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PubMed1995

Primary structure of a coagulant enzyme, bilineobin, from Agkistrodon bilineatus venom.

The amino acid sequence and disulfide bridge location of the coagulant enzyme, named bilineobin, isolated from the venom of Agkistrodon bilineatus was determined by Edman sequencing of the peptides derived from digests with cyanogen bromide, clostripain, Staphylococcus aureus V8 protease, trypsin, and chymotrypsin. This enzyme has a molecular weight of 57,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, bilineobin consists of 235 amino acids and has a calculated molecular weight of 26,481. The enzyme contains fucose, GlcNAc, galactose, mannose and NeuAc and six N-linked glycosylation consensus sites. The carboxyterminal amino acid, proline, was determined using carboxypeptidase Y. The six disulfide bonds of bilineobin link Cys78 to Cys234, Cys120 to Cys188, Cys178 to Cys203, Cys7 to Cys141, Cys152 to Cys167, and Cys28 to Cys44. The amino acid sequence similarity to flavoxobin (T.C. Shieh et al., 1988, J. Biochem (Tokyo) 103, 596-605) and batroxobin (N. Itoh et al., 1987, J. Biol. Chem. 262, 3132-3135) was 67%. The deglycosylated enzyme more rapidly generated fibrinopeptide A than native bilineobin.

Agkistrodon↗
PubMed1994

Characterization of one novel venom protease with beta-fibrinogenase activity from the Taiwan habu (Trimeresurus mucrosquamatus): purification and cDNA sequence analysis.

Several fibrinogenolytic proteases were isolated from the venom of Taiwan habu, Trimeresurus mucrosquamatus, a snake species belonging to the Crotalidae family. One protease with strong fibrinogenolytic activity was further purified to homogeneity through multiple-step chromatographies including ion-exchange chromatography, gel permeation and reversed-phase HPLC. In vitro, the purified enzyme cleaved beta-chain of fibrinogen molecules efficiently and showed relatively lower activity on alpha-chain, with almost no activity on gamma-chain even after a long period of incubation. Further characterization indicated that it is a single-chain polypeptide with molecular weight of about 28,000. Its stability at high temperatures (> 90 degrees C) distinguished it from the previously reported venom fibrinogenases. N-Terminal sequence analysis revealed that it is similar to batroxobin and ancrod, which were shown to possess either fibrinogen-clotting or antithrombotic effect. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh venom glands of the same snake species to facilitate the cloning and sequencing of this important fibrinogenase. Sequencing several positive clones corresponding to the coding sequence of the enzyme revealed the existence of a family of novel thrombin-like fibrinogenases in the Taiwan habu, which are heat-stable and may be useful as strong antithrombotic agents.

Amino Acid Sequence↗
PubMed2001

Cloning and functional expression of the mucrosobin protein, a beta-fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu).

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5'-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His(41), Asp(84) and Ser(179) in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific beta-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquire a functional snake venom protein with therapeutic potential.

Amino Acid Sequence↗
PubMed1989

On the significance of animal experiments in toxinology.

In increasing areas of the world, there is considerable controversy over the use of animals in scientific research. Ethical, scientific and economic reasons require a reduction in animal experiments as well as animal numbers, a refinement of existing methods and, wherever possible, a replacement of in vivo methods by in vitro test systems. By scoping on papers published in Toxicon between 1980 and 1986, the current situation of animal experimentation in toxinology is evaluated. Fundamental research and antivenin production are reviewed and special reference is given to the experiments involved in drug registration by the example of batroxobin, a purified serine proteinase from Bothrops moojeni venom used as a defibrinogenating agent. Although the ethical question is the most important motif, a reduction in the number of animal experiments can be achieved more pragmatically: the scientific community must learn to constantly ask whether the intention behind an experimental series justifies the use of animals or not. This learning process can be supported by special educational programmes at university level, by making financial support dependent on questionnaires concerning the planned animal experimentation, by including respective questions in the application files for grants, and by ethical guidelines that form an integral part in the instruction for authors of scientific journals.

Animal Experimentation↗
PubMed1993

Some properties of a capillary permeability-increasing enzyme-2 from the venom of Agkistrodon caliginosus (Kankoku-Mamushi).

