Batroxobin binds fibrin with higher affinity and promotes clot expansion to a greater extent than thrombin.

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bß-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ' variant with an extended γ-chain. Thrombin binds to the γ'-chain and forms a higher affinity interaction with γA/γ'-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 µM) even though it does not interact with the γ'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 µM, the α(17-51) and Bß(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.

A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding.

INTRODUCTION: We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin. MATERIALS AND METHODS: We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories. RESULTS: Increasing the thrombin concentration improved release of FpA from the patient's fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient's fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment-thrombin complex became unstable due to the missing AαE11 residue. CONCLUSIONS: Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment-thrombin complex formation.

Radioimmunoassay of human fibrinopeptide B and kinetics of fibrinopeptide cleavage by different enzymes.

Thrombin converts fibrinogen to fibrin monomer by cleaving fibrinopeptides A and B (FPA and FPB) from the amino terminal ends of the A (alpha) and B (beta) chains. A radioimmunoassay capable of measuring the A peptide in human blood as an index of thrombin action in vivo has been described previously. This paper describes the development of a radioimmunoassay for FPB and the use of both assays in the demonstration of distinctive patterns of cleavage of the amino terminal ends of the A (alha) and B (beta) chains of fibrinogen by various enzymes. Antisera were raised in rabbits to a synthetic analogue of FPB coupled to bovine serum albumin. FPB analogue was couple to desaminotyrosine and radiolabeled with 125I by the chloramine-T technique. The radiolabeled peptide was bound by the antiserum, and binding was inhibited by synthetic or native FPB. Unbound tracer was separated from bound tracer by charcoal adsorption. The senistivity of the assay was such that 50% inhibition of binding of the tracer was caused by 1.25 ng of the native FPB. Fibrinogen was treated with thrombin, plasmin, trypsin, Reptilase, and an extract of the venom from Ancistrodon contortrix contortrix (ACC). After ethanol precipitation and centrifugation, dialysates of enzymatically altered fibrinogen were assayed for FPA and FPB. The action of thrombin on fibrinogen resulted in a rapid release of FPA and a slower release of FPB. Plasmin cleaved a segment(s) of the B (beta) chain which included FPB but cleaved no detectable FPA-containing material for the first 2 h of incubation. In the case of plasmin-treated fibrinogen, the dialysates had been further treated with thrombin before being assayed for FPA and FPB. Trypsin rapidly cleaved both peptides, the B before the A. Reptilase cleaved only FPA in 24 h. ACC cleaved FPB at a rapid rate, with a slowere cleavage of FPA. The distinctive cleavage patterns produced by the serine proteases may be useful in interpreting the levels of FPA and FPB measured in human blood and in studying the generation of FPA and FPB in clinical blood samples.

Dysfibrinogenemia associated with liver disease.

To test the possibility that a functionally abnormal fibrinogen may exist in some patients with liver disease, we studied the plasma and purified fibrinogens of five patients whose plasma thrombin times were prolonged at least 40% over normal controls. In no patient was there evidence of disseminated intravascular coagulation and/or fibrinolysis. No abnormalities were detected by immunoelectrophoresis of plasmas or purified fibrinogens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced patient fibrinogens showed normal mobility and amount of Aalpha, Bbeta, and gamma chains. Alkaline polyacrylamide gel electrophoresis and gradient elution, DEAE-cellulose chromatography of admixtures of radio-iodinated patient (125)I-fibrinogen and normal (131)I-fibrinogen showed identical mobility in the gel and simultaneous elution from the column, respectively. Thrombin and Reptilase (Abbott Scientific Products Div., Abbott Laboratories, South Pasadena, Calif.) times of purified patient fibrinogens were prolonged, and calcium ions improved but did not completely correct these defects. Increasing amounts of thrombin progressively shortened the clotting times of patient fibrinogens but not to the level of normal. Addition of equal amounts of patient fibrinogen to normal fibrinogen resulted in a prolongation of the thrombin time of the normal protein. Thrombin-induced fibrinopeptide release was normal. Fibrin monomers prepared from patient plasmas and purified fibrinogens demonstrated impaired aggregation at low (0.12) and high (0.24) ionic strength. These studies demonstrate that some patients with liver disease and prolonged plasma thrombin times have a dysfibrinogenemia functionally characterized by an abnormality of fibrin monomer polymerization.

