Identification of molecular sites on factor VII which mediate its assembly and function in the extrinsic pathway activation complex

Factor VII-VIIa, in association with tissue factor, participates in the complex which initiates blood coagulation through the extrinsic pathway. To identify functional domains on factor VII which mediate the activation of factor X, 16 synthetic peptides corresponding to 55% of the primary structure were assayed for their ability to inhibit factor VII function. Factor Xa formation was inhibited by eight of the peptides in a dose-dependent manner. Kinetic analyses indicated noncompetitive inhibition of factor X activation by seven of these peptides. Peptide-(347-361) inhibited factor Xa cleavage of a chromogenic substrate by a competitive mechanism and was excluded from further analysis in this study. Among the seven inhibitory peptides which have the ability to prevent the factor VIIa-tissue factor-mediated conversion of factor X to factor Xa, peptide-(285-305) was most inhibitory, with a Ki value of 2.4 microM. The Ki values were in the range of 42-65 microM for peptides-(44-50), -(194-214), -(208-229), and -(376-390). The least inhibitory peptides were at positions 170-178 and 330-340, with a Ki value greater than 200 microM. Polyclonal antibodies were raised against four of these peptides; and when antisera were assayed by a solid-phase radioimmunoassay, they bound not only to their respective immunizing peptides, but also to factor VII. The Fab fragments of specific IgG preparations, affinity-purified on a factor VII-agarose column, inhibited the rate of factor X activation in a dose-dependent manner. Six of the seven inhibitory peptides represent amino acid sequences within the heavy chain of factor VII, and the remaining one corresponds to a sequence within the light chain. The corresponding regions in the x-ray crystal structure of chymotrypsin represented by the six heavy chain inhibitory peptides are found to be located in three distinct regions, one region located spatially distal to the active site and the other two regions located relatively closer to the active site and the substrate-binding pocket. The results suggest that at least three specific regions in the heavy chain and one region in the light chain of factor VII mediate its interaction with the factor X activation complex.     

Molecular recognition sites on factor Xa which participate in the prothrombinase complex.

Coagulation factor X, when activated to factor Xa by proteolytic cleavage, itself becomes an active serine protease which participates as a component of the macromolecular prothrombinase complex along with factor Va, phospholipid, and calcium ions. To identify specific structural regions on factor Xa responsible for mediating its function in activating prothrombin, we used 21 synthetic peptides corresponding to 65% of the primary structure of factor X as potential inhibitors of prothrombin activation. Using purified components, thrombin formation was inhibited by seven peptides in a dose-dependent noncompetitive manner. Antibodies to selected inhibitory peptides affinity purified on a factor Xa-agarose column inhibited thrombin formation in a dose-dependent manner, indicating that the corresponding regions on factor Xa are surface-exposed. Kinetic analyses varying the order of reagent addition suggested that peptides 211-222, 254-269, and 263-274 were highly effective in preventing the factor Xa-factor Va interaction. Peptides 275-287 and 415-425 were considered to derive from a distal region involved in substrate binding, based upon mixed inhibition kinetic analyses and assuming that inhibitory peptides not inhibitory in factor Va binding are related to a specific region of substrate interaction. Cross-linking studies confirmed that peptides 263-274 and 263-276 could bind specifically to the light chain of factor V/Va. These findings provide the basis for further pursuing the precise definition of interactive sites on factor Xa using site-directed mutagenesis and molecular modeling.     

Degradation of coagulation proteins by an enzyme from Malayan pit viper (Akistrodon rhodostoma) venom

Three hydrolases from the crude venom of the Malayan pit viper (Akistrodon rhodostoma) can be differentiated. The first, which we designate ARH alpha, is the well-known fibrinogenolytic enzyme ancrod. The second, ARH beta, which has not been described previously, is identified by its electrophoretic mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by its ability to hydrolyze H-D-phenylalanyl-L-piperyl-L-arginyl-rho-nitroanilide, and by inhibition of its activity by diisopropyl phosphorofluoridate. The third, ARH gamma, also previously not described, has been purified by using gel permeation and ion-exchange chromatography and preparative PAGE. Chemical, electrophoretic, and hydrodynamic data indicate that it is a single-chain, nonglobular glycoprotein with a molecular weight of 25,600. ARH gamma catalyzes the degradation of several plasma vitamin K dependent coagulation factors, including factor IX, factor X, prothrombin, and protein C. The products are electrophoretically similar to factor IXa beta, factor Xa, thrombin, and activated protein C, respectively. However, these products contain little or no enzymatic activity. ARH gamma-degraded factor IX, factor X, prothrombin, and protein C can be subsequently activated by factor XIa, Russell's viper venom X coagulant protein, crude taipan snake venom, and thrombin, respectively. The N-terminal sequence of the peptides resulting from the ARH gamma digest of porcine factor IX shows that at least three bonds are hydrolyzed: (1) at position 152, seven residues from the Arg145-Ala146 factor XIa cleavage site; (2) at position 167 within the factor IX activation peptide; and (3) at position 177, three residues from the Arg180-Val181 factor XIa cleavage site. The degradation of factor IX by ARH gamma is not affected by several serine protease inhibitors. ARH gamma catalyzes the degradation of both the heavy and light chains of porcine factor VIII which results in the inability of thrombin to activate factor VIII. ARH gamma also catalyzes the degradation of porcine antithrombin III which abolishes its ability to inhibit thrombin. These findings may have relevance to studies of hemostatic derangements following envenomation by this snake. Additionally, several novel coagulation factor derivatives have been generated for structure-function studies.     

