Effect of the small molecule plasminogen activator inhibitor-1 (PAI-1) inhibitor, PAI-749, in clinical models of fibrinolysis.

BACKGROUND: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. OBJECTIVE: To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis. METHODS: In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. RESULTS: There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P < 0.0001). PAI-749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t-PA. Inhibition of PAI-1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). CONCLUSIONS: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems.

Effect of tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis.

OBJECTIVE: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI-039), an orally bioavailable antagonist of PAI-1, in rat models of thrombosis. METHODS AND RESULTS: Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI-039 was administered orally 90 min before injury (1-30 mg kg(-1)). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI-039 administration. PAI-039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg(-1), respectively. Time to occlusive thrombosis was increased from 18.2 +/- 4.6 min in controls to 32.5 +/- 8.7 (P = ns), 46.1 +/- 7.0 (P < 0.05), and 41.6 +/- 11.3 min (P < 0.05) in the respective PAI-039 treatment groups. In the vena cava protocol, PAI-039 pretreatment significantly reduced thrombus weight at PAI-039 doses of 3, 10 and 30 mg kg(-1). When PAI-039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI-039 doses of 3, 10 and 30 mg kg(-1). PAI-039 (10, 30 and 100 mg kg(-1)) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI-039 had no effect on circulating, low-levels of PAI-1 activity. In contrast, circulating PAI-1 activity increased 5-fold following the induction of vascular injury, which was completely neutralized by PAI-039. CONCLUSIONS: PAI-039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state.

gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.

Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans.

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.

Antithrombotic efficacy of thrombin inhibitor L-374,087: intravenous activity in a primate model of venous thrombus extension and oral activity in a canine model of primary venous and coronary artery thrombosis.

The small molecule direct thrombin inhibitor L-374,087 was characterized across species in an in vitro activated partial thromboplastin clotting time (aPTT) assay and in vivo in rhesus monkey and dog thrombosis models. In vitro in rhesus, dog, and human plasma, L-374,087 concentrations eliciting 2-fold increases in aPTT were 0.25, 1.9, and 0.28 microM, respectively. In anesthetized rhesus monkeys, 300 microgram/kg bolus plus 12 microgram/kg/min and 300 microgram/kg bolus plus 30 microgram/kg/min L-374,087 i.v. infusions significantly reduced jugular vein thrombus extension, with both regimens limiting venous thrombus extension to 2-fold that of baseline thrombus mass compared with a 5-fold extension observed in the vehicle control group. Antithrombotic efficacy in the rhesus with the lower-dose regimen was achieved with 2.3- to 2.4-fold increases in aPTT and prothrombin time. In a conscious instrumented dog model of electrolytic vessel injury, the oral administration of two 10 mg/kg L-374,087 doses 12 h apart significantly reduced jugular vein thrombus mass, reduced the incidence of and delayed time to occlusive coronary artery thrombosis, and significantly reduced coronary artery thrombus mass and ensuing posterolateral myocardial infarct size. Antithrombotic efficacy in the dog was achieved with 1.6- to 2.0-fold increases in aPTT at 1 to 6 h after oral dosing with L-374,087. These results indicate significant antithrombotic efficacy against both venous and coronary arterial thrombosis with L-374,087 with only moderate elevations in aPTT or prothrombin time. The oral efficacy of L-374,087 characterizes this compound as a prototype for the further development of orally active direct thrombin inhibitors.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis.

The importance of thrombin in arterial and venous thrombosis renders thrombin inhibition an important therapeutic target. Identification of novel inhibitors requires an appropriate animal model. We modified a previously reported rat arterial thrombosis model to allow simultaneous assessment of the arterial and venous antithrombotic efficacies of heparin, hirudin, hirulog, a novel thrombin inhibitor H-(N-Me-D-Phe)-Pro-L-trans-4-aminocyclohexyl-Gly-[CO-CO]-NHCH3+ ++ (L-370,518) and the factor Xa inhibitor tick anticoagulant peptide in rabbits. Thrombosis was induced through application of 70% ferric chloride to the femoral artery and jugular vein. Incidence of occlusion, thrombus weight, aPTT and plasma inhibitor concentrations were determined. Heparin was efficacious in preventing arterial and venous occlusive thrombosis but at a dose that profoundly elevated aPTT. On a molar dosing basis, the approximate order of potency of the thrombin and factor Xa inhibitors was similar in artery and vein: hirudin>tick anticoagulant peptide>hirulog> or =L-370,518. Data suggested that compounds tended to be more potent in preventing venous thrombosis than arterial. This thrombin-dependent model is an economical and efficient approach to arterial and venous antithrombotic efficacy screening that eliminates variabilities encountered when multiple model/multiple animal strategies are employed.

