrinogen degradation and in vitro plasminogen activation in plasma samples obtained during infusion of recombinant t-PA in patients with thromboembolic disease. Thus, MA-1C8 is a useful tool for discriminating between in vivo and in vitro fibrinolysis during thrombolytic therapy with t-PA.     

Comparison of intravenous bolus injection or continuous infusion of recombinant single chain urokinase-type plasminogen activator (saruplase) for thrombolysis. A canine model of combined coronary arterial and femoral venous thrombosis

The thrombolytic efficacy of recombinant unglycosylated full length single chain urokinase-type plasminogen activator (rscu-PA, saruplase), applied either as single intravenous bolus or as a continuous infusion over 60 minutes, was studied in 5 randomized blinded groups of 5 dogs with combined copper coil induced coronary artery thrombosis and 125I-fibrin labeled femoral vein clots. Infusion of 1 mg/kg recu-PA (group I) induced coronary recanalization in 4 of 5 dogs and 98 +/- 1% (mean +/- SEM) venous clot lysis. Bolus injection of 1 mg/kg recu-PA (group II) caused reflow in 3 of 5 dogs and 88 +/- 5 percent venous clot lysis. Infusion of 0.5 mg/kg rescu-PA (group III) achieved reflow in 3 of 5 dogs and 52 +/- 6% venous clot lysis. Bolus injection of 0.5 mg/kg rscu-PA (group IV) induced reflow in 4 of 5 dogs and 48 +/- 12% venous clot lysis. Placebo infusion (group V) was associated with late recanalization in 1 of 5 dogs and 18 +/- 8% venous clot lysis. Coronary artery reocclusion after reflow was not observed in groups I and II, but occurred in 2 of 3 animals in group III and in 3 of 4 animals in group IV (P = .02). The time to reflow in responsive animals was 22 +/- 5 minutes with infusion of 0.5 or 1 mg/kg rscu-PA and 14 +/- 1 minute with bolus injection of 0.5 or 1 mg/kg (P = .14). Depletion of fibrinogen and alpha 2-antiplasmin to less than 25% of baseline levels was observed in the 5 dogs given 1 mg/kg rscu-PA by bolus and in 3 of the 5 dogs given 1 mg/kg rscu-PA via infusion, but in none of the dogs that received 0.5 mg/kg rscu-PA (P less than .001). Plasma clearance rates were 170 +/- 44 and 230 +/- 30 mL/minute after bolus injection and 190 +/- 47 and 310 +/- 56 mL/minute during infusion of rscu-PA for the 1 mg/kg and 0.5 mg/kg doses respectively. Thus, intravenous bolus injection of rscu-PA (saruplase) appears to be equipotent to an infusion over 60 minutes for both coronary and venous thrombolysis. This animal model of combined arterial and venous thrombolysis may be useful for the evaluation of new thrombolytic strategies.     

Regulation by alpha 2-antiplasmin and fibrin of the activation of plasminogen with recombinant staphylokinase in plasma

The effects of alpha 2-antiplasmin and fibrin on the activation of plasminogen by recombinant staphylokinase (STAR) were studied in an effort to elucidate further the molecular basis of the fibrin- specificity of this fibrinolytic agent. In purified systems consisting of 1.5 mumol/L intact or low-M(r) plasminogen and 3 mumol/L alpha 2- antiplasmin, at 37 degrees C and in the absence of fibrin, STAR did not induce plasminogen activation and plasmin-alpha 2-antiplasmin complex (PAP) formation. Addition of a purified fibrin clot (30% vol at a concentration of 3 mg/mL) to mixtures containing intact plasminogen caused approximately 40% plasminogen activation within 2 hours, whereas in mixtures containing low-M(r) plasminogen, no activation was observed. In contrast, 10 nmol/L streptokinase (SK) induced 74% to 100% plasminogen activation within 2 hours in mixtures containing either intact or low-M(r) plasminogen, in both the absence and the presence of fibrin. In citrated human plasma in the absence of fibrin, 30 nmol/L STAR did not induce measurable plasminogen activation and PAP formation (< 1.5% within 2 hours), whereas addition of a plasma clot (12% vol) resulted in complete clot lysis and conversion of 19% +/- 8% of the plasminogen to PAP within 2 hours. Addition of a second plasma clot produced 23% +/- 2% additional plasminogen activation. Equipotent concentrations for plasma clot lysis of SK (100 nmol/L) induced 54% +/- 11% plasminogen activation in the absence and 49% +/- 16% in the presence of fibrin. Addition of 50 mmol/L 6-aminohexanoic acid (6-AHA) abolished the effect of fibrin on plasminogen activation with STAR, but not on activation with SK. In alpha 2-antiplasmin-depleted human plasma in the absence of fibrin, 30 nmol/L STAR did not induce fibrinogen breakdown (> 90% residual fibrinogen after 6 hours), whereas 30 nmol/L preformed plasmin-STAR complex induced extensive fibrinogen degradation (70% within 20 minutes). Thus, in the absence of fibrin, alpha 2- antiplasmin inhibits the activation of plasminogen by STAR, by preventing generation of active plasmin-STAR complex. Fibrin stimulates plasminogen activation by STAR via mechanisms involving the lysine- binding sites of plasminogen, probably by facilitating the generation of plasmin-STAR complex and by delaying its inhibition at the clot surface.     