Coagulation of plasma from the chicken (Gallus domesticus): phospholipids influence clotting rates induced by components from Russell's viper venom

Added phospholipid failed to accelerate chicken-plasma coagulation, induced by high concentrations of crude Russell's viper venom; however, similarly induced coagulation of canine and human plasma proceeded more rapidly when phospholipid was added. Phospholipid reduced clotting times of canine, human and also chicken plasma when partially purified factor X-activating enzyme from Russell's viper venom was the inducing agent. In the absence of added phospholipid, preincubation of chicken plasma with factor V-activating enzyme from Russell's viper venom accelerated factor X-activating-enzyme-induced coagulation. Preincubation of chicken plasma with the factor V-activating enzyme slowed factor X-activating-enzyme-induced coagulation in the presence of added phospholipid.     

A novel heparin-dependent inhibitor of activated protein C that potentiates consumptive coagulopathy in Russell's viper envenomation

The activation of coagulation factors V and X by Russell's viper venom (RVV) has been implicated in the development of consumptive coagulopathies in severely envenomed patients. However, factor Va is prone to inactivation by activated protein C (APC), an important serine protease that negatively regulates blood coagulation. It is therefore hypothesized that APC may be down-regulated by some of the venom components. In this study, we managed to isolate a potent Kunitz-type APC inhibitor, named DrKIn-I. Using chromogenic substrate, DrKIn-I dose-dependently inhibited the activity of APC. Heparin potentiated the inhibition and reduced the IC(50) of DrKIn-I by 25-fold. DrKIn-I, together with heparin, also protected factor Va from APC-mediated inactivation. Using surface plasmon resonance, DrKIn-I exhibited fast binding kinetics with APC (association rate constant = 1.7 × 10(7) M(-1) s(-1)). Direct binding assays and kinetic studies revealed that this inhibition (K(i) = 53 pM) is due to the tight binding interactions of DrKIn-I with both heparin and APC. DrKIn-I also effectively reversed the anticoagulant activity of APC and completely restored the thrombin generation in APC-containing plasma. Furthermore, although the injection of either DrKIn-I or RVV-X (the venom factor X-activator) into ICR mice did not significantly deplete the plasma fibrinogen concentration, co-administration of DrKIn-I with RVV-X resulted in complete fibrinogen consumption and the deposition of fibrin thrombi in the glomerular capillaries. Our results provide new insights into the pathogenesis of RVV-induced coagulopathies and indicate that DrKIn-I is a novel APC inhibitor that is associated with potentially fatal thrombotic complications in Russell's viper envenomation.     

Interactions and inhibition of blood coagulation factor Va involving residues 311-325 of activated protein C.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide-(311-325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa-1-stage coagulation assays in normal and in protein S-depleted plasma with 50% inhibition at 13 microM peptide. In a system using purified clotting factors, peptide-(311-325) inhibited APC-catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 microM peptide. However, peptide-(311-325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]alpha 1-antitrypsin. Peptide-(311-325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 microM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose-dependent manner to immobilized peptide-(311-325). Peptide-(311-315) inhibited the binding of factor Va to immobilized APC or factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)     

New insights into the functions and N-glycan structures of factor X activator from Russell's viper venom

