Molecular cloning and expression of a functional snake venom serine proteinase,with platelet aggregating activity,from the Cerastes cerastes viper

The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze alpha-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42). This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.     

Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH₂-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 × 10⁻³ mol/L and 4.09 × 10⁻³ μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.     

Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities

Two venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major crotalid snake species in Taiwan. The purified enzymes showed a strong beta-fibrinogenolytic activity, cleaving the beta-chain of fibrinogen molecules specifically. They also showed strong kallikrein-like activity in vitro, releasing bradykinin from kininogen. The purified enzymes did not coagulate human plasma, yet decreasing fibrinogen levels in plasma and prolonging bleeding without formation of fibrin clots, indicating that both proteases have specificities different from thrombin and the thrombin-like proteases of snake venom reported previously. They also exhibit amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and found to be more stable when compared with ancrod and thrombin. Intravenous injection of either protease was shown to lower blood pressure in experimental rats. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases.     

Coagulopathy after snake bite by Bothrops neuwiedi: case report and results of in vitro experiments

Coagulation studies were performed in a patient who had been bitten by a snake of the species Bothrops neuwiedi. The patient presented with hemorrhagic necrosis at the envenomization site and considerable bleeding from venous puncture sites. He developed a severe defibrination syndrome with a clottable fibrinogen level of approximately 0.1 g/l. Fibrinogen was not measurable by clotting time assay. Fibrin degradation products were greatly elevated. Treatment with antivenom caused an anaphylactic reaction within ten minutes and serum sickness after three days. In vitro experiments revealed that B. neuwiedi venom directly activates Factors II and X, but does not activate Factor XIII. In vivo consumption of Factor XIII after B. neuwiedi envenomization is ascribed to the action of Factor IIa. At low venom concentrations clotting is initiated by activation of prothrombin by the venom either directly or via Factor X activation. Treatment with heparin might be beneficial in coagulopathy secondary to snake bite by reducing circulating active thrombin. The venom contains thrombin-like proteases which cause slow clotting of fibrinogen, and plasmin-like components causing further proteolysis of fibrinogen and fibrin. Antivenom has no effect on the proteolytic action of the snake venom. The in vivo effects of antivenom are presumably caused by acceleration of the elimination of venom components from the circulation. Intravenous administration of antivenom caused normalization of blood coagulation parameters within 48 h.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.     

Calcineurin B subunit acts as a potential agent for preventing cardiac ischemia/reperfusion injury

Calcineurin B subunit (CnB) is the regulatory subunit of calcineurin (Cn), a Ca²⁺/calmodulin-dependent serine/threonine protein phosphatase. It has been reported that mice deleting the CnB gene lose nearly all Cn activity and show poor tolerance to cardiac stress; CnB gene expression is downregulated in the hearts of rats that have suffered ischemia/reperfusion (I/R) injury. Therefore, we wonder whether injection of exogenous CnB protein can prevent the rats from suffering I/R injury. In cardiomyocytes, fluorogenic labeling shows that exogenous CnB quickly enters the cell. Pretreatment of cardiomyocytes with CnB reduces apoptosis in response to hypoxia/reoxygenation injury (an in vitro model mimicking ischemia/reperfusion injury), and CsA reverses this effect by inhibiting Cn activity. Furthermore, CnB upregulates Bcl-2 and Bcl-XL expression in the process of hypoxia/reoxygenation injury, which may contribute to protecting cardiomyocytes against apoptosis. In vivo experiments shows that pretreatment with CnB improves cardiac contractile function and reduces the frequency of arrhythmias induced by global I/R injury. These findings reveal a novel function for CnB protein in cardiac stress response and suggest a possible application of CnB in coronary disease therapy.     

Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.     

Purification of a protein C activator from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix)

A protease has been purified by ion-exchange chromatography from the venom of Agkistrodon contortrix contortrix (Southern copperhead snake) that can activate the vitamin K dependent protein, protein C. The apparent molecular weight of this protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 20,000 under nonreducing conditions. Incubation of this protease with plasma resulted in a prolongation of the clotting time and a time-dependent increase in amidolytic activity. Incubation of the protease with purified protein C resulted in an increase in both amidolytic and anticoagulant activity. The protease had no inhibitory effect on thrombin, factor V, fibrinogen, or factor X. It had slight clotting activity toward fibrinogen. The apparent Km of the protease for protein C was 0.28 microM. Calcium ions were observed to inhibit protein C activation with an apparent Ki of 0.2 mM. Ethylenediaminetetraacetic acid, diisopropyl fluorophosphate, and soybean trypsin inhibitor were observed to inhibit the venom protease. These results suggest that the venom of the Southern copperhead snake contains a protease that is a specific activator of protein C.     