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.     

Coronary thrombolysis with recombinant single-chain urokinase-type plasminogen activator in patients with acute myocardial infarction

Seventeen patients with acute transmural myocardial infarction and angiographically confirmed complete coronary occlusion were treated with heparin combined with intravenous single-chain urokinase-type plasminogen activator (scu-PA), obtained by expression of the cDNA encoding mature human scu-PA in Escherichia coli. In eight patients, recombinant scu-PA (rscu-PA) was given as a 10 mg bolus followed by 30 mg over 1 hr. Recanalization was obtained in six patients, but with persistent delayed opacification of the vessel in four of these patients. During infusion, a plateau level of rscu-PA antigen in plasma of 3.4 micrograms/ml (median value, range 1.4 to 5.5) was reached. At the end of the infusion the alpha 2-antiplasmin level had decreased to 54% (median, range 22% to 82%) of the preinfusion level, the fibrinogen level to 89% (median, range 26% to 101%), and fibrinogen degradation products (FDPs) to 20 micrograms/ml (median, range 8 to 387). In nine patients, rscu-PA was administered as a 10 mg bolus followed by 60 mg over 1 hr. This resulted in recanalization with normal distal filling of the vessel in seven patients, within 46 +/- 17 min (mean +/- SD). During infusion the concentration of rscu-PA in plasma increased to a median value of 7.4 micrograms/ml (range 4.0 to 13.3). At the end of the infusion the alpha 2-antiplasmin level was 22% of baseline (range 5% to 47%), the fibrinogen level 45% (range 4% to 94%), and the concentration of FDPs 87 micrograms/ml (range 6 to 1034). No significant bleeding or short-term side effects were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin- specific antibody fused to single-chain urokinase

The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single- chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady- state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody- targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.     

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.     

Thrombolytic properties of human tissue-type plasminogen activator, single-chain urokinase-type plasminogen activator, and synergistic combinations in venous thrombosis models in dogs and rabbits

Thrombolysis with single and combined four-hour intravenous (IV) infusions of recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator of 54,000 molecular weight (mol wt) (rscu-PA), and rscu-PA-32 kD, an rscu-PA derivative of 32,000 mol wt was studied in a femoral vein thrombosis model in the dog and in a jugular vein thrombosis model in the rabbit. In both species, the dose-response curves were linear, and no systemic activation of the fibrinolytic system or fibrinogen breakdown was observed. The steady-state levels of rt-PA-, rscu-PA-, and rscu-PA-32 kD-related antigens in plasma were proportional to the infusion rates. In the dog model, 25% lysis was obtained with 0.11 mg/kg rt-PA, 0.8 mg/kg rscu-PA, and 0.37 mg/kg rscu-PA-32 kD. Combinations of rt-PA and rscu-PA were 2.6 times more active (P less than .005) than anticipated on the basis of their pharmacologic additive effects, whereas combinations of rt-PA and rscu-PA-32 kD were 2.7 times more active (P less than .05). In the rabbit model, 25% lysis was obtained with 0.24 mg/kg rt-PA, 0.75 mg/kg rscu-PA, and 1.25 mg/kg rscu-PA-32 kD. Combinations of rt-PA and rscu-PA have a fivefold synergistic interaction, but surprisingly no synergism was observed between rt-PA and rscu-PA-32 kD. This study shows that synergism between rt-PA and rscu-PA occurs both in rabbits and dogs in a relatively narrow concentration range that allows a fractional reduction of the total equipotent dose by a factor of 2.5-fold to fivefold. Combination therapy is not associated with systemic fibrinolytic activation. This range of synergistic interaction, although limited, may be useful in devising the best thrombolytic therapy for patients with thromboembolic disease.     