The coagulation factor X activator from Russell's viper venom (RVV-X) is a heterotrimeric glycoprotein. In this study, its three subunits were cloned and sequenced from the venom gland cDNAs of Daboia siamensis. The deduced heavy chain sequence contained a C-terminal extension with four additional residues to that published previously. Both light chains showed 77-81% identity to those of a homologous factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X could strongly bind protein S, in addition to factors X and IX. This might inactivate protein S and potentiate the disseminated intravascular coagulation syndrome elicited by Russell's viper envenomation. The N-glycans released from each subunit were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans, one on each light chain and four on the heavy chain, showed a heterogeneous pattern, with a combination of variable terminal fucosylation and sialylation on multiantennary complex-type sugars. Amongst the notable features were the presence of terminal Lewis and sialyl-Lewis epitopes, as confirmed by western blotting analyses. As these glyco-epitopes have specific receptors in the vascular system, they possibly contribute to the rapid homing of RVV-X to the vascular system, as supported by the observation that slower and fewer fibrinogen degradation products are released by desialylated RVV-X than by native RVV-X.     

Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain.

Human coagulation factor V is an integral component of the prothrombinase complex. Rapid activation of prothrombin is dependent on the interactions of this nonenzymatic cofactor with factor Xa and prothrombin in the presence of calcium ions and a phospholipid or platelet surface. Factor V is similar structurally and functionally to the homologous cofactor, factor VIII, which interacts with factor IXa to accelerate factor X activation in the presence of calcium and phospholipids. Both of these cofactors, when activated, possess homologous heavy and light chains. Binding to anionic phospholipids is mediated by the light chains of these two cofactors. In bovine factor Va, a phosphatidylserine-specific binding site has been localized to the amino-terminal A3 domain of the light chain. In human factor VIII, on the other hand, a region within the carboxyl-terminal C2 domain of the light chain has been shown to interact with anionic phospholipids. We have constructed a series of recombinant deletion mutants lacking domain-size fragments of the light chain of human factor V (rHFV). These mutants are expressed and secreted as single-chain proteins by COS cells. Thrombin and the factor V activator from Russell's viper venom process these deletion mutants as expected. The light chain deletion mutants possess essentially no procoagulant activity, nor are they activated by treatment with factor V activator from Russell's viper venom. Deletion of the second C-type domain results in essentially complete loss of phosphatidylserine-specific binding whereas the presence of the C2 domain alone (rHFV des-A3C1, which lacks the A3 and C1 domains of the light chain) results in significant phosphatidylserine-specific binding. The presence of the A3 domain alone (rHFV des-C1C2) does not mediate binding to immobilized phosphatidylserine. Increasing calcium ion concentrations result in decreased binding of recombinant human factor V and the mutant rHFV des-A3C1 to phosphatidylserine, similar to previous studies with purified plasma factor V and phospholipid vesicles. These results indicate that human factor V, similar to human factor VIII, possesses a phosphatidylserine-specific binding site within the C2 domain of the light chain.     

Expression of human factor X with normal biological activity in human embryonic kidney cells

Summary Human embryonic kidney cells (293) were transfected with a construct containing human factor X cDNA and selected for G418 resistance. The level of expression of recombinant factor X in serum-free medium was 4 to 5 g/ml. Purified recombinant factor X had a molecular size identical to that of normal plasma factor X. Amino-terminal sequencing revealed normal processing cleavages. The -carboxy Glu and -OH Asp content of the recombinant factor X was close to 90% of the expected levels of these post-translational residues. The specific activity of recombinant factor X was about 95% of that of plasma factor X in three plasma-based clotting assays. This report demonstrates that 293 cells can produce a high level of biologically active factor X and describes a visual criterion for verifying the transfection process.Abbreviations FX factor X - rFX recombinant factor X - DMEM Dulbecco's modified Eagle's medium - RVV-X Russell's viper venom - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - Gla -carboxy glutamic acid     

Identification of anti-TNFalpha peptides with consensus sequence

Phage displayed peptide library was used to select tumor necrosis factor alpha (TNFalpha) binding peptides. After three sequential rounds of biopanning, some linear TNFalpha-binding peptides were identified from a 12-mer peptide library. A consensus sequence (L/M)HEL(Y/F)(L/M)X(W/Y/F), where X might be variable residue, was deduced from sequences of these peptides. The phages bearing these peptides showed specific binding to immobilized TNFalpha, with over 80% of phages bound being competitively eluted by free TNFalpha. To confirm the binding activity and to explore further functional properties, three peptides with typical structure were selected and expressed as GST-fused protein. These recombinant peptides effectively competed for [125I]TNFalpha binding to TNFR1 in a dose-dependent manner, with IC(50) from 10 to 160 microM. Furthermore, the GST-fused derivatives showed inhibitory effects on TNFalpha-induced cytotoxicity. Taken together, these data demonstrate that the TNFalpha-binding peptides are effective antagonists of TNFalpha and the deduced motif might be useful in development of novel low molecular weight anti-TNFalpha drugs.     