Role of 'B-b' knob-hole interactions in fibrin binding to adsorbed fibrinogen.

BACKGROUND: The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs 'A' and 'B' exposed by thrombin cleavage and polymerization holes 'a' and 'b' present in fibrinogen and fibrin. Although structural studies have defined the 'A-a' and 'B-b' interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin. OBJECTIVES: We designed experiments to determine the affinities of knob-hole interactions, either 'A-a' alone or 'A-a' and 'B-b' together. METHODS: We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing 'A' knobs, desA-NDSK, or both 'A' and 'B' knobs, desAB-NDSK. RESULTS: The desA- and desAB-NDSK fragments bound to fibrinogen with statistically similar K(d)'s of 5.8 +/- 1.1 microm and 3.7 +/- 0.7 microm (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic 'A' knob peptide GPRP and synthetic 'B' knob peptides GHRP and AHRPY, inhibited the binding of desA- and/or desAB-NDSK. CONCLUSIONS: The peptide inhibition findings show both 'A-a' and 'B-b' interactions participate in desAB-NDSK binding to fibrinogen, indicating 'B-b' interactions can occur simultaneously with 'A-a'. Furthermore, 'A-a' interactions are much stronger than 'B-b' because the affinity of desA-NDSK was not markedly different from desAB-NDSK.

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.

BACKGROUND: Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. OBJECTIVES: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. METHODS: We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. RESULTS: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. CONCLUSION: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Simulating fibrin clotting time.

The clotting time (CT) of fibrinogen mixed with thrombin decreased, then increased with increasing fibrinogen levels. By contrast, log CT decreased monotonically with respect to the log level of activating enzyme (thrombin or reptilase). Here, the CT was determined over a large range of fibrinogen concentration (to 100 mg ml(-1)) at a fixed level of enzyme. A new parameter, [Fib]min, the minimal fibrinogen concentration required for thrombin or reptilase-instigated phase change (coagulation), was determined as [Fib]min = 0.2 +/- 0.05 microM fibrinogen. A dynamic simulation program (Stella) was employed to organize simulations based on simple and complex coagulation mechanisms, which generated CT values. The successful simulation aimed at forming [Fib]min and "recognized" the binding of unreacted fibrinogen with intermediate fibrin protofibrils. The "virtual data" mimicked the biphasic experimental CT values over a wide range of concentrations. Fibrinogen appeared to act in three modalities: as a thrombin substrate; as a precursor of fibrin; and as a competitor for fibrin protofibrils. The optimized simulation may provide a basis for predicting CT in more complex systems, such as pathological plasmas or whole blood or at high concentrations encountered with fibrin sealant.

Ontogenetic Variation in Biological Activities of Venoms from Hybrids between Bothrops erythromelas and Bothrops neuwiedi Snakes.

Lance-headed snakes are found in Central and South America, and they account for most snakebites in Brazil. The phylogeny of South American pitvipers has been reviewed, and the presence of natural and non-natural hybrids between different species of Bothrops snakes demonstrates that reproductive isolation of several species is still incomplete. The present study aimed to analyze the biological features, particularly the thrombin-like activity, of venoms from hybrids born in captivity, from the mating of a female Bothrops erythromelas and a male Bothrops neuwiedi, two species whose venoms are known to display ontogenetic variation. Proteolytic activity on azocoll and amidolytic activity on N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) were lowest when hybrids were 3 months old, and increased over body growth, reaching values similar to those of the father when hybrids were 12 months old. The clotting activity on plasma diminished as hybrids grew; venoms from 3- and 6-months old hybrids showed low clotting activity on fibrinogen (i.e., thrombin-like activity), like the mother venom, and such activity was detected only when hybrids were older than 1 year of age. Altogether, these results point out that venom features in hybrid snakes are genetically controlled during the ontogenetic development. Despite the presence of the thrombin-like enzyme gene(s) in hybrid snakes, they are silenced during the first six months of life.

Effect of calcium and synthetic peptides on fibrin polymerization.

