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.

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.

A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function.

The aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95-98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

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.

Reptilase time (RT).

The reptilase time is a functional plasma clotting assay, which is based on the enzymatic activity of batroxobin. By specifically cleaving fibrinogen A from fibrinogen, batroxobin leads to the formation of a stable fibrin clot. The time, starting from the addition of batroxobin to the plasma sample, until clot formation is the reptilase time and is given in seconds. Clot formation can be detected manually or on automated coagulation systems. Reference values for healthy adults are 18-22 s. Healthy newborns may have a slightly prolonged reptilase time of up to 24 s. In addition to other coagulation assays, the reptilase time is usually performed to confirm or to exclude the suspicion of dysfibrinogenemias. The reptilase time is independent of thrombin generation disturbances or disturbances in the action of thrombin on fibrinogen. Therefore, it can be used to confirm heparin contamination or to obtain similar information as with the thrombin clotting time in heparinized and hemophiliac patients.

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.

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.

[Recombinant batroxobin expressed highly in Pichia pastoris].

Methylotrophic yeast, Pichia pastoris was used to express recombinant batroxobin, and a technology route of producing recombinant protein was finally established. We synthesized batroxobin gene artificially by means of recursive PCR. pPIC9-batroxobin was constructed and transformed into Pichia pastoris GS115 (his4). Recombinant batroxobin was expressed in yeast engineering strain and it was purified from the culture supernatant. 10 mg of recombinant batroxobin was purified from 1 liter fermentation media, it exhibited specific activity of 238 NIH units/mg and had molecular weight of 30.55 kD. The purified recombinant protein converted fibrinogen into fibrin clot in vitro, and shortened bleeding time in vivo. This study laid a foundation of development of hemostatic of recombinant snake venom thrombin-like enzyme.

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.

Binding of synthetic B knobs to fibrinogen changes the character of fibrin and inhibits its ability to activate tissue plasminogen activator and its destruction by plasmin.

Synthetic peptides corresponding to the amino-terminal sequence of the beta chain of fibrin increase the turbidity of fibrin clots, whether they are generated by the direct interaction of thrombin and fibrinogen or by the reassociation of fibrin monomers. The turbidity of batroxobin-induced clots, which are characteristically "fine," is increased even more dramatically. Pentapeptides are more effective than tetrapeptides. Surprisingly, the same peptides also delay fibrinolysis, whether activated by exogenously added plasmin or from the fibrin-enhanced stimulation of tissue plasminogen activator (tPA) activation of plasminogen. The peptides have only a very slight effect on the plasmic hydrolysis of a chromogenic peptide, either by the direct addition of plasmin or by plasmin generated from plasminogen by tPA. The synthetic peptides mimicking the B knobs appear to exert their action in two ways. First, they render fibrin less vulnerable to attack by plasmin. Second, they delay the fibrin activation of tPA. The latter is attributed to their ability to prevent the binding of the authentic B knob, which itself is located at the end of a flexible 50-residue tether and which needs time to find its elusive "hole". We propose that, when after a while the tethered knob does become inserted, it locks the betaC domain in a conformation that allows access to tPA-plasminogen-binding sites, whereas the untethered synthetic knobs restrict the fibrin to a conformation in which those sites remain inaccessible. Thus, although the interaction involving the A knob and gammaC hole is the basis for the polymerization of fibrin, the comparable but delayed interaction involving the B knob and the betaC hole is ultimately directed at preparing the clot for its eventual destruction.

Recombinant BbetaArg14His fibrinogen implies participation of N-terminus of Bbeta chain in desA fibrin polymerization.

We synthesized BbetaArg14His fibrinogen with histidine substituted for arginine at the Bbeta thrombin-cleavage site. This substitution led to a 300-fold decrease in the rate of thrombin-catalyzed fibrinopeptide B (FpB, Bbeta 1-14) release, whereas the rate of FpA release was normal with either thrombin or the FpA-specific enzyme, batroxobin. Both thrombin- and batroxobincatalyzed polymerization of BbetaArg14His fibrinogen were significantly impaired, with a longer lag time, slower rate of lateral aggregation, and decreased final turbidity. Moreover, desA monomer polymerization was similarly impaired, demonstrating that the histidine substitution itself, and not the lack of FpB cleavage, caused the abnormal polymerization of BbetaArg14His fibrin. Scanning electron microscopy showed BbetaArg14His fibrin fibers were thinner than normal (BbetaArg14His, approximately 70 nm; normal, approximately 100 nm; P <.0001), as expected from the decreased final turbidity. We conclude that the N-terminus of the Bbeta chain is involved in the lateral aggregation of normal desAprotofibrils and that the Arg-->His substitution disrupts these interactions in BbetaArg14His fibrinogen.

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.

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.

The contribution of residues 192 and 193 to the specificity of snake venom serine proteinases.

Snake venom serine proteinases, which belong to the subfamily of trypsin-like serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that of the less specific enzyme trypsin. One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence identity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom, specifically activates plasminogen to plasmin like tissue-type plasminogen activator (t-PA), even though it exhibits only 23% sequence identity with t-PA. This study shows that TSV-PA, t-PA, and batroxobin are quite different in their specificity toward small chromogenic substrates, TSV-PA being less selective than t-PA, and batroxobin not being efficient at all. The specificity of TSV-PA, with respect to t-PA and batroxobin, was investigated further by site-directed mutagenesis in the 189-195 segment, which forms the basement of the S(1) pocket of TSV-PA and presents a His at position 192 and a unique Phe at position 193. This study demonstrates that Phe(193) plays a more significant role than His(192) in determining substrate specificity and inhibition resistance. Interestingly, the TSV-PA variant F193G possesses a 8-9-fold increased activity for plasminogen and becomes sensitive to bovine pancreatic trypsin inhibitor.

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.

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.

Hemostatic activity of the bovine parotid gland glycoconjugates.

The bovine parotid gland was studied by means of biochemical analyses and the glycoconjugates extracted were used to investigate the activity on the human hemostatic system. Thromboelastography was unable to reveal anticoagulant properties. Conversely, the Thrombin Time (TT) was prolonged in a statistically significant way and with dose-coupling response. Reptilase Time (RT) was affected by the highest concentration of extract suggesting that the bovine parotid glycoconjugates alter the fibrinogen polymerization.

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.