Purification and biochemical characterization of atroxase, a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom

Crotalus atrox venom contains a variety of proteases which render fibrinogen incoagulable and solubilize fibrin. One of these proteases was purified by using ion-exchange and gel permeation liquid chromatography. The protease, called atroxase, consists of a single nonglycosylated polypeptide chain with a molecular weight of 23,500 and an isoelectric point of pH 9.6. Amino acid analysis indicates atroxase to contain 206 residues with no sulfhydryl groups. Metal analysis found zinc and potassium at 1 mol/mol of protein, and calcium at 0.3 mol/mol of protein. Proteolytic activity is inhibited by ethylenediaminetetraacetate and alpha 2-macroglobulin. Maximal proteolytic activity occurs at pH 9.0 and 55 degrees C. Proteolytic specificity, using oxidized insulin B chain, is similar to that of several hemorrhagic toxins found within the same venom, yet atroxase shows no hemorrhagic activity and exhibits low lethality when tested on Swiss Webster mice. Atroxase, an A alpha, B beta fibrinogenase, cleaves the A alpha chain of fibrinogen first followed by the B beta chain and shows no effect on the gamma chain. The nonspecific action of the enzyme results in the extensive hydrolysis of fibrinogen which releases a variety of fibrinopeptides. Fibrin solubilization appears to occur primarily from the hydrolysis of alpha-polymer and unpolymerized alpha and beta chains. Although crude venom induces platelet aggregation, atroxase demonstrated no ability to induce or inhibit aggregation.     

Blood coagulation induced by the venom of Bothrops atrox. 2. Identification, purification, and properties of two factor X activators

We have characterized and purified the two components of the venom of Bothrops atrox that activate the coagulation factor X. Activator 1 and activator 2 were separated by ion-exchange chromatography but otherwise presented similar characteristics. They consist of a heavy polypeptide of Mr 59,000 and either one or two light chains forming a doublet of Mr 14,000-15,000. They are inactive on synthetic substrates and on prothrombin or fibrinogen and thus appear to act specifically on factor X. They are not sensitive to inhibitors of serine proteases or thiol esterases. The activation of factor X is activated by Ca2+ ions with a Hill coefficient of 2.4 and is inhibited by Hg2+, Ba2+, and Cd2+. Its pH dependency suggests that the activity depends on the ionization of a group with an apparent pK of 6.9. We studied the cleavage of purified bovine factor X by B. atrox activators and compared it to that obtained with the factor X activator from Vipera russelli venom. Like the physiological activators, the venom's activators cleave the heavy chain of factor X, producing the activated factor Xa alpha. They produce however two other cleavages: one near the N-terminal end of the heavy chain of factor X, generating factor Xmu, and a second one located at one extremity of the heavy chain of factor Xa alpha, generating factor Xav.     

Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum

A fibrinolytic enzyme was isolated from a marine green alga, Codium divaricatum, and designated C. divaricatum protease (CDP). This protease effectively hydrolyzed fibrinogen A alpha chain, while it had very low hydrolyzing efficiency for B beta and gamma chains. This property was similar to that of alpha-fibrinogenase isolated from snake venom. Protease activity peaked at pH 9, and was completely inhibited by diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), identifying it as a serine protease. Its molecular form was single polypeptide structure and molecular weight was estimated as 31,000 by SDS-PAGE. Fibrinogen clotting enzyme was also identified in a fraction by ion-exchange chromatography. Analysis of clots formed by the enzyme and by thrombin by SDS-PAGE showed that the fibrinogen clotting enzyme would act like thrombin and have high substrate specificity.     

Blood coagulation induced by Bothrops atrox venom: identification and properties of a factor X activator

The coagulating activity of Bothrops atrox venom was investigated in vitro with purified fibrinogen, with normal plasma and with plasma deficient in various factors of the coagulation cascade. This study indicated that Bothrops atrox venom possesses a thrombin-like action caused by one or several serine proteases sensitive to DFP treatment, but that its clotting action is in fact mainly due to components insensitive to DFP which activate prothrombin and factor X (Stuart factor). We partially purified the DFP insensitive activator of factor X from Bothrops atrox venom and found it to be a protein of molecular weight 77,000. Analysis of the purified fraction by electrophoresis on polyacrylamide gel in the presence of SDS showed that it is mainly composed of one heavy polypeptide chain (65,000) and one light doublet (12 - 13,000). This activator is calcium-dependent and catalyzes in vitro the conversion of purified factor X into factor X alpha. By its molecular properties, it resembles the factor X activator from Vipera russellii venom rather than physiological activators.     

Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.     