Capillary permeability-increasing enzyme-2 (CPI-enzyme-2) consists of a single polypeptide chain with an isoelectric point of pH 3.5. The enzyme is composed of 369 amino acid residues, based on a mol. wt of 44,000, and contains 20.3% carbohydrate. Both arginine ester hydrolytic and capillary permeability-increasing activities of the enzyme were inhibited by treatment with diisopropylfluorophosphate, indicating that the enzyme is a serine proteinase. The N-terminal amino acid sequence shows homology with that of CPI-enzyme-1 and similarity to those of batroxobin or kallikrein-like enzyme from other snake venoms.

Agkistrodon↗
PubMed1994

Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurus okinavensis (himehabu snake) venom which release fibrinopeptides A and B.

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavenis (himehabu snake) venom, released specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for alpha-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI = 8.1) than okinaxobin I (pI = 5.4). The N-terminal sequence was highly similar to those of okinaxobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like alpha-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to alpha-thrombin.

Amino Acid Sequence↗
PubMed1982

Fibrinogen Bondy: a new case of dysfibrinogenemia. Isolation of the abnormal fibrinogen molecules.

In a 81 year old health woman, gross abnormalities of fibrin formation led to the discovery of an abnormal fibrinogen named fibrinogen Bondy. Clottability of purified fibrinogen Bondy was only 53% compared to 95-98% for normal fibrinogen. Functional studies revealed (i) delayed coagulation by thrombin and batroxobin (Reptilase), (ii) incomplete release of fibrino-peptides A and B, (iii) poor fibrin monomer aggregation, (iv) delayed fibrin proteolysis by plasmin. Electrophoretic mobility of fibrinogen Bondy, its three chains and the products of fibrin cross-linking, was normal. Fibrinogen NH2-terminal residues of fibrinogen Bondy were found to be normal. The presence of Ala, in addition to Gly and Tyr in the fibrin clot and its supernatant, showed that a part of fibrinogen molecules was not clotted, i.e. either copolymerised with fibrin or remaining in solutions. Gel filtration of the supernatant allowed the separation of both soluble complexes and fibrinogen. This fibrinogen population was shown to be unclottable by thrombin and to inhibit clotting of normal fibrinogen.

Aged↗
PubMed1982

Arachidonic acid induces human platelet-fibrin retraction: the role of platelet cyclic endoperoxides.

Arachidonic acid (0.2-0.8 mM) retracts clots formed in human citrated platelet-rich plasma by batroxobin. Extracellular calcium ions, but not the secretion of ADP by platelets, are required. AA-induced clot-retraction requires cyclo-oxygenase but not thromboxane synthetase activity since the retraction is inhibited by aspirin but not by selective inhibitors of thromboxane synthesis. The data indicate that endogenous cyclic endoperoxides mediate the retraction. Moreover, intact endoperoxide/thromboxane receptors also seem to be necessary because clot retraction is inhibited by thromboxane receptor antagonists.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1985

Crosslinking of fibrinogen to immobilized DesAA-fibrin.

DesAA-fibrin Sepharose was produced by treating fibrinogen-Sepharose with batroxobin. DesAA-fibrin Sepharose was mixed with different concentrations of fibrinogen and at different ratios, and incubated with preactivated FXIII. After 2 hours at 37 degrees C, the Sepharose beads were separated by centrifugation and non-crosslinked fibrinogen was removed by twice times washing with guanidinium chloride, pH 4.1. Under these experimental conditions specific crosslinking of fibrinogen to immobilized desAA-fibrin by FXIIIa was found. These results support the concept of a specific interaction between fibrinogen and fibrin involving polymerization which enables FXIIIa to crosslink fibrin to fibrinogen being in an half-staggered overlap position but not in DD-long contact.

Fibrin↗
PubMed1994

At high heparin concentrations, protamine concentrations which reverse heparin anticoagulant effects are insufficient to reverse heparin anti-platelet effects.

