Proexosite-1 on prothrombin is a factor Va-dependent recognition site for the prothrombinase complex

Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of prothrombin recognition by factor Xa in the prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54-65) peptide suggested that although the peptide inhibits the prothrombinase activation of the wild type zymogen with a KD of 0.5-0.7 microm, it is ineffective in inhibiting the activation of mutant zymogens (KD = 2-30 microm). These results suggest that basic residues of proexosite-1 on prothrombin are factor Va-dependent recognition sites for factor Xa in the prothrombinase complex.     

Role of basic residues of the autolysis loop in the catalytic function of factor Xa

The autolysis loop of factor Xa (fXa) has four basic residues (Arg(143), Lys(147), Arg(150), and Arg(154)) whose contribution to protease specificity of fXa has not been examined. Here, we substituted these basic residues individually with Ala in the fX cDNA and expressed them in mammalian cells using a novel expression/purification vector system. Following purification to homogeneity and activation by the factor X activator from Russell viper venom, the mutants were characterized with respect to their ability to assemble into the prothrombinase complex to activate prothrombin and interact with target plasma fXa inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin. We show that all mutants interacted with factor Va with normal affinities and exhibited wild-type-like prothrombinase activities toward prothrombin. Lys(147) and Arg(154) mutants were inhibited by TFPI approximately 2-fold slower than wild type; however, both Arg(143) and Arg(150) mutants were inhibited normally by the inhibitor. The reactivities of Arg(143) and Lys(147) mutants were improved approximately 2-fold with antithrombin in the absence but not in the presence of heparin cofactors. On the other hand, the pentasaccharide-catalyzed reactivity of antithrombin with the Arg(150) mutant was impaired by an order of magnitude. These results suggest that Arg(150) of the autolysis loop may specifically interact with the activated conformation of antithrombin.     

Purification and characterization of a prothrombin activator from the venom of the Australian brown snake, Pseudonaja textilis textilis

A simple procedure, involving chromatography on concanavalin A-Sepharose and gel filtration, has been developed for the purification of a prothrombin activator from the venom of the Australian brown snake Pseudonaja textilis textilis. The prothrombin activator, which is a major venom component, is a high molecular weight protein (Mr greater than or equal to 200,000) which yields a number of subunits when examined by SDS-PAGE. It is related antigenically to the venom prothrombin activator of the taipan Oxyuranus scutellatus. P. textilis prothrombin activator is able to coagulate citrated plasma, warfarin plasma, and Factor V- and Factor X-deficient plasmas; to convert purified human prothrombin to thrombin; and to hydrolyse the peptide p-nitroanilide substrate S-2222. Calcium ions and phospholipids had little if any effect on the rates of coagulation of citrated plasma or S-2222 hydrolysis catalysed by this enzyme.     

Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake)

The prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake) was purified by gel filtration on Sephadex G-200 and ion-exchange chromatography on QAE-Sephadex. The activator is a large protein with a molecular weight of approximately 300,000, which is composed of subunits of Mr 110,000 and 80,000 and two disulfide-linked polypeptides of Mr 30,000. One or both of these Mr 30,000 subunits contain the active site. The venom activator readily converts Factor Xa-specific chromogenic substrates and is also able to activate prothrombin (Km = 166 microM, Vmax = 2.5 mumol of prothrombin activated per min/mg of venom). Gel electrophoretic analysis of prothrombin activation indicates that the venom activator randomly cleaves the Arg274-Thr275 and Arg323-Ile324 bonds of prothrombin since both thrombin and meizothrombin are formed as reaction products. Venom-catalyzed prothrombin activation is not affected by bovine Factor Va but is greatly stimulated by phospholipids plus Ca2+ ions. This stimulatory effect is explained by a decrease of the Km for prothrombin. In the presence of 50 microM phospholipid vesicles (25% phosphatidylserine/75% phosphatidylcholine; mole/mole), the Km is 0.34 microM and the Vmax is 7.1 mumol of prothrombin activated per min/mg of venom. The purified venom activator contains gamma-carboxyglutamic acid residues which presumably function in the interaction between the venom activator and phospholipids. Treatment of the activator with 0.8 M NaSCN strongly reduces its ability to activate prothrombin but has no effect on its amidolytic activity. The prothrombin-converting activity of the NaSCN-treated activator can be restored with bovine Factor Va. During prolonged gradient gel electrophoresis, the Mr 300,000 activator dissociates into smaller subunits. This causes a loss of the prothrombin-converting activity, while the amidolytic activity is recovered in a protein with an apparent molecular weight of 57,000. This protein can, however, rapidly activate prothrombin in the presence of Factor Va or in the presence of a protein component of Mr 220,000 that also migrates on the gel. These results suggest that the prothrombin activator from the O. scutellatus venom is a multimeric protein complex consisting of a Factor Xa-like enzyme and a Factor Va-like cofactor.     

Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus

The prothrombin activator present in the venom of the mainland tiger snake (Notechis scutatus scutatus) was purified to homogeneity by gel chromatography on Sephadex G-200 followed by ion-exchange chromatography on SP-Sephadex. The venom activator has an apparent molecular weight of 54,000. It consists of a heavy chain (Mr = 32,000) and a light chain (Mr = 23,000) held together by one or more disulfide bridges. The active site is located at the heavy chain region of the molecule. The venom activator contains 8 gamma-carboxyglutamic acid residues/molecule. Gel electrophoretic analysis of prothrombin activation indicates that the venom activator is capable of cleaving both the Arg 274-Thr 275 and Arg 323-Ile 324 bonds of bovine prothrombin. The order of bond cleavage appears to be random since prethrombin-2 and meizothrombin occur as intermediates during prothrombin activation. Prothrombin activation by the venom activator alone is very slow. This is explained by the unfavorable kinetic parameters for the reaction (Km for prothrombin = 105 microM, Vmax = 0.0025 nmol of prothrombin activated per min/microgram of venom activator). Phospholipids plus Ca2+ and Factor Va greatly stimulate venom-catalyzed prothrombin activation. In the presence of 50 microM phospholipid vesicles composed of 20 mol % phosphatidylserine and 80 mol % phosphatidylcholine, the Km drops to 0.2 microM, whereas there is hardly any effect on the Vmax. Factor Va causes a 3,500-fold increase of the Vmax (8.35 nmol of prothrombin activated per min/microgram of venom activator) and a 10-fold decrease of the Km (9.5 microM). The most favorable kinetic parameters are observed in the presence of both 50 microM phospholipid and Factor Va (Km = 0.16 microM, Vmax = 27.9 nmol of prothrombin activated per min/microgram of venom activator). These changes of the kinetic parameters explain the stimulatory effects of Factor Va and phospholipid on venom-catalyzed prothrombin activation. The venom activator slowly converts the Factor Xa-specific chromogenic substrates CH3SO2-D-leucyl-glycyl-L-arginine-p-nitroanilide and N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginyl-p-nitroani lide hydrochloride. Factor Va causes a 7-fold stimulation of chromogenic substrate conversion by the venom activator. This stimulation appears to be the result of the formation of a tight 1:1 complex between the venom activator and Factor Va.     

The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1.

The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis. Basic residues in anion-binding exosite-I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38-60) based on the PAR1 extracellular domain. Compared with wild-type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38-60 (k(cat)/K(m) = 1.8 x 10(6) and 4.6 x 10(6) vs 9.2 x 10(7) M(-1).s(-1)), whereas the remaining mutations of the anion-binding exosite-I of thrombin had a less pronounced effect, with k(cat)/K(m) values ranging from 3.3 x 10(7) M(-1). s(-1) (R77(a)Q) to 5.8 x 10(7) M(-1).s(-1) (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38-60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion-binding exosite-I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.     