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K(m) and V(max) values of Bafibrinase towards fibrin were determined as 0.24 μM and 2.8 μmol/min, respectively. It showed a K(m) value of 0.139 mM towards a chromogenic substrate for plasmin (D-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,β-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

Purification, Properties, and N-Terminal Amino Acid Sequence of a Kallikrein-like Enzyme from the Venom of Lachesis muta rhombeata (Bushmaster)

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0–6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N-α-tosyl-L-Arg methyl esterase (955.38 units/mg) and N-BZ-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A-α and B-β chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.     

Differential effects of endotoxin and fibrinogen degradation products (FDPS) on liver synthesis of fibrinogen and albumin: evidence for the involvement of a novel monokine in the stimulation of fibrinogen synthesis induced by FDPS

1. Administration of endotoxin or fibrinogen degradation products (FDPs) in rats increase fibrinogen synthesis comparable to that found during the acute phase response. 2. An increased fibrinogen synthesis is also found in co-cultures of hepatocytes with peripheral blood mononuclear cells upon administration of endotoxin or FDPs, but not in primary cultures of hepatocytes alone. 3. However, the increased synthesis of fibrinogen by FDPs is not accompanied by a decreased albumin synthesis, as in the case of stimulated fibrinogen synthesis induced by endotoxin in vivo and in co-cultures of hepatocytes with peripheral blood mononuclear cells, or induced by monocytic products in vivo and in primary cultures of hepatocytes alone. 4. Since IL-1 and/or IL-6 could not be accounted for the stimulation of fibrinogen synthesis without a decreased albumin synthesis, a novel monokine produced by mononuclear cells upon FDP administration might be involved.     

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta)

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.     

Sulfatide can markedly enhance thrombogenesis in rat deep vein thrombosis model

Although sulfatide (galactosylceramide I3-sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), it has been administered to animals without subsequent thrombus formation. We recently found that sulfatide binds to fibrinogen and thus disturbs fibrin formation in vitro, suggesting its possible role as an anticoagulant rather than as a coagulant. We therefore examined the in vivo effects of sulfatide on thrombogenesis by using a rat deep vein thrombosis model in which thrombus is induced by ligating the inferior vena cava. Sulfatide and gangliosides were each separately administered to rats 1 min before the vein ligation, and after 3 h, sulfatide but not gangliosides markedly (P < .001) enhanced the thrombogenesis. A kinetic turbidmetric assay of plasma coagulation initiated by CaCl2 in the wells of a microtiter plate revealed that coagulation was also markedly accelerated in the presence of sulfatide but not gangliosides, the results of which seemed to be very consistent with those of the in vivo experiments. Because sulfatide could not induce thrombosis without vein ligation in rats, the enhancement of thrombogenesis by sulfatide in the in vivo model might require endothelial damage and/or venous congestion, both of which could be induced by vein ligation.     

Structural and functional significance of disulfide bonds in saxatilin,a 7.7 kDa disintegrin

Saxatilin is a 7.7 kDa disintegrin that belongs to a family of homologous protein found in several snake venoms. Six disulfide bond locations of the disintegrin were determined by enzymatic cleavage and matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Functional implications of the disulfide bonds related to the biological activity of saxatilin were investigated with recombinant protein species produced by site-directed mutagenesis of saxatilin. Several lines of experimental evidence indicated that three disulfide bonds, Cys21-Cys35, Cys29-Cys59, and Cys47-Cys67, of the disintegrin are closely associated with its biological function such as its ability to block the binding of integrin GPIIb-IIIa and alpha(v)beta(3) with fibrinogen and extracellular matrix. Those disulfide linkages were also revealed to be important for maintaining the functional structure of the protein molecule. On the other hand, the disulfide bridges of Cys6-Cys15 and Cys8-Cys16 do not appear to be critical for the molecular structure and function of saxatilin.     

Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper)

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.     

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.