Prevention of coronary thrombosis with subthrombolytic doses of tissue-type plasminogen activator

To determine whether tissue-type plasminogen activator (t-PA) may prevent coronary thrombosis or accelerate the lysis of clot formed under conditions in which increased concentration of the activator is present before thrombosis, clot lysis studies were undertaken in vitro and in vivo. In vitro, exogenous t-PA (6 to 100,000 ng/ml) accelerated the lysis of clot in a dose-dependent fashion when the clot was formed either from whole plasma or from euglobulin fractions (n = 316 determinations). Adding t-PA before clot formation shortened the time to lysis by at least threefold with euglobulin fractions and by at least 10-fold with whole plasma clots, which is consistent with the presence of inhibitors of fibrinolysis in whole plasma and with the binding of t-PA to nascent fibrin. In an intact dog preparation of coronary thrombosis (n = 25), occlusive thrombus formation was prevented when t-PA was present in subthrombolytic concentrations (430 to 1200 ng/ml, n = 5). Occlusive thrombus formation occurred after only discontinuation of the t-PA infusion and clearance of t-PA. Lower concentrations of t-PA (147 to 427 ng/ml, n = 6) significantly delayed occlusion (26 +/- 6.5 vs 7.8 +/- 2.8 min for controls). In animals with t-PA concentrations of less than 140 ng/ml (n = 4), the time to occlusion was unaltered (7.7 +/- 4.5 min). The present study demonstrates that t-PA present before clot formation inhibits thrombosis or accelerates thrombolysis depending on concentration, and that subthrombolytic doses of t-PA can prevent thrombus formation in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrin fragment D-dimer and fibrinogen B beta peptides in plasma as markers of clot lysis during thrombolytic therapy in acute myocardial infarction

The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the "Thrombolysis in Myocardial Infarction II" (TIMI II) trial. Cross-linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1-42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15-42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1-42 in the fibrinopeptide beta 15-42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15-42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1-42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin-specific tag ELISA may provide more accurate information when correlated with clinical endpoints.     

FDP D-dimer induces the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a human promonocytic leukemia cell line.

We studied the effect of fibrinogen degradation products D, E, and D-dimer on a human promonocytic leukemia cell line, NOMO-1. After exposure to a 10(-5)-mol/L fragment D or D-dimer, the cells displayed macrophage-like characteristics, such as adherence to plastic surfaces, and showed approximately a twofold increase in response to the nitroblue tetrazolium reduction test. The secretion of interleukin-1 alpha (IL-1 alpha) into the medium was markedly stimulated by a 10(-5)-mol/L fragment D, E, and D-dimer, whereas a significant increase in IL-1 beta secretion was observed only in D-dimer-stimulated cells. In addition, D-dimer induced a rapid increase in urokinase-type plasminogen activator on day 1 (0.52 +/- 0.02 ng/mL v 0.07 +/- 0.01 ng/mL in the control culture) and a slow increase in plasminogen activator inhibitor-2 on day 5 (3.9 +/- 1.6 ng/mL v 1.2 +/- 0.2 ng/mL in the control culture). An increase in tissue factor (TF) was also demonstrated on the cell surface of NOMO-1 cells exposed to fragment D or D-dimer by indirect immunofluorescence using an anti-TF monoclonal antibody. Scatchard plot analysis showed that fragment D and D-dimer bound to the NOMO-1 cells with a kd of 3.3 nmol/L and 2.7 nmol/L, respectively. These results suggest that fragment D-dimer specifically stimulates cells of monocyte-macrophage lineage to secrete key substances that regulate blood coagulation, fibrinolysis, and inflammation.     

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.     

Increased immunoreactivity of plasma after fibrinolytic activation in an anti-DD ELISA system. Role of soluble crosslinked fibrin polymers

After addition of a low concentration of thrombin to normal plasma, a progressive and significant increase in crosslinked fibrin polymers was found by sodium dodecyl sulfate agarose gel electrophoresis, reaching 27% of total fibrinogen and fibrin before gel formation. As measured by enzyme-linked immunosorbent assay with a monoclonal antibody specific for an epitope near the gamma gamma crosslink site, increased immunoreactivity of plasma did not occur after adding thrombin despite formation of crosslinked fibrin polymers, which indicates that the antibody does not recognize the epitope in the polymers. Addition of tissue-type plasminogen activator (t-PA) to plasma resulted in a more rapid degradation of fibrin polymers than of fibrinogen, indicating that the fibrin specificity of t-PA is retained with soluble fibrin. Coincident with degradation of plasma crosslinked fibrin polymers, plasma DD immunoreactivity increased 70-fold from 50.3 +/- 4.5 (mean +/- SD) to 3,560 +/- 1,235 ng/ml. The presence of increased crosslinked fibrin polymers produced by adding thrombin to plasma significantly increased maximum immunoreactivity after t-PA-induced degradation to 18,500 +/- 11,780 ng/ml. The increase in DD immunoreactivity was dependent on t-PA concentration; no elevation occurred below 0.01 micrograms/ml, and maximal increases occurred above 100 micrograms/ml. Analysis of gel electrophoretic patterns of thrombin and t-PA-treated plasma samples suggests that the DD reactivity of t-PA-treated plasma is mainly due to degradation of soluble crosslinked fibrin polymers. Our findings indicate that plasmic degradation of soluble fibrin polymers in plasma may be an important source of fragment DD during thrombolytic therapy.