Anticoagulant activity of M-LAO,L-amino acid oxidase purified from Agkistrodon halys blomhoffii,through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.     

The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway

Prolixin-S is a salivary anticoagulant of the blood-sucking insect, Rhodnius prolixus, and known as an inhibitor of the intrinsic Xase. We report here its inhibitory mechanisms with additional important anticoagulation activities. We found prolixin-S specifically bound to factor IX/IXa in the presence of Ca(2+) ions. Light scattering and surface plasmon resonance studies showed that prolixin-S interfered with factor IX/IXa binding to the phospholipid membrane, indicating that prolixin-S inhibit Xase activity of factor IXa by interference with its Xase complex formation. Furthermore, reconstitution experiments showed that prolixin-S binding to factor IX strongly inhibited factor IXa generation by factor XIa. We also found that prolixin-S inhibited factor IXa generation by factor VIIa-tissue factor complex and factor IXalpha generation by factor Xa. These results suggest that prolixin-S inhibits both intrinsic and extrinsic coagulations by sequential inhibition of all coagulation pathways in which factor IX participates. It was also suggested that prolixin-S may bind to factor IX/IXa by recognizing conformational change of the Gla domain induced by Ca(2+) binding.     

A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.     

A novel anticoagulant protein from Scapharca broughtonii

An anticoagulant protein was purified from the edible portion of a blood ark shell, Scapharca broughtonii, by ammonium sulfate precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G- 75, DEAE-Sephacel, and Biogel P-100. In vitro assays with human plasma, the anticoagulant from S. broughtonii, prolonged the activated partial thromboplastin time (APTT) and inhibited the factor IX in the intrinsic pathway of the blood coagulation cascade. But, the fibrin plate assay did not show that the anticoagulant is a fibrinolytic protease. The molecular mass of the purified S. broughtonii anticoagulant was measured to be about 26.0 kDa by gel filtration on a Sephadex G-75 column and SDSPAGE under denaturing conditions. The optimum activity in the APTT assay was exhibited at pH 7.0-7.5 and 40-45 degrees C in the presence of Ca(2+).     

Anticoagulant mechanism of factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis

Anticoagulant mechanism of the coagulation factor IX/factor X-binding protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis was investigated. IX/X-bp had no effect on the amidase activity of factor Xa measured with a synthetic peptide substrate Boc-Leu-Gly-Arg-pNA. Prothrombin activation by factor Xa without cofactors, such as factor Va and phospholipids, was only slightly influenced by IX/X-bp. However, prothrombin activation by factor Xa in the presence of factor Va resulted in IX/X-bp inhibiting the increase of k(cat) of thrombin formation through inhibition of interaction between factor Xa and factor Va. IX/X-bp also inhibited the decrease of K(m) for thrombin formation through interaction with phospholipids. Thus, IX/X-bp appears to act as an anticoagulant protein by inhibiting the interaction between factor Xa and its cofactors in the prothrombinase complex by binding to the Gla domain of factor Xa.     

Rheological approach to the analysis of blood coagulation in endothelial cell-coated tubes: Activation of the intrinsic reaction on the erythrocyte surface

Coagulation of blood in cultured endothelial cell-coated tubes was examined using a theological technique. Coagulation of recalcified, platelet-free plasma in contact with an endothelial cell monolayer did not occur within the experimental time period (more than 150 min). The endothelial cell surface did not activate the intrinsic coagulation reaction or the extrinsic coagulation reaction initiated by tissue factor. The time of onset of coagulation in platelet-free plasma supplemented with erythrocytes was nearly the same as that of whole blood (31.2 ± 5.5 min), which was shorter than that for platelet-rich plasma (54.3 ± 14.3 min) and platelet-free plasma supplemented with granulocytes (58.3 ± 6.3 min). In factor VII-, XI- or XII- deficient, platelet-free plasma supplemented with erythrocytes, the time of onset of coagulation was about 30 min. The coagulation of factor IX-deficient, platelet-free plasma supplemented with erythrocytes, however, did not occur within the experimental time period. These data suggest that activation of factor IX on the erythrocyte surface is capable of activating the intrinsic coagulation system.     

