Crystal structure of RVV-X: an example of evolutionary gain of specificity by ADAM proteinases

Russell's viper venom factor X activator (RVV-X) is a heterotrimeric metalloproteinase with a mammalian ADAM-like heavy chain and two lectin-like light chains. The crystal structure of RVV-X has been determined at 2.9 A resolution and shows a hook-spanner-wrench-like architecture, in which the metalloproteinase/disintegrin region constitutes a hook, and the lectin-like domains constitute a handle. A 6.5nm separation between the catalytic site and a putative exosite suggests a docking model for factor X. The structure provides a typical example of the molecular evolution of multi-subunit proteins and insights into the molecular basis of target recognition and proteolysis by ADAM/adamalysin/reprolysin proteinases.     

Allosteric aptamers controlling a signal amplification cascade allow visual detection of molecules at picomolar concentrations

A broadly applicable homogeneous detection system has been developed. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition (precipitation) of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF(165)) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF(165), reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus, triggering the cascade and enabling the phase transition. As few as 5 fmol of VEGF(165) could be detected by the naked eye within an hour. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout and can be modified to detect molecules to which aptamers can be obtained.     

Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains.

We determined the complete amino acid sequence of RVV-X, the blood coagulation factor X activating enzyme, isolated from Russell's viper venom and studied structure-function relationships. RVV-X (M(r) 79,000) consists of a disulfide-bonded two-chain glycoprotein with a heavy chain of M(r) 59,000 and a light chain of heterogeneous M(r) 18,000 (LC1) and 21,000 (LC2). These chains were separated after reduction and S-pyridylethylation, and the isolated major component LC1 was used for sequence analysis. The heavy chain consists of 427 residues containing four asparagine-linked oligosaccharides, and its entire sequence was similar to that of the high molecular mass hemorrhagic protein, HR1B, isolated from the venom of Trimere-surus flavoviridis. The heavy chain contains three distinct domains, metalloproteinase, disintegrin (platelet aggregation inhibitor)-like and unknown cysteine-rich domains. On the other hand, light chain LC1 consists of 123 amino acid residues containing one asparagine-linked oligosaccharide and shows sequence homology similar to that found in the so-called C-type (Ca(2+)-dependent) lectins. Therefore, RVV-X is a novel metalloproteinase containing a mosaic structure with distintegrin-like, cysteine-rich, and C-type lectin-like domains. RVV-X potently inhibits collagen- and ADP-stimulated platelet aggregations, probably via its distintegrin-like domain, although this domain does not contain the Arg-Gly-Asp sequence which is conserved in various venom distintegrins and which is thought to be one of the interaction sites for platelet integrins. Our findings also indicate that snake venom factor IX/factor X-binding protein with a C-type lectin structure (Atoda, H., Hyuga, M., and Morita, T. (1991) J. Biol. Chem. 266, 14903-14911) inhibits RVV-X-catalyzed factor X activation; hence, the light chain of RVV-X probably participates in recognizing some portion of the zymogen factor X.     

Purification and properties of an activating enzyme of blood clotting factor X from the venom of Cerastes cerastes

An activator of blood coagulation factor X was found in the venom of the horned viper Cerastes cerastes, and was purified by gel filtration, ion-exchange chromatography and chromatofocussing. The activator is a protein composed of a heavy and a light polypeptide chain linked by disulfide bonds. Two subforms of the activator were found. Both contained a heavy chain of Mr 58000 and are distinguished from each other by the presence of two different light chains of Mr 17700 and 15000. The activator appears to cleave the bond in the factor X molecule that is also cleaved by factor IXa. Factor X activation by the activator is strongly stimulated by Ca2+. The kinetic parameters for the activation reaction have been determined. A Km for factor X of 19.2 nM and a Vmax of 0.11 pmol of Xa/min per ng venom were found.     

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.     

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.     

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.     

Factor X activator from Vipera lebetina snake venom,molecular characterization and substrate specificity

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6–9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly – factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly – factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg⁵²-Ile⁵³ bond in the heavy chain of human factor X and the Arg²²⁶-Val²²⁷ bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

A sensitive microsphere coagulation ELISA for Escherichia coli O157:H7 using Russell's viper venom

A microsphere coagulation enzyme-linked immunosorbent assay (MC-ELISA) using Russell's viper venom factor X activator (RVV-XA) is described for the detection of Escherichia coli O157:H7. This microtitre plate assay comprises a standard sandwich immunoassay incorporating RVV-XA as the enzyme label. Coagulation substrate together with polystyrene microspheres are added to the wells of the microtitre plate. RVV-XA initiates the coagulation cascade causing formation of an artificial clot of polystyrene microspheres bound together with fibrin. As few as 10(2) E. coli O157 in a well (10(3) per ml) can be detected within 3 h. The assay is two orders of magnitude more sensitive than a standard ELISA and is a generic technique with the potential for widespread use in sandwich immunoassays.     