The contribution of amino acid region ASP695-TYR698 of factor V to procofactor activation and factor Va function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K(i) values of approximately 6.3 and approximately 5.3 microm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp-Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC(50) of 1.6 microm (with a K(D) for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --> Lys, Tyr(696) --> Phe, Asp(697) --> Lys, and Tyr(698) --> Phe (factor V(2K2F)) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa (factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.     

An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.     

Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: role of neurotoxic and procoagulant effects in venom toxicity

The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A(2) (PLA(2)), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA(2)s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey.     

Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg306 and Arg506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg306 and Arg506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 microm prothrombin, whereas half-maximal inhibition was obtained at 0.7 microm prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.     

A control switch for prothrombinase: characterization of a hirudin-like pentapeptide from the COOH terminus of factor Va heavy chain that regulates the rate and pathway for prothrombin activation

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg(271) and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg(320) (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695-699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg(320). These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg(320) and Arg(271), respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg(271) of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.     

The Gla domain of human prothrombin has a binding site for factor Va

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.     

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.     

The biological properties of venoms from juvenile and adult taipan (Oxyuranus scutellatus) snakes.

1. The biological properties of four venom pooled samples from adult taipan (Oxyuranus scutellatus) snakes and one pooled venom sample from six juvenile taipan snakes (11 months old) were compared. 2. The intravenous LD50 (median lethal dose), procoagulant activity and enzymatic activities of the juvenile venom were not significantly different from those of the adult venoms. 3. The juvenile and adult venoms exhibited similar polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns, indicating that they possessed a similar protein composition. 4. The results suggest that there is no significant age-dependency in the biological properties of taipan venom.     

Crystal structures of prethrombin-2 reveal alternative conformations under identical solution conditions and the mechanism of zymogen activation

Prethrombin-2 is the immediate zymogen precursor of the clotting enzyme thrombin, which is generated upon cleavage at R15 and separation of the A chain and catalytic B chain. The X-ray structure of prethrombin-2 determined in the free form at 1.9 ? resolution shows the 215-217 segment collapsed into the active site and occluding 49% of the volume available for substrate binding. Remarkably, some of the crystals harvested from the same crystallization well, under identical solution conditions, diffract to 2.2 ? resolution in the same space group but produce a structure in which the 215-217 segment moves >5 ? and occludes 24% of the volume available for substrate binding. The two alternative conformations of prethrombin-2 have the side chain of W215 relocating >9 ? within the active site and are relevant to the allosteric E*-E equilibrium of the mature enzyme. Another unanticipated feature of prethrombin-2 bears on the mechanism of prothrombin activation. R15 is found buried within the protein in ionic interactions with E14e, D14l, and E18, thereby making its exposure to solvent necessary for proteolytic attack and conversion to thrombin. On the basis of this structural observation, we constructed the E14eA/D14lA/E18A triple mutant to reduce the level of electrostatic coupling with R15 and promote zymogen activation. The mutation causes prethrombin-2 to spontaneously convert to thrombin, without the need for the snake venom ecarin or the physiological prothrombinase complex.     

Structural and functional characterization of a prothrombin activator from the venom of Bothrops neuwiedi

A prothrombin activator from the venom of Bothrops neuwiedi was purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on a Zn2+-chelate column. The overall purification was about 200-fold, which indicates that the prothrombin activator comprises about 0.5% of the crude venom. The venom activator is a single-chain protein with an apparent molecular weight of 60 kDa. It readily activated bovine prothrombin with a Km of 38 microM and a Vmax of 120 mumol prothrombin activated per min per mg of venom activator. Venom-catalyzed prothrombin activation was not accelerated by the so-called accessory components of the prothrombinase complex, phospholipids plus Ca2+ and Factor Va. Gel-electrophoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg-323-Ile-324 bond of bovine prothrombin, since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze commercially available p-nitroanilide substrates and its prothrombin-converting activity was not inhibited by benzamidine, phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethyl ketone and soy-bean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline rapidly destroyed the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could be partially restored by the addition of an excess CaCl2. These results indicate that the venom activator remarkably differs from Factor Xa and that the enzyme is not a serine proteinase, but likely belongs to the metalloproteinases. The structural and functional properties of the venom prothrombin activator from B. neuwiedi are similar to those reported for the venom activator from Echis carinatus.     