Human fibrinogen was subjected to limited proteolytic attack by thrombin, batroxobin or Agkistrodon contortrix thrombin-like enzyme, yielding desAB-, desA- or desB-fibrin monomers, respectively. Turbidity curves demonstrated that, with all three enzymes, the polymerization process was strongly accelerated by increasing the calcium concentration from 10(-5) M to 10(-4) M. Synthetic peptide Gly-His-Arg (5 mM), an analogue of the aminoterminal sequence of fibrin beta-chain, inhibited aggregation of desB-fibrin monomers at physiological calcium concentration whereas it enhanced aggregation of desA- and desAB-fibrin monomers at calcium concentrations below 10(-4) M. On the other hand, Gly-Pro-Arg (1 mM) corresponding to the amino-terminus of fibrin alpha-chain, dramatically inhibited aggregation of both desA- and desB-fibrins, but it only moderately affected the polymerization of thrombin-induced monomers. We conclude that the observed effects of Gly-Pro-Arg and Gly-His-Arg are not due solely to their competition with fibrin amino-termini for the respective binding sites in the D-domain, but rather reflect conformational changes in fibrin monomers which affect the polymerization process.

Fibrinogen Matsumoto V: a variant with Aalpha19 Arg-->Gly (AGG-->GGG). Comparison between fibrin polymerization stimulated by thrombin or reptilase and fibrin monomer polymerization.

Fibrinogen Matsumoto V (M-V) is a dysfibrinogen identified in a 52-year-old woman with systemic lupus erythematous. The triplet AGG encoding the amino acid residue Aalpha19 was replaced by GGG, resulting in the substitution of Arg-->Gly. Residue Aalpha19 has been shown to be one of the most important amino acids in the so-called 'A' site or alpha-chain knob. The thrombin-catalyzed release of fibrinopeptide A from M-V fibrinogen was only slightly delayed yet release of fibrinopeptide B was significantly delayed. Both thrombin-catalyzed fibrin polymerization and fibrin monomer polymerization were markedly impaired compared to normal fibrinogen. In addition, reptilase-catalyzed fibrin polymerization of M-V was much more impaired than thrombin-catalyzed fibrin polymerization. These results indicate 'B' and/or 'b' site of M-V fibrinogen play a more important role in thrombin-catalyzed fibrin polymerization than that of normal control fibrinogen.

Mechanism of thrombin inhibition by antithrombin and heparin cofactor II in the presence of heparin.

The kinetics of thrombin inhibition by antithrombin (AT) and heparin cofactor II (HC II) were analysed as a function of the heparin concentration, from 10(-9) to 10(-4) M. The initial concentrations of inhibitor (l) and thrombin (E) were set at equimolar levels (CI = CE = 10(-8) M). The experimental data indicate that the reaction of thrombin inhibition was second-order both in the absence and in the presence of heparin, and that the apparent rate constant increased at heparin concentrations ranging from 10(-9) to 10(-6) M and decreased at higher concentrations. The data fit with the kinetic model established by Jordan et al. [J. Biol. Chem. 1979, 254, 2902-2913] for the catalysis of the thrombin-AT reaction by a low-molecular-weight heparin fraction. In this model, heparin (H) binds quickly to the inhibitor (I) and forms a heparin-inhibitor complex (HI), which is more reactive than the free inhibitor towards thrombin, leading to the formation of an inactive inhibitor-thrombin complex (I*E) and the release of free heparin, in a second step which is rate limiting. KH,I, the dissociation constant of HI, and k, the second-order rate constant of free thrombin inhibition by HI, were found to be 3.7 x 10(-7) M and 1.3 x 10(9) M-1 min-1, respectively, for AT, compared to a KH,I of 2.0 x 10(-6) M and k of 6.4 x 10(9) M-1 min-1 for HC II. These data indicate that heparin-HC II complex reactivity is greater than that of the heparin-AT complex towards thrombin, whereas heparin affinity is stronger for AT. At heparin concentrations higher than 10(-6) M, the decrease in the reaction rate was in keeping with the formation of a heparin-thrombin complex (HE), whose inactivation by the heparin-inhibitor complex (HI) is slower than that of the free protease.

Prevalence and clinical implications of heparin-associated false positive tests for serum fibrin(ogen) degradation products.