Characterization of the two-step pathway for inhibition of thrombin by alpha-ketoamide transition state analogs.

The interaction of thrombin with several potent and selective alpha-ketoamide transition state analogs was characterized. L-370, 518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (Ki = 90 pM) and selective (>10(4)-fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed that k-1, the rate constant for dissociation of EI1, was comparable to k2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s-1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nM) largely due to an increase in k-1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1' N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K-1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pM) than L-370,518 due to its 16-fold higher k2 and 10-fold lower k-2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K-1 value for its alpha-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the Ki value for L-372,011 and the K-1 value for its alpha-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the alpha-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the alpha-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.

Discovery and development of the novel potent orally active thrombin inhibitor N-(9-hydroxy-9-fluorenecarboxy)prolyl trans-4-aminocyclohexylmethyl amide (L-372,460): coapplication of structure-based design and rapid multiple analogue synthesis on solid support.

Early studies in these laboratories of peptidomimetic structures containing a basic P1 moiety led to the highly potent and selective thrombin inhibitors 2 (Ki = 5.0 nM) and 3 (Ki = 0.1 nM). However, neither attains significant blood levels upon oral administration to rats and dogs. With the aim of improving pharmacokinetic properties via a more diverse database, we devised a resin-based route for the synthesis of analogues of these structures in which the P3 residue is replaced with a range of lipophilic carboxylic amides. Assembly proceeds from the common P2-P1 template 7 linked via an acid-labile carbamate to a polystyrene support. Application of the methodology in a repetitive fashion afforded several interesting analogues out of a collection of some 200 compounds. Among the most potent of the group, N-(9-hydroxy-9-fluorenecarboxy)-prolyl trans-4-aminocyclohexylmethyl amide (L-372,460 8, Ki = 1.5 nM), in addition to being fully efficacious in a rat model of arterial thrombosis at an infusion rate of 10 micrograms/kg/min, exhibits oral bioavailability of 74% in dogs, and oral bioavailability of 39% in monkeys with a serum half-life of just under 4 h. On the basis of its favorable biological properties, inhibitor 8 has been subject to further evaluation as a possible treatment for thrombogenic disorders.

L-374,087, an efficacious, orally bioavailable, pyridinone acetamide thrombin inhibitor.

Replacement of the amidinopiperidine P1 group of 3-benzylsulfonylamino-6-methyl-2-pyridinone acetamide thrombin inhibitor L-373,890 (2) with a mildly basic 5-linked 2-amino-6-methylpyridine results in an equipotent compound L-374,087 (5, Ki = 0.5 nM). Compound 5 is highly selective for thrombin over trypsin, is efficacious in the rat ferric chloride model of arterial thrombosis and is orally bioavailable in dogs and cynomolgus monkeys. The structural basis for the critical importance of both methyl groups in 5 was confirmed by X-ray crystallography.

C6 modification of the pyridinone core of thrombin inhibitor L-374,087 as a means of enhancing its oral absorption.

1 (L-374,087) is a potent, selective, efficacious, and orally bioavailable thrombin inhibitor that contains a core 3-amino-2-pyridinone moiety. Replacement of the C6 pyridinone methyl group of 1 by a propyl group gave 5 (L-375,052), which retained all the excellent properties of 1, and also yielded higher plasma levels after oral dosing in dogs and rats.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Potent noncovalent thrombin inhibitors that utilize the unique amino acid D-dicyclohexylalanine in the P3 position. Implications on oral bioavailability and antithrombotic efficacy.