Inhibition of prothrombin activation by bothrojaracin, a C-type lectin from Bothrops jararaca venom

Bothrojaracin is a potent and specific alpha-thrombin inhibitor (Kd approximately 0.6 nM) isolated from Bothrops jararaca venom. It binds to both of thrombin's anion-binding exosites (1 and 2), thus inhibiting the ability of the enzyme to act upon several natural macromolecular substrates, such as fibrinogen, platelet receptor, protein C, and factor V. Additionally, bothrojaracin interacts with prothrombin (Kd approximately 30 nM), as previously determined by a solid-phase assay. However, there is no information concerning the effect of this interaction on prothrombin activation and whether the binding of bothrojaracin can occur in plasma. Here, we show that bothrojaracin specifically interacts with prothrombin in human plasma. It is an effective anticoagulant after activation of the intrinsic pathway of blood coagulation, and analysis of prothrombin conversion in plasma shows that bothrojaracin strongly reduces alpha-thrombin formation. To determine whether this effect is due exclusively to inhibition of feedback reactions involving the thrombin-induced activation of factors V and VIII, we analyzed the effect of bothrojaracin on the activation of purified prothrombin by Oxyuranus scutellatus venom. As with plasma, bothrojaracin greatly inhibited thrombin formation, suggesting a direct interference in the prothrombin activation by the enzyme found in this venom (scuterin, a prothrombin activator described as a factor Xa/factor Va-like complex). Altogether, we suggest that bothrojaracin exerts its anticoagulant effect in plasma by two distinct mechanisms: (1) it binds generated thrombin and inhibits exosite 1 dependent activities such as fibrinogen clotting and factor V activation, and (2) it interacts with prothrombin and decreases its proteolytic activation. Thus, bothrojaracin may be useful in the search for thrombin inhibitors that bind both the zymogen and the active enzyme.     

Probing the molecular basis of factor Xa specificity by mutagenesis of the serpin, antithrombin.

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa approximately 3-4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (approximately 2-3x10(2) M(-1)s(-1)). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300-500-fold. A similar 10(4)-10(5)-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4-P4' residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4-P4' binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.     

Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara

In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.     

Incorporation of factor Va into prothrombinase is required for coordinated cleavage of prothrombin by factor Xa

Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320. Factor Va, along with phospholipid and Ca2+, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg320. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor VaFF/MI). The present studies were undertaken to determine whether loss of activity can be attributed to selective loss of efficiency at one or both of the two prothrombin-activating cleavage sites. Kinetic constants for the overall activation of prothrombin by prothrombinase assembled with saturating concentrations of recombinant mutant factor Va were calculated, prothrombin activation was assessed by SDS-PAGE, and rate constants for both cleavages were analyzed from the time course of the concentration of meizothrombin. Prothrombinase assembled with factor VaFF/MI had decreased k(cat) for prothrombin activation with Km remaining unaffected. Prothrombinase assembled with saturating concentrations of factor VaFF/MI showed significantly lower rate for cleavage of plasma-derived prothrombin at Arg320 than prothrombinase assembled with saturating concentrations of wild type factor Va. These results were corroborated by analysis of cleavage of recombinant prothrombin mutants rMz-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A), which can be cleaved only at Arg320 or Arg271, respectively. Time courses of these mutants indicated that mutations in the factor Xa binding site of factor Va reduce rates for both bonds. These data indicate that the interaction of factor Xa with the heavy chain of factor Va strongly influences the catalytic activity of the enzyme resulting in increased rates for both prothrombin-activating cleavages.