Molecular recognition in the activation of human blood coagulation factor X

Factor X can be activated by the extrinsic activation complex (factor VIIa:tissue factor), the intrinsic activation complex (factor IXa:factor VIIIa) and by an enzyme from Russell's viper venom (RVV-X). To identify the regions on the surface of factor X that mediate its association with these three activators, we have prepared 21 synthetic peptides representing 65% of the primary structure of factor X. Only 3 of the 21 peptides inhibited the rate of factor X activation, indicating the regions represented by these three peptides are involved in factor X association. Using purified components, the rate of factor Xa formation was inhibited in a dose-dependent manner by these three peptides with the same relative potency of inhibition in each of the activation systems. The observed relative potencies were: peptide 267-283 greater than or equal to peptide 284-303 greater than peptide 417-431. Kinetic analyses indicated that the three peptides inhibited factor X activation in a non-competitive manner, and in mixed inhibitor assays the peptides were shown to be mutually exclusive of one another. In coagulation-based assays, the potency of inhibition by each peptide was decreased. However, in Russell's viper venom-X-initiated assays peptide 417-431 was the best inhibitor. Fab fragments of antibodies raised to these peptides and affinity purified on factor X-agarose columns inhibited both the purified and coagulation-based assays in a dose-dependent manner. Using the x-ray crystal structure of chymotrypsinogen as a model, these three peptides were found to be located spatially close to one another on the surface of factor X and opposite to the region where factor X is cleaved for activation. These data are consistent with a model in which the three activators combine with factor X through a recognition site composed of multiple loci that is distal to the potential cleavage site. This interaction aligns the active sites of these three enzymes in the correct orientation to cleave factor X at the same arginyl-isoleucyl bond.     

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.     

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.     

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.     

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.     

烙铁头蛇毒对体外血凝及纤溶的影响

行体外血凝及纤溶观察结果表明：①烙铁头蛇毒能明显延长凝血活酶时间（ＰＴＴ）、凝血酶原时间（ＰＴ）及蝰蛇毒磷脂时间（ＲＶＶＣＴ）,显示出较强的抗凝活性;②对人纤维蛋白原及纤维蛋白有较强的溶解作用,这种作用不完全是单一的活化素样作用,还有脆浆素样作用     

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.     

Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site

Human blood coagulation factor X has two N-linked oligosaccharides at Asn³⁹ and Asn⁴⁹ residues and two O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues in the region of the factorX activationpeptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn³⁹ and Asn⁴⁹ residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri-and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Ac-26 residue. In addition, the structures of O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues were suggested to be disialylated Gal/3GalNAc sequences by their component analyses.Abbreviations Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose - HPLC high-performance liquid chromatography - NDV Newcastle disease virus - Neu5Ac 5-N-acetylneuraminic acid - ODS octadecylsilyl - PA pyridylamino - RVV-X Russell's viper venom factor X activator - TBS Tris-buffered saline - XAP factor X activation peptide.     

Bovine coagulation factor V visualized with electron microscopy. Ultrastructure of the isolated activated forms and of the activation fragments

Single chain bovine factor V (Mr = 330,000) was isolated and visualized by means of high resolution transmission electron microscopy of negatively stained samples. Both factor Va, activated by thrombin or by the factor V activator from Russell's viper venom, and the isolated fragments, D (Mr = 105,000), C1 (Mr = 150,000), and F1F2 (Mr = 72,000), were studied. Single chain factor V appeared as a multidomain structure with three globular domains of similar size (diameter approximately 80 A), and oriented around a somewhat larger central domain (diameter approximately 140 A). The distance between the center of the molecule and the center of each of the peripheral domains was 120 A and the maximum length of factor V was 300 A. The structure was essentially identical with that recently shown for human single chain factor V (Dahlb?ck, B. (1985) J. Biol. Chem. 260, 1347-1349). Isolated thrombin-activated factor Va (containing fragments D and F1F2) was composed of two domains of similar size, each of which was approximately 80 A in diameter and corresponded in size and shape to the peripheral domains seen in intact factor V. The isolated activation fragment C1 appeared as an irregular structure with an approximate diameter of 140 A and corresponded in size and shape to the larger central domain in intact factor V. The activator from Russell's viper venom only cleaves the bond(s) between C1 and F1F2, which results in two fragments, a larger fragment (Mr = 220,000) bearing the D, E, and C1 region and a smaller one corresponding to the F1F2 fragment. The venom-activated factor Va in the electron microscope demonstrated a multidomain structure similar in size and shape to that obtained with intact factor V. A model for factor V and the molecular events involved in activation is proposed.     

Structural requirements for expression of factor Va activity

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.