Effects of starvation and different culture conditions on the phospholipid content of isolated pancreatic islets

Three forms of the normal human plasma fibrinogen gamma-chain which differ in molecular weight have been purified. Plasma fibrinogen was separated by ion exchange chromatography on DEAE-Sephacel into three populations of molecules, each with a unique gamma-chain composition. Following reduction and S-carboxymethylation, the fibrinogen polypeptide chains in each chromatographic peak were separated by ion exchange chromatography on DEAE-Sephacel and identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A alpha, B beta and smallest gamma-chain (gamma 50) eluted at progressively higher ionic strengths, but the elution positions of A alpha, B beta and gamma 50 chains were identical for fibrinogen from each of the three different chromatographic fractions. The unique gamma chain of fibrinogen in the second chromatographic peak (gamma 55) eluted at an ionic strength higher than that of the gamma 50 chain, while the largest gamma-chain (gamma 57.5), which was contained only in the third chromatographic peak of fibrinogen, eluted at the highest ionic strength. The higher ionic strengths needed to elute fibrinogen in the second and third peaks was paralleled by the higher ionic strengths needed to elute the gamma-chains unique to them, suggesting that the gamma-chain composition of the three fibrinogen fractions accounted for their differential binding to the ion exchange resin. Following desialation with neuraminidase, the differences in electrophoretic mobilities between the three gamma-chain forms was maintained, indicating that differential migration on SDS-polyacrylamide gel electrophoresis was not due to variation in sialic acid content.     

Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the A alpha and gamma chains of frog fibrinogen are each coded by a single mRNA species. The A alpha mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the A alpha precursor polypeptide. The gamma chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the B beta subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger B beta mRNA is ca, fivefold more abundant that the smaller species.     

Conservation of human fibrinogen conformation after cleavage of the B beta chain NH2 terminus

Human fibrinogen exposed to protease III from Crotalus atrox venom is cleaved near the NH2 terminus of the B beta chain yielding a species of Mr 325,000 (Fg325) with impaired thrombin clottability. The derivative was compared with intact fibrinogen in a number of ways to determine whether the functional defect resulted from a conformational change or from the loss of a polymerization site. NH2-terminal amino acid sequencing of isolated A alpha, B beta, and gamma chains showed that Fg325 contained intact A alpha and gamma chains, but differed from fibrinogen by the absence of the first 42 residues of the B beta chain. Fibrinopeptide A was present and was cleaved at the same rate in both fibrinogen and Fg325. The rate and extent of A alpha and gamma cross-linking by factor XIIIa was also indistinguishable. In contrast, the thrombin-catalyzed coagulation of Fg325 was 46% less in extent and 180-fold slower than observed for intact fibrinogen. A conformational comparison of Fg325 and fibrinogen was made using immunochemical and spectroscopic approaches. Antisera specific for different regions of the fibrinogen molecule were used to characterize the epitopes in Fg325. The only significant differences were found in the NH2-terminal region of the B beta chain, probed with antiserum to B beta 1-118. The conformational similarity of Fg325 and fibrinogen was confirmed by the identity of both near and far UV CD spectra of the two proteins. Structural, functional, and immunochemical results imply that cleavage of 42 NH2-terminal residues from the B beta chain is not accompanied by a measurable conformational change. The residues of this B beta chain segment, which are evidently located on the surface of the molecule, in conjunction with the NH2-terminal part of the A alpha chain appear to play an important role in the expression of a fibrin polymerization site.     

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.     

Separation and characterization of two polypeptide chains from the 7S cross-linking domain of basement-membrane (type IV) collagen.

The long-form 7S domain of human placental type IV collagen was prepared and after reduction, denaturation and aminoethylation, was separated into its subunits. The monomer subunit was further separated into two polypeptide chains of Mr about 25 000. From compositional data and CNBr peptide patterns it was shown that the two chains were different. Furthermore, all subunits contained both chains, thus supporting a proposed subunit structure for the 7S domain and a chain composition [alpha 1(IV)]2 alpha 2(IV) for the type IV molecule.     

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.     

Studies on the assembly and secretion of fibrinogen.

cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.     

Separation of both the Bbeta- and the gamma-polypeptide chains of human fibrinogen into two main types which differ in sialic acid content

The gamma- and Bbeta-polypeptide chains of purified human fibrinogen have each been resolved into two major species: gammaL and gammaR and BbetaL and BbetaR. These molecular variants, separable on CM-cellulose, differ from each other in sialic acid content: approximately 2 residues of sialic acid per molecule of polypeptide chain for the L species to 1 residue of sialic acid per molecule for the R species. The two types of each polypeptide are demonstrable in preparations of fibrinogen from single donors as well as in pooled fibrinogen. The L and R forms of the gamma chains or the Bbeta chains do not differ in their electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting that they are similar in molecular weight. They are also indistinguishable in polyacrylamide gels in the presence of urea at pH 2.7. Maps of ninhydrin-positive tryptic peptides of the L and R forms of the gamma chain displayed differences within a small group of peptides which have been shown to contain the sialic acid residues present in the gamma-polypeptides. No differences between the peptide maps of BbetaL and BbetaR chains were obvious. A larger ratio of L/R in the gamma and Bbeta chains of dysfibrinogenemia fibrinogen "Zürich II" than in those of normal fibrinogen explains the higher content of sialic acid measured in the native Zürich II fibrinogen molecule.     