An electrophoretic method has been applied to characterize specific fibrinogen and fibrin degradation products (FDP) in 135 serum samples from 59 consecutive patients having a positive latex agglutination test for serum FDP in the evaluation of consumption coagulopathy. In 20 of 135 positive samples, the principal fibrinogen derivatives present were not degradation products of fibrinogen or fibrin but were instead residual fibrinogen or fibrin monomer and polymers (FFMP) due to incomplete clotting. Heparin exposure was common in patients with positive FDP tests occurring in 29 of 59 patients (49%) with 81 of 135 samples (60%). Heparin exposure by parenteral administration or catheter was significantly correlated with a false positive serum FDP test because of residual FFMP occurring in 19 of 81 (23%) samples from heparin-exposed patients but in only 1 of 54 (2%) samples from patients without exposure (P less than 0.005). Treatment of the false positive samples with reptilase, an enzyme unaffected by heparin, resulted in complete removal of the residual FFMP, and in vitro experiments demonstrated that heparin-containing plasma samples could be completely clotted with either reptilase or protamine sulfate plus thrombin. Survey of 20 regional laboratories showed that only 10% used reptilase or protamine sulfate to prepare serum if heparin exposure had occurred and that this was done in only 22 of 5,049 (0.4%) of samples in the last calendar year. Greater attention should be given to proper preparation of serum from heparin-exposed patients, and physicians should be aware of the possibility of falsely positive or falsely elevated serum FDP tests in evaluation of consumption coagulopathy in heparin-exposed patients.

Expression of cDNA for batroxobin, a thrombin-like snake venom enzyme.

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the fusion protein with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batroxobin with enzymatic activity was obtained by the refolding of recombinant batroxobin.

Defibrinogenation with benzoyl-batroxobin.

The B. moojeni thrombic protease, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about one hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effects of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.

Comparison of the sequence of fibrinopeptide A cleavage from fibrinogen fragment E by thrombin, atroxin, or batroxobin.

In order to investigate the sequence of fibrinopeptide release from the amino terminal end of a dimeric fibrinogen-derived substrate by thrombin or batroxobins, we studied their effects on plasmic fragment E1, a core fragment from the central domain of fibrinogen containing both A alpha chain fibrinopeptide A (FPA) sequences. Isoelectric focussing (IEF) was employed as a means of resolving des A-fragment E1, from which one FPA had been cleaved, from des AA-fragment E1 resulting from the loss of both FPA's. Using densitometric gel scanning for quantification of the levels of intact fragment E1, des A-fragment E1, and des AA-fragment E1, in mixtures incubated with enzyme for various periods of time, we found similar catalytic rate constants (k1, k2) for release of the first fibrinopeptide A, (FPA1) or the second, (FPA2) from fragment E1, with either thrombin or batroxobin (k2:k1 ratios of 1.10 +/- 0.42, 1.34 +/- 0.26 respectively). Atroxin released FPA2 more slowly than FPA1 with a k2:k1 ratio of 0.34 +/- 0.1. Th finding that the cleavage of FPA2 by Atroxin is three-fold slower than thrombin and almost four-fold slower than batroxobin, suggest that batroxobin and thrombin cleavage of FPA2 may be cooperative in nature. However, the cooperativity in the cleavage sequence is insufficient to markedly suppress the evolution of intermediate des A fragment E species during early and intermediate phases of FPA cleavage from fragment E.

Isolation and anticoagulant properties of polysaccharides of Typha Augustata and Daemonorops species.

Polysaccharides were isolated from Dragon's Blood (DB), a red resinous secretion from the fruits of Daemonorops species and from pollens of Typha Augustata (TA) by alcohol precipitation followed by deaminoethylaminoethyl (DEAE) cellulose chromatography. The purified polysaccharides of DB and TA gave specific rotations of [alpha]D20 64 and [alpha]D20 of 80 and their molecular weights by membrane osmometry were approximately 25000 and approximately 30000 respectively. The effect of addition of the polysaccharides on coagulation of pooled normal human plasma was investigated. They were found to accelerate the recalcified plasma times at concentrations below 100 micrograms/ml while inhibiting them at higher concentrations. Their procoagulant properties were due to their effects on the activation of Hageman factor (XII) while the anticoagulant effect was mainly directed towards fibrinogen. Studies of the mechanism of the anticoagulant effect showed that addition of TA polysaccharide inhibited the rate of release of fibrinopeptides by thrombin and also the aggregation of fibrin monomers.

Human fibrinogen heterogeneities: determination of the major Aalpha chain derivatives in blood.