Processing of snake venom l-amino acid oxidase during intracellular transport

The isoelectrofocusing patterns of l-amino acid oxidase (LAO) from venom gland homogenates and of the secreted venom of Vipera palaestinae have been compared. The LAO isozyme profile of actively synthesizing gland spans over a wider range of pIs (4.8–6.0) and includes more variants as compared with the profile of the secreted venom. A basic shift of the isoelectrofocusing pattern of LAO obtained by treatment of the gland homogenate or the venom with neuraminidase indicates that sialic acid residues are responsible for the changes in the electronegativity of the isozymes. Analyses of subcellular fractions show that the microsomal fraction of the venom gland homogenate exhibits the highest multiplicity of molecular forms of LAO, whereas the fraction including the secretory granules has an isozyme profile similar to the venom. Double labelling experiments show that the newly synthesized LAO include isozymes which span over a wide range of pIs, whereas later on labelling of the more acidic isozymes is prominent. The results obtained may suggest that the sialic acid residues which are attached to LAO during its transport serve as “markers” for secretion.     

Coagulation factor IX regulates cell migration and adhesion in vitro

Coagulation factor IX is thought to circulate in the blood as an inactive zymogen before being activated in the coagulation process. The effect of coagulation factor IX on cells is poorly understood. This study aimed to evaluate the effects of intact coagulation factor IX and its cleavage fragments on cell behavior. A431 cells (derived from human squamous cell carcinoma), Pro5 cells (derived from mouse embryonic endothelial cells), Cos7 cells, and human umbilical vein endothelial cells were utilized in this study. The effects of coagulation factor IX and its cleavage fragments on cell behavior were investigated in several types of experiments, including wound‐healing assays and modified Boyden chamber assays. The effect of coagulation factor IX depended on its processing; full‐length coagulation factor IX suppressed cell migration, increased adhesion to matrix, and enhanced intercellular adhesion. In contrast, activated coagulation factor IX enhanced cell migration, suppressed adhesion to matrix, and inhibited intercellular adhesion. An activation peptide that is removed during the coagulation process was found to be responsible for the activity of full‐length coagulation factor IX, and the activity of activated coagulation factor IX was localized to an EGF domain of the coagulation factor IX light chain. Full‐length coagulation factor IX has a sedative effect on cells, which is counteracted by activated coagulation factor IX in vitro. Thus, coagulation factor IX may play roles before, during, and after the coagulation process.     

Ca(2+) -induced binding of anticoagulation factor II from the venom of Agkistrodon acutus with factor IX

Anticoagulation factor II (ACF II), a coagulation factor X- binding protein from the venom of Agkistrodon acutus has both anticoagulant and hypotensive activities. Previous studies show that ACF II binds specifically with activated factor X (FXa) in a Ca(2+) -dependent manner and inhibits intrinsic coagulation pathway. In this study, the inhibition of extrinsic coagulation pathway by ACF II was measured in vivo by prothrombin time assay and the binding of ACF II to factor IX (FIX) was investigated by native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR). The results indicate that ACF II also inhibits extrinsic coagulation pathway, but does not inhibit thrombin activity. ACF II also binds with FIX with high binding affinity in a Ca(2+) -dependent manner and their maximal binding occurs at about 0.1 mM Ca(2+) . ACF II has similar binding affinity to FIX and FX as determined by SPR. Ca(2+) has a slight effect on the secondary structure of FIX as determined by circular dichroism spectroscopy. Ca(2+) ions are required to maintain in vivo function of FIX Gla domain for its recognition of ACF II. However, Ca(2+) at high concentrations (>0.1 mM) inhibits the binding of ACF II to FIX. Ca(2+) functions as a switch for the binding between ACF II and FIX. ACF II extends activated partial thromboplastin time more strongly than prothrombin time, suggesting that the binding of ACF II with FIX may play a dominant role in the anticoagulation of ACF II in vivo.     