Combined effects of heparin and protamine on plasma clot structure and platelet function were studied. Anticoagulant effects were monitored as changes in aPTT. Clot structure was defined in terms of fibrin fiber mass/length ratio (mu) and clot elastic modulus (EM). Platelet function was studied utilizing platelet aggregation and platelet force development (PFD) measurements. Heparin (1 U/ml) prolonged the aPTT from 30 to > 300 seconds, reduced PFD from 5,100 to 0 dynes, decreased mu (in batroxobin-induced gels) from 1.36 to 1.08 x 10(13) daltons/cm and decreased clot EM from 9,600 to 2000 dynes/cm2. Varying amounts of protamine reversed these effects: 16 micrograms/ml normalized the aPTT, 20 micrograms/ml normalized PFD, 32 micrograms/ml corrected mu, and 20 micrograms/ml returned EM to baseline. At high heparin concentrations (4 U/ml), protamine concentrations which corrected anticoagulant effects were inadequate to reverse antiplatelet effects. A protamine concentration of 40 micrograms/ml normalized the aPTT and mu, but 140 micrograms/ml of protamine was required to reverse heparin suppression of force development and clot elastic modulus. Excess protamine inhibited clotting and platelet function. In plasma containing 1 u heparin/ml, 140 micrograms protamine/ml reduced PFD by 83%, prolonged the aPTT by 63%, and reduced clot EM by 75%. In heparin free plasma, > 75 micrograms protamine/ml prolonged the aPTT. Thus, platelet function and clot structure are sensitive to protamine during heparin neutralization, and anti-platelet effects of heparin may persist when the aPTT is completely corrected. Excess protamine inhibits platelet function and compromises clot structure.

Blood Coagulation↗
PubMed1996

On the occurrence of thrombin-like enzymes in mosquitoes.

A thrombin-like enzyme has been identified in mosquitoes and partially purified. The enzyme preparation displays on SDS gel electrophoresis a major protein band of about 22 kDa and one minor of about 28 kDa. The enzyme preparation cleaves a synthetic substrate (S-2238) for thrombin with Km 113 mumol/L (as compared to 3 mumol/L for thrombin). The enzyme clots fibrinogen and plasma. During activation of fibrinogen, fibrinopeptide A (but scarcely fibrinopeptide B) is released. The enzyme is not inhibited by hirudin and activates (if at all) factor XIII differently from thrombin. Predominantly gamma-dimers are formed in the cross linking process. As compared to thrombin a larger extent of activation is required to induce gelation (clotting) by the mosquito enzyme. At a given clotting time the enzyme produces tighter gel structures than thrombin. In its action the enzyme resembles the snake venom enzyme, batroxobin.

Aedes↗
PubMed1983

Inhibition of fibrin polymerization by fragment d is affected by calcium, Gly-Pro-Arg and Gly-His-Arg.

Fibrinopeptides A and B were removed from purified human fibrinogen by bovine thrombin, whereas the snake venom protease batroxobin only split fibrinopeptide A from fibrinogen. Aggregation of the resulting desAB- and desA-fibrin monomers was evaluated by recording the turbidity of incubation mixtures. Fibrin assembly was strongly accelerated by increasing the calcium concentration from 10(-5) to 10(-3) M. Fragment D was obtained from fibrinogen by proteolytic degradation with plasmin in the presence of Ca2+. At a 4-fold molar concentration relative to fibrinogen, fragment D dramatically inhibited fibrin polymerization at up to 10(-4) M Ca2+. This anticlotting activity was, however, much less pronounced at 10(-3) M Ca2+. The thrombin clotting time, measured on human plasma, was prolonged by fragment D in a dose-dependent manner. In citrate-containing plasma, the fibrinogen clotting was significantly delayed by an equimolar concentration of fragment D. In barium sulfate-adsorbed oxalated plasma, containing 2.5 mM Ca2+, the same amount of fragment D hardly affected fibrin polymerization. We conclude that fragment D has no important anticlotting effect under physiological conditions. The synthetic peptide Gly-Pro-Arg, corresponding to the amino-terminal sequence of the fibrin alpha-chain, inhibited aggregation of both desA-fibrin and desAB-fibrin at 10(-3) M Ca2+. The inhibition of desAB-fibrin polymerization by Gly-Pro-Arg was abolished at 10(-5) M Ca2+. In addition, Gly-Pro-Arg depressed the anticlotting activity of fragment D at low calcium concentration. An analogue of the amino-terminus of fibrin beta-chain, Gly-His-Arg, strongly accelerated aggregation of desA-fibrin monomers, but only moderately enhanced polymerization of desAB-fibrin monomers at 10(-5) M Ca2+, both in the presence and in the absence of fragment D. This activating effect of Gly-His-Arg was abolished at 10(-3) M Ca2+. It is suggested that the binding of calcium, Gly-His-Arg, and possibly also Gly-Pro-Arg, induces a conformational change in fibrin monomers and thus accelerates the polymerization process.

Animals↗
PubMed1998

Vivostat system autologous fibrin sealant: preliminary study in elective coronary bypass grafting.