Intrinsic pathway activation of factor X and its activation peptide-deficient derivative, factor Xdes-143-191.

The role of the activation peptide in determining the substrate specificity of intrinsic pathway factor X (fX) activation was studied by using a novel derivative of fX in which 49 residues were removed enzymatically from the NH2 terminus of the 52-residue activation peptide by an enzyme from the venom of the snake Agkistrodon rhodostoma. The modified protein, designated fXdes-143-191, is inactive but is activated to alpha-fXa by either the intrinsic fX activation complex (intrinsic fXase) composed of factor IXa beta, thrombin-activated factor VIII (fVIIIaIIa), and phospholipid vesicles or by the fX coagulant protein from Russell's viper venom (RVV-XCP). Both the Km and kcat for the activation of fX by RVV-XCP were greater than for fXdes-143-191, resulting in less than a 2-fold difference in the catalytic efficiency (kcat/Km) suggestive of nonproductive binding of fXdes-143-191 to RVV-XCP. The activation of each substrate by intrinsic fXase revealed that the kcat was 100-fold greater for fX than fXdes-143-191 (16 and 0.16 s-1, respectively), although there was no detectable difference in Km (60 and 80 nM, respectively). Activations by fIXa beta/phospholipid in the absence of fVIIIaIIa also revealed a difference in kcat but not Km, but the difference in kcat was smaller (kcat of 0.007 and 0.002 s-1 and Km of 220 and 170 nM for fX and fXdes-143-191, respectively). Analysis of product versus time curves demonstrated that fVIIIaIIa promotes formation of the actyl-enzyme intermediate during fX activation. We conclude that the activation peptide plays a critical role during acyl-enzyme formation that is most pronounced in the presence of fVIIIaIIa. The absence of Km differences suggests that residues NH2-terminal to P3 do not contribute to the initial formation of the enzyme-substrate complex.     

Analysis of the generation and inhibition of activated coagulation factor X in pure systems and in human plasma

The overall generation and inhibition of human factor Xa have been studied in pure systems and plasma to determine the kinetic characteristics of inhibition during factor Xa generation. Generation curves were measured amidolytically in a pure system containing factor X and antithrombin, which was activated with the factor X-activating enzyme of Russell's viper venom (RVV-X). The measured change in factor Xa level with time was fitted to a 3-parameter 2-exponential model to determine apparent first-order rates of inhibition. With antithrombin at 4.5 microM, the inhibition rate constant thus obtained was very close to the known rate of inhibition of exogenous enzyme. Factor Xa generation curves were also analyzed in plasma; however, to reduce interference in the assay of thrombin, congenitally prothrombin-deficient plasma was used containing 0.5 microM D-Phe-Pro-Arg-chloromethylketone. In plasma, factor Xa generated in the presence of phospholipid and Ca2+ ions by RVV-X, factor IXa, or tissue factor was inhibited more slowly than exogenous enzyme. The reduction was particularly severe with tissue factor activation, where the rate was 0.04-0.06 min-1. This protection by tissue factor was also observed in pure systems and apparently required factor VII.     

The use of acetylated factor X to prevent feedback activation of factor VIII during factor X activation: a tool for kinetic studies

The modification of human factor X by 2-sulfo-N-succinimidyl acetate was investigated and shown to produce a factor X species which, when activated, has no activity toward factor VIII. Acylation of factor X (0.9 microM) was carried out in the presence of 1 mM calcium at different reagent concentrations and pH values at 22 degrees C for time courses up to 1 h. Optimal modification was achieved using 0.3 mM reagent at pH 8.0 for 30 min. The modified zymogen, acetylated factor X, is activated at full rates by factor IXa/VIIIa and by the factor X-activating protein of Russell's viper venom. The activated product, acetylated Xa, has an enhanced amidolytic activity (110%) but has almost no detectable clotting activity (0.1%). More importantly, we have shown that acetylated Xa, in contrast to native Xa, does not activate factor VIII. This allows accurate quantitation of factor VIII activation without complications due to positive feedback reactions. We have demonstrated this in an examination of the activation of factor VIII by factor IXa.     

Intracellular receptors for activated protein kinase C. Identification of a binding site for the enzyme.