Amphipathic peptides can act as an anticoagulant by competing with phospholipid membranes for blood coagulation factors

An ideal amphipathic peptide (IAP), composed of simply lysine and leucine residues in a 1:2 ratio (K₇L₁₅), specifically prolongs in vitro coagulation assays that use phospholipids, such as the activated partial thromboplastin time (APTT). The main hypothesis of the present work is that IAP’s anticoagulant effect occurs by competing with phospholipid membranes in in vitro coagulation reactions. We verified this hypothesis by employing different phospholipid-dependent coagulation assays, such as the APTT, the dilute prothrombin time (dPT) and the dilute Russell viper venom time (dRVVT) with both low and high amounts of phospholipids. We show that coagulation times are prolonged by IAP in a concentration-dependent manner, and that this prolongation is abrogated by adding excess phospholipid, demonstrating a phospholipid dependence for this inhibition. Using an ELISA-based binding assay, we show IAP inhibits the binding of one of the vitamin K-dependent coagulation factors, factor X, to phospholipid membranes. This is further confirmed with fluorescence spectroscopy, where the interaction of IAP and factor X is inhibited by phospholipid. In summary, this work demonstrates that IAP can act as an anticoagulant by impairing the interaction of coagulation factors with phospholipid membranes and provides a paradigm for the development of novel anticoagulants.     

Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.     

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg⁵⁰⁶ and Arg³⁰⁶ and of factor VIIIa (FVIIIa) by cleavage at Arg³³⁶ and Arg⁵⁶². To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K_D of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg⁵⁰⁶ and not Arg³⁰⁶ and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg⁵⁰⁶ cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg⁵⁰⁶ interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.     

Feasibility and Safety of Local Treatment with Recombinant Human Tissue Factor Pathway Inhibitor in a Rat Model of Streptococcus pneumoniae Pneumonia

Pulmonary coagulopathy is intrinsic to pulmonary injury including pneumonia. Anticoagulant strategies could benefit patients with pneumonia, but systemic administration of anticoagulant agents may lead to suboptimal local levels and may cause systemic hemorrhage. We hypothesized nebulization to provide a safer and more effective route for local administration of anticoagulants. Therefore, we aimed to examine feasibility and safety of nebulization of recombinant human tissue factor pathway inhibitor (rh-TFPI) in a well-established rat model of Streptococcus (S.) pneumoniae pneumonia. Thirty minutes before and every 6 hours after intratracheal instillation of S. pneumonia causing pneumonia, rats were subjected to local treatment with rh-TFPI or placebo, and sacrificed after 42 hours. Pneumonia was associated with local as well as systemic activation of coagulation. Nebulization of rh-TFPI resulted in high levels of rh-TFPI in bronchoalveolar lavage fluid, which was accompanied by an attenuation of pulmonary coagulation. Systemic rh-TFPI levels remained undetectable, and systemic TFPI activity and systemic coagulation were not affected. Histopathology revealed no bleeding in the lungs. We conclude that nebulization of rh-TFPI seems feasible and safe; local anticoagulant treatment with rh-TFPI attenuates pulmonary coagulation, while not affecting systemic coagulation in a rat model of S. pneumoniae pneumonia.     

Functional characterization of human blood coagulation factor XIa using hybridoma antibodies

During the initiation of intrinsic coagulation factors XI and XIa interact intimately with several other coagulation proteins (factor XIIa, high Mr kininogen, and factor IX) as well as with the platelet surface. To help elucidate these complex intramolecular interactions, we have prepared a collection of monoclonal antibodies directed against various epitopes in factor XI. We have utilized these reagents to isolate factor XI and the light chain of factor XIa on affinity columns, and to probe structure-function relationships involved in the interactions of factor XIa with factor IX. The isolated light chain of factor XIa retained greater than 90% of its amidolytic activity against the oligopeptide substrate pyro-Glu-Pro-Arg-pNA (S-2366), but only 3.8% of its clotting activity in a factor XIa assay and 1% of its factor IX activating activity in an activation peptide release assay. This suggests that regions of the heavy chain are required for development of coagulant activity and specifically for the interaction of factor XIa with factor IX. To test this hypothesis, the effects of three of the monoclonal antibodies (5F4, 1F1, and 3C1) on the function of factor XIa were examined. The results show that in a clotting assay the light chain-specific antibody (5F4) inhibits 100% of the factor XIa activity, whereas of the heavy chain-specific antibodies, one (3C1) inhibits 75% and another (1F1) only 17%. Similarly in the factor IX activation peptide release assay, antibody 5F4 inhibits 100% of the factor XIa activity, whereas 3C1 inhibits 75% and 1F1 inhibits 33%. We conclude that regions located in the heavy chain, in addition to those in the light chain, are involved in the interaction of factor XIa with factor IX and in the expression of the coagulant activity of factor XI.     