Most plasma fibrinogen molecules, when analyzed by Na Dod SO4 polyacrylamide gel electrophoresis migrate in three major positions, designated bands I, II, and III, respectively. Band I reflects the presence of intact molecules, whereas bands II and III represent molecules containing catabolic Aalpha chain fragments. In order to establish the Aalpha chain composition of band II molecules, we analyzed plasma fraction I-6 which consists amost exclusively of material migrating in this band position. Analyses of ancrod- and of reptilase-treated samples by Na Dod SO4 polyacrylamide gel electrophoresis and gel scanning densitometry permitted assessment of the Aalpha chain composition of fraction I-6. We found that an Aalpha chain derivative, termed Aalpha/4, comprises more than 70% of the Aalpha chain population of I-6. Consistent with this finding and with studies showing that band II molecules were the most abundant species of catabolic derivatives in plasma, Aalpha/4 was shown to be the most abundant of the Aalpha chain (core) fragments in blood.

Analysis of reptilase and thrombin-induced changes in fibrinogen subunits by isoelectric focussing.

Isoelectric focussing (IEF) in 6M urea/4% polyacrylamide gel of reduced human fibrinogen and the fibrins formed by reptilase and thrombin gave a complex multibanded pattern. Similar results were obtained on IEF in urea/agarose gels containing excess mercaptoethanol. To characterize these multiple bands on the basis of molecular weight secondary electrophoreses into a discontinuous SDS-polyacrylamide gel system were carried out perpendicular to the initial IEF's. At least five major Alpha chains ranging in pI from 7.14 to 7.60 were found in fibrinogen. These were shifted 0.40 pI units towards the cathode after the action of reptilase or thrombin due to the release of electronegative fibrinopeptide A species. A similar change occurred with the three main B beta chains with initial pI's of 6.81 to 7.13, but only after thrombin action. Gamma dimer showed no significant difference in pI from its component chains 6.05 to 6.17.

Fibrinogen Stony Brook II: partial characterization of a heterozygously transmitted peptide A anomaly.

We describe preliminary studies of a new familial dysfibrinogen, fibrinogen Stony Brook II, present in a propositus and his mother. Both presented with a history of unexplained and chronic joint swelling following trauma, and the propositus suffered recurrent knee haemarthroses following arthroscopic surgery. Prolonged plasma thrombin times required a 3-5 fold excess of normal plasma for correction. Isolated fibrinogen displayed prolonged clotting times and delayed onset of clot turbidity. Also, an abnormal peptide could be released by thrombin but not by batroxobin along with approximately half the expected amounts of normal A peptide. Assessed by its thrombin release and by its early HPLC retention position, the abnormal peptide suggests a possible A alpha-16-Arg----His substitution. The data suggests an association in these probands between this heterozygously transmitted anomaly and the apparently impaired healing in hypovascular sites.

The cleavage and inactivation of plasminogen activator inhibitor type 1 and alpha2-antiplasmin by reptilase, a thrombin-like venom enzyme.

Reptilase, defibrase and ancrod are thrombin-like venom enzymes that cleave fibrinogen to release fibrinopeptide-A and generate fibrin monomers. Although these enzymes decrease fibrinogen levels in vivo, presumably by enhancing fibrinolytic activity, the mechanism has not been identified. In the present study, we analyzed their effects on the inhibitors of fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) was cleaved at its C-terminus by reptilase and lost its specific activity. Alpha2-antiplasmin (alpha2-AP) was cleaved both at the Pro19-Leu20 peptide bond and at its C-terminus by reptilase, and also lost its specific activity. The apparent second-order rate constants (mol/l per min per Batroxobin unit) were 0.22 for the cleavage of PAI-1 (3.2 micromol/l) and 0.19 for that of alpha2AP (6.4 micromol/l), which were approximately 200-fold lower than that (47.0) for the cleavage of fibrinogen (1.1 micromol/l). Neither defibrase nor ancrod cleaved and inactivated these inhibitors. Only reptilase enhanced euglobulin clot lysis in vitro at high concentration, due probably to PAI-1 inactivation. Since all these three enzymes enhance fibrinolysis similarly during defibrination therapy, the neutralization or inactivation of the inhibitors of fibrinolysis appeared not to represent the main mechanism for the enhancement.