In an effort to prepare orally bioavailable analogs of our previously reported thrombin inhibitor 1, we have synthesized a series of compounds that utilize the unique amino acid D-dicyclohexylalanine as a P3 ligand. The resulting compounds are extremely potent and selective thrombin inhibitors, and the N-terminal Boc derivative 8 exhibited excellent oral bioavailability and pharmacokinetics in both rats and dogs. The des-Boc analog 6 was not orally bioavailable in rats. The high level of oral bioavailability observed with 8 appears to be a direct function of its increased lipophilicity versus other close analogs. Although increased lipophilicity may serve to increase the oral absorption of tripeptide thrombin inhibitors, it also appears to have detrimental effects on the antithrombotic properties observed with the compounds. Compound 6 performed extremely well in our in vivo antithrombotic assay, while the much more lipophilic but essentially equipotent analog 8 performed poorly. We have found that in general with this series of thrombin inhibitors as well as with other unreported series, increased lipophilicity and the associated increases in plasma protein binding have detrimental effects on 2X APTT values and subsequent performance in in vivo antithrombotic models.

Vampire bat salivary plasminogen activator evokes minimal bleeding relative to tissue-type plasminogen activator as assessed by a rabbit cuticle bleeding time model.

Cuticle bleeding time (CBT) measurements in anesthetized rabbits were performed to assess the potential bleeding risks which may accompany the administration of tissue-type plasminogen activator (tPA) or vampire bat salivary plasminogen activator (BatPA). The dose of BatPA or tPA used in this study, 42 nmol/kg, was previously shown to be efficacious using a rabbit femoral artery thrombosis model (Gardell et al, Circulation 84:244, 1991). CBT was determined by severing the apex of the nail cuticle and monitoring the time to cessation of blood flow. CBT was minimally elevated (1.6-fold, p = NS) following bolus intravenous administration of BatPA; in contrast, bolus intravenous administration of tPA dramatically elevated CBT (6.2-fold, p < 0.05). Rabbits treated with tPA, but not BatPA, displayed profound activation of systemic plasminogen and consequent degradation of Factor VIII and fibrinogen. Elevations in CBT after the administration of tPA were reversed by the replenishment of plasma Factor VIII activity to 40% of control, but were unaffected by complete replenishment of plasma fibrinogen. The results of this study suggest that the administration of BatPA, at a dose that promotes thrombolysis, may evoke a minimal bleeding risk, relative to an equi-efficacious dose of tPA. In addition, the tPA-provoked proteolytic consumption of Factor VIII may be a key contributor to the heightened bleeding risk.

Comparison of the bleeding potential of vampire bat salivary plasminogen activator versus tissue plasminogen activator in an experimental rabbit model.

BACKGROUND: Vampire bat salivary plasminogen activator (Bat-PA) has significantly greater fibrin specificity than any of the fibrinolytic agents currently in clinical use. This study tests the hypothesis that avoiding fibrinogen depletion may protect against the hemorrhage induced by plasminogen activator treatment. METHODS AND RESULTS: Bat-PA was compared with tissue-type plasminogen activator (TPA) in a randomized, prospective, and blinded study using a rabbit ear puncture model of fibrinolytic bleeding. The two agents were used at equimolar dosages (42 nmol/kg) that yielded similar thrombolytic efficacies in a rabbit femoral artery thrombosis model. Both Bat-PA and TPA prolong primary bleeding to double the baseline values, from between 2.1 and 2.3 minutes to between 4.8 and 5.2 minutes. Rebleeding from hemostatically stable sites during the 3-hour observation period occurred equally often with Bat-PA and TPA, 31% from preinjection sites and 23% to 25% from postinjection sites. The lag time between the time of plasminogen activator injection and the onset of rebleeding was likewise the same for both agents, most occurring at 41 to 57 minutes. However, a greater number of prolonged primary or rebleeding occurrences continued for longer than 10 minutes (63% versus 36%) or longer than 30 minutes (30% versus 10%) after Bat-PA than TPA injection. Animals treated with TPA showed a dramatic decrease in plasma fibrinogen and factor VIII concentrations, but those in the Bat-PA treatment group showed only a slight decrease from control values. CONCLUSIONS: The results indicate that fibrinolytic bleeding after plasminogen activator infusion into rabbits did not correlate with the intensity of the plasma proteolytic state. If anything, Bat-PA usage was associated with a higher proportion of more protracted fibrinolytic bleeding episodes, despite the relatively mild lytic state in comparison with that induced by TPA.