Protein kinase C (PKC) isozymes comprise a family of cytosolic enzymes that translocate to different intracellular sites on activation. We have recently characterized at least two intracellular receptor proteins for PKC (termed RACKs for receptors for activated C-kinase) in the Triton-insoluble material of the particulate fraction from neonatal rat heart. Here, we identify a sequence that appears to resemble the PKC binding site on these RACKs. A peptide (peptide I) with the sequence KGDYEKILVALCGGN bound PKC, and binding was markedly increased in the presence of PKC activators. Furthermore, peptide I inhibited PKC binding to RACKs in a dose-dependent manner. These data suggest that these RACKs have a common PKC binding sequence. Since peptide I inhibited PKC binding to RACKs in vitro, it may be a useful tool to inhibit PKC translocation and subsequent function in vivo.     

Allosteric changes in thrombin's activity produced by peptides corresponding to segments of natural inhibitors and substrates.

Acidic synthetic peptides corresponding to segments of several nonhomologous proteins (hirudin, residues 54-65; heparin cofactor II, residues 54-75; and fibrinogen, residues 410-427 of the gamma B-chain) inhibit thrombin's cleavage of fibrinogen without blocking the enzyme's active site. Here, we examined effects of these peptides on thrombin's cleavage of protein C and small peptides. Activation of protein C by thrombin in the absence of calcium was inhibited by all of the peptides. Maximal inhibition was 60%, and no greater inhibition was produced by higher peptide concentrations. This differed from progressive inhibition of protein C activation by increasing peptide concentrations in the presence of thrombomodulin and calcium. Potencies of the peptides were in the order hirudin-(54-65) greater than heparin cofactor II-(54-75) greater than gamma B-chain-(410-427). Sulfation of the tyrosine residue in hirudin-(54-65) increased its potency about 10-fold, similar to changes in anticlotting activity. The peptides were activators rather than inhibitors of the cleavage of small chromogenic substrates. In the presence of the peptides, the affinity of thrombin for the substrates S-2366 (pyro-Glu-Pro-Arg-4-nitroanilide), Chromozyme TH (tosyl-Gly-Pro-Arg-4-nitroanilide), and S-2251 (D-Val-Leu-Lys-4-nitroanilide) increased 1.5-2-fold with little change in the Vmax of substrate cleavage. Potencies of peptides in these allosteric effects on thrombin was in the same order as for their other effects. The similar actions of these nonhomologous peptides, which are believed to bind to thrombin's anion-binding exosite, suggest that binding of any peptide to this site exerts the same allosteric effect on thrombin's active site. Interactions of these peptides with thrombin may serve as models for regulation of thrombin's interactions with natural substrates and inhibitors.     

A family with heterozygous factor X Friuli defect outside Friuli

Three members of the same family were found to have a clotting defect consistent with the diagnosis of heterozygous factor X Friuli disorder. The main features of the defect were a mild prolongation of prothrombin time and partial thromboplastin time, but a normal Stypven-Cephalin clotting time. Factor X activity was 40-50% of normal using tissue thromboplastin, but was perfectly normal using Russell's viper venom and cephalin. Using chromogenic substrate S-2222 the level was 30% of normal. Immunologically, factor X was normal. Bleeding manifestations were mild if any. The hereditary pattern was autosomal. The family comes from an area far away from Friuli and represents the first example of factor X Friuli discovered outside the Friuli.     

Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa

Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.     

Dendroaspis natriuretic peptide and its functions in pig ovarian granulosa cells

Dendroaspis natriuretic peptide (DNP), a 38-amino acid peptide, was isolated from the venom of green mamba. It has structural and functional similarities to the other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP is present in pig ovarian granulosa cells and to define its biological functions. The serial dilution curves of extracts of granulosa cells and follicular fluid were parallel to the standard curve of DNP, and a major peak of molecular profile of both extracts by HPLC was synthetic DNP. The concentration of DNP was 7.51+/-1.46 pg/10(7) cells and 24.81+/-2.38 pg/ml in granulosa cells and follicular fluid, respectively. Natriuretic peptides increased cGMP production in the purified membrane of granulosa cells with a rank order of potency of C-type natriuretic peptide (CNP)>atrial natriuretic peptide (ANP)=DNP. mRNAs for natriuretic peptide receptor-A (NPR-A), NPR-B and NPR-C were detected by RT-PCR. The binding site of (125)I-DNP was also observed in granulosa cell layer by in vitro autoradiography. Synthetic DNP inhibited the secretion of ANP from granulosa cells in a concentration-dependent manner and the potency was similar to CNP. The concentration of DNP and CNP, which inhibited the secretion of ANP by 50%, was about 1 nM. Increases in production of cGMP in granulosa cells were observed by DNP or CNP. Therefore, these results show the existence of DNP system and the cross-talk between natriuretic peptides in pig ovarian granulosa cells.     

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.