The size of human factor VIII heterodimers and the effects produced by thrombin

The heterodimeric structure of factor VIII was demonstrated by two approaches. First, the native molecular weights of several partially purified fractions of factor VIII were determined by measurement of Stokes radii and sedimentation coefficients to be approx. 237 500, 201 000 and 141 000. These measured molecular weights correlated with those derived from polypeptide chain composition, in which each molecule would consist of a doublet polypeptide of Mr 83 000/81 000 plus one predominant high-Mr polypeptide of either 146 000, 120 000 or 93 000. In addition, immunoadsorption using a monoclonal antibody specific for the light-chain doublet removed all of the heavy chains. Separation of the heavy chains from the light chain by EDTA further illustrated the non-covalent nature of the heterodimers. All forms had coagulant activity which was potentiated 13-15-fold by an equimolar amount of human alpha-thrombin. Thrombin converted the Mr 83 000/81 000 doublet to one of Mr 73 000/71 000, and cleaved the largest polypeptides to a transient intermediate form of Mr 93 000 which was further cleaved to polypeptides of Mr 51 000 and 43 000. Potentiation of coagulant activity was correlated with proteolytic cleavage of either or both the doublet and the Mr 93 000 polypeptides. These data indicate that human factor VIII purified from plasma consists of a group of heterodimers, composed of a light chain of Mr 83 000 (81 000) and a heavy chain which varies in size between Mr 170 000 and 93 000, each form of which is similarly potentiated and cleaved by thrombin.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.     

Multiple proteases from Streptomyces moderatus: I. Isolation and purification of five extracellular proteases

The presence of multiple proteases in the culture filtrate of Streptomyces moderatus was detected. After preliminary purification by ammonium sulfate precipitation and decolorization using DEAE-cellulose, the fractionation of various proteases was carried out using CM-trisacryl cation-exchange chromatography. By this procedure, four different protease fractions (Fr.) were separated (Fr. I, II, III, and IV). The first fraction was further separated into two different proteolytically active fractions (Fr. Ia and Fr. Ib) by DEAE-trisacryl anion-exchange chromatography. Fraction Ia was purified further by affinity chromatography on N-carbobenzoxy-d-phenylalanyl triethylenetetramine-Sepharose 4B. The second fraction (Fr. Ib) was purified by gel filtration on Ultrogel AcA 44. For the purification of the other protease fractions (Fr. II, III, and IV) single-step affinity chromatography methods were employed. Protease fractions II and III were purified by ϵ-aminocaproyl-4-(4-aminophenylazo)phenylarsonic acid Sepharose 4B and protease fraction IV was purified on ϵ-aminocaproyl trialanine-Sepharose 4B. All five proteases purified were found to be apparently homogeneous by gel electrophoretic methods.     

Characterization of a protein C activator from Agkistrodon contortrix contortrix venom

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.     

A novel P-I class metalloproteinase with broad substrate-cleaving activity, agkislysin, from Agkistrodon acutus venom

The venom of Viperdae is a rich source of metalloproteinases, which have potential clinical applications for lowering plasma fibrinogen or dissolving thrombi. Recently, we purified a novel proteinase from Formosan Agkistrodon acutus venom using DEAE-Sephadex A-50 and Mono-Q HR 5/5 column chromatography. The purified getatinolytic component, agkislysin, is a 22kDa-monomeric protein without Asn-linked sugar. Functional characterization showed that agkislysin possessed both fibronectin- and type IV collagen-cleaving activities. In addition, agkislysin preferentially cleaved the Aalpha chain of fibrinogen, followed by the Bbeta chain, while the gamma chain was finally affected. Furthermore, agkislysin was also capable of cleaving prothrombin into various fragments, as well as suppressing ristocetin-induced platelet aggregation by hydrolyzing von Willebrand factor. However, the proteolytic activity of agkislysin was slightly enhanced by divalent metal ions and completely inhibited by chelating agents, but not serine proteinase inhibitor. These findings suggest that agkislysin is a P-I class metalloproteinase with unique biological properties.     

Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom.

By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.