Characterization and expression of L-amino acid oxidase of mouse milk

l-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with l-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His other 11 amino acids tested and produced H(2)O(2) in a dose- and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N- and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H(2)O(2) using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.     

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity,a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host''s immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.     

Identification of an anticoagulant peptide that inhibits both fXIa and fVIIa/tissue factor from the blood-feeding nematode Ancylostoma caninum

Factor VIIa-tissue factor complex (fVIIa/TF) and factor XIa (fXIa) play important roles in the initiation and amplification of coagulation, respectively. They may be good targets for the development of novel anticoagulants to treat and prevent thromboembolic disease. In this study, we cloned, expressed and identified a novel anticoagulant peptide, AcaNAP10, from the blood-feeding nematode Ancylostoma caninum. AcaNAP10 showed potent anticoagulant activity and doubled the activated partial thromboplastin and prothrombin times at estimated concentrations of 92.9 nM and 28.8 nM, respectively. AcaNAP10 demonstrated distinct mechanisms of action compared with known anticoagulants. It inhibited fXIa and fVIIa/TF with IC₅₀ values of 25.76 ± 1.06 nM and 123.9 ± 1.71 nM, respectively. This is the first report on an anticoagulant that can inhibit both fXIa and fVIIa/TF. This anticoagulant peptide may be an alternative molecule for the development of novel anticoagulants.     

Heparinoid-active two sulfated polysaccharides isolated from marine green algae Monostroma nitidum

Two sulfated polysaccharides WF1 and WF3 were isolated from marine green algae Monostroma nitidum, and their structural characteristics were determined. Anticoagulant activities of WF1 and WF3 were evaluated by assays of the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), antithrombin and anticoagulation factor Xa activities. The results showed that WF1 and WF3 had similar high contents of rhamnose, whereas their sulfate contents, sulfation positions, molecular sizes and linkage patterns of rhamnose residues were different. The bioassay results demonstrated that WF1 and WF3 had high anticoagulant activities, and were potent thrombin inhibitors mediated by heparin cofactor II, especially WF3. They also hastened thrombin and coagulation factor Xa inhibition by potentiating antithrombin III, but at a lower effectiveness. The differences of anticoagulant activities between WF1 and WF3 were directly due to their structural features discrepancy.     

IgE-induced blood coagulation alterations in the rabbit: consumption of coagulation factors XII, XI, and IX in vivo.

Intravenous administration of BSA into 3-month-old rabbits producing detectable anti-BSA antibody only of the IgE class of immunoglobulin induced a variety of intravascular blood coagulation alterations observed in the plasma 15 min after antigen challenge included: a) the intravascular consumption of intrinsic blood coagulation factors XII, XI, and IX and possibly the reduction in clottable fibrinogen; b) a significant prolongation of the activated partial thromboplastin time but not the prothrombin time; and c) the production of an inhibitor affecting the last stage of blood coagulation. The observed blood coagulation alterations were not caused by the manipulative procedures utilized, the presence of anti-BSA, IgG or IgM antibody, histamine-induced alterations in the vascular endothelium or the development of hypotensive shock. It is proposed that specific IgE antibody can induce directly or indirectly the activation of intrinsic blood coagulation in vivo.     

Isolation of a venom factor devoid of proteolytic activity from Taiwan Habu (Trimeresurus mucrosquamatus): N-Terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca²⁺ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.