BACKGROUND: The Vivostat System is a medical device for the preparation of an autologous fibrin sealant from 120 mL of the patient's blood in the operating room. The system is fully automated and microprocessor controlled and is made up of three components: an automated processor unit, an automated applicator unit, and a disposable, single-patient-use unit, which includes a preparation set and a Spraypen applicator. The biochemical process is initiated by batroxobin, which acts upon the fibrinogen in the patient's plasma. The completion of the process depends entirely on endogenous thrombin in producing the sealant. METHODS: Twenty-four volunteer patients undergoing elective primary coronary artery bypass grafting were randomized to either conventional hemostasis (control group) or the use of Vivostat fibrin sealant as an adjunct to conventional hemostasis. The patients were followed up at 1 month and 1 year. RESULTS: The preparation process was completed in 30 minutes. No safety issues associated with the use of the sealant were identified. From 120 mL of the patient's blood the yield of fibrin sealant was 4.5 mL (range, 3.9 to 4.8 mL). There was a favorable trend toward lower amounts of chest tube drainage in the Vivostat group. In the Vivostat group, 1 of 11 patients (9%) required a perioperative transfusion and in the control group 3 of 12 patients (25%) required a perioperative transfusion. CONCLUSIONS: It is possible to prepare autologous fibrin sealant with the Vivostat system in 30 minutes. No exogenous thrombin is required. The sealant has no known adverse effects and may prove to be a useful adjunct to hemostasis in cardiothoracic surgery.

Adult↗
PubMed1986

Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides.

The structural basis of the wide variability of the physical properties of fibrin clots and the process of assembly of the clot were investigated by electron microscopy of fibers formed under various ionic conditions. In addition, highly specific proteolytic enzymes from different snake venoms were used to remove selectively only the A (batroxobin) or the B (venzyme) fibrinopeptides from fibrinogen, in contrast to thrombin, which removes both pairs. Fibers produced by cleavage of only the B fibrinopeptides displayed a characteristic band pattern indistinguishable from that of fibers formed upon removal of either the A fibrinopeptides alone or of both pairs. Computer modeling studies suggest that there is a unique molecular packing that gives rise to this fibrin band pattern. These findings imply that the release of either fibrinopeptide triggers similar modes of aggregation; the intermolecular binding sites can be localized to particular molecular domains. The diameters of fibers formed with each condition of enzyme, pH, salt concentration, and temperature were measured from electron micrographs. All fibers, except for those produced at both high ionic strength and pH, had about the same average diameter of 85 +/- 13 nm. The degree of lateral aggregation of the fibers themselves varied greatly, however; fibers aggregated more readily with cleavage of both pairs of fibrinopeptides and at lower pH and salt concentrations. The formation of such thick fiber bundles increases the stability of the clot and its resistance to proteolytic dissolution.

Fibrin↗
PubMed1988

Clots of beta-fibrin. Viscoelastic properties, temperature dependence of elasticity, and interaction with fibrinogen-binding tetrapeptides.

Clots of human beta-fibrin, in which only (or predominantly) the B fibrinopeptide is released, were formed at 14 degrees C by copperhead venom procoagulant enzyme (CVE or venzyme), at pH 8.5, ionic strength 0.45. The shear modulus of elasticity increased slowly and after several days attained a constant value, which was lower than those of alpha-fibrin or alpha beta-fibrin under the same conditions. Before studying the temperature dependence of elasticity, the CVE was then inhibited by introducing phenyl methyl sulfonyl chloride (PMSF) by diffusion. With increasing temperature, the modulus decreased progressively from 5 degrees C to nearly zero at 35 degrees and was essentially reversible with temperature change; recovery of elasticity after change from 34.5 degrees to 14 degrees required approximately 2 d but was considerably faster than the initial buildup of elasticity by CVE at 14 degrees. Creep and creep recovery measurements on unligated clots showed creep rates and irrecoverable deformation that were similar in magnitude to those of alpha-fibrin clots formed with batroxobin and much larger than those of alpha beta-fibrin clots formed with thrombin, under the same conditions. During creep and creep recovery, the differential modulus or compliance remained constant, showing that there was no permanent structural damage, and if network strands are severed in slow flow, they must rejoin in new configurations. Introduction (by diffusion) of the tetrapeptides Gly-His-Arg-Pro (GHRP) and Gly-Pro-Arg-Pro (GPRP), which resemble the B and A binding sites on the E domain of fibrin respectively, reduced the shear modulus and increased the creep rate of beta-fibrin clots to an extent similar to the effect of GPRP on alpha beta-fibrin, much more than that of GHRP on alpha beta-fibrin, but much less than that of GPRP on a-fibrin. A ligated beta-fibrin clot formed with Factor XIIIa (in which the activating thrombin had been neutralized by hirudin) showed essentially perfect elastic behavior, with no creep and with complete recovery after removal of stress, and was inert to GHRP.

Blood Coagulation↗
PubMed2001

Purification and characterization of calobin II, a second type of thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).

In our previous report, we purified and cloned the gene of a thrombin-like enzyme, calobin, from the venom of Agkistrodon caliginosus (Hahn, B.S., Yang, K.Y., Park, E.M., Chang, I.M., Kim, Y. S., 1996. Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper). J. Biochem. 119, 835-843.). During the purification of calobin, a second type of thrombin-like protease was found and it was purified using Affi-Gel Blue and Mono-S cation-exchange chromatography. It was identified as a serine protease with a molecular weight of 41, 000 on SDS-PAGE and its isoelectric point was determined to be 7.4 by isoelectric focusing. It showed little azocaseinolytic and fibrinolytic activity. However, this enzyme acted on fibrinogen to form fibrin with a specific activity of 7,587 NIH equivalent units and also exhibited arginine esterase activity. Amino acid sequencing of the N-terminal region established a primary structure composed of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Arg-Phe-Leu-Val-Ala-X -Tyr. This sequence was entirely consistent with that of calobin and showed a high homology with other thrombin-like enzymes, such as ancrod, batroxobin and gyroxin. Based on the biochemical properties such as molecular weight and isoelectric point, we can demonstrate a second thrombin-like protein showing a high potent clotting activity from the venom of Korean viper.

Agkistrodon↗
PubMed2002

Aprotinin counteracts heparin-induced inhibition of platelet contractile force.

BACKGROUND: Aprotinin interferes with heparin binding to platelets and decreases blood loss during cardiopulmonary bypass (CPB). Heparin abolishes platelet force during CPB, and the extent of platelet force recovery after protamine administration appears to correlate with blood loss. This study assessed the effect of aprotinin on heparin suppression of platelet force. METHODS: Platelet force was measured using the Hemodyne Hemostasis Analyzer. Clots were formed from platelet-rich plasma (PRP) by the addition of batroxobin and 10 mM CaCl(2). Clotting conditions included pH 7.4, ionic strength 0.15 M, fibrinogen level 1 mg/ml and 75,000 platelets/microl. RESULTS: After 1200 s of clotting, force was reduced from 7110+/-1190 to 450+/-450 dyn by 0.2 U/ml of heparin. Platelet force in aprotinin [20 microg/ml (140 KIU/ml)] containing PRP was not suppressed by heparin addition (7480+/-2410 dyn). Aprotinin [40 microg/ml (280 KIU/ml)] addition to previously heparinized plasma counteracted heparin force suppression. Aprotinin (40 microg/ml) increased platelet force from 5630 to 11,138+/-562 in PRP devoid of heparin. Aprotinin did not affect thrombin activity, fibrin structure, platelet aggregation or secretion. CONCLUSIONS: Aprotinin counteracts heparin suppression of platelet force and enhances platelet force in the absence of heparin. Aprotinin-heparin-platelet interactions may help explain aprotinin's ability to reduce blood loss during CPB.

Adenosine Triphosphate↗
PubMed1989

Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom.

The amino acid sequence of a protease, protein C activator, from Agkistrodon contortrix contortrix venom was determined. Peptide fragments obtained by chemical or enzymatic cleavage of the S-carboxymethylated protein were purified by gel filtration and reverse-phase high-performance liquid chromatography. The present study demonstrates that protein C activator from A. contortrix contortrix venom is a trypsin-type serine protease that is composed of 231 residues with a molecular weight of 25,095 for the polypeptide portion of the molecule. By analogy to the mammalian serine proteases, the catalytic triad in venom protein C activator consists of His-40, Asp-85, and Ser-177. The protein also contains three N-linked glycosylation sites at Asn-21, Asn-78, and Asn-129. The amino acid sequence of protein C activator exhibits a high degree of sequence identity with other snake venom proteases: 73% with batroxobin, 68% with flavoxobin, and 55% with Russell's viper venom factor V activator.

Amino Acid Sequence↗