Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor X(a) in the absence of factor V(a) and in the absence and presence of membranes.

Activation of prothrombin by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X(a) of human prothrombin was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-1) s(-1), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X(a), (2) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X(a) for rapid conversion to thrombin.     

Cooperative roles of factor V(a) and phosphatidylserine-containing membranes as cofactors in prothrombin activation

Activation of prothrombin, as catalyzed by the prothrombinase complex (factor X(a), enzyme; factor V(a) and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzII(a)) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2). V(max), K(m), or V(max)/K(m) for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzII(a) and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 microm, whereas MzII(a) proteolysis was saturable. First order rate constants for formation of MzII(a) and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-V(a)-X(a) complex (i.e. "channeling") also was adjusted because both the membranes and factor V(a) have been shown to cause channeling. k(cat), K(m), or k(cat)/K(m) values were reported for one lipid concentration, for which all X(a) was likely incorporated into a X(a)-V(a) complex on a PS membrane. Comparing previous results, which were obtained either with factor V(a) (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002) Biochemistry 41, 935-949), with results presented here we conclude that both factor V(a) and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor V(a) has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzII(a) versus Pre2 pathway; and 3) PS membranes cause approximately 50% of the substrate to be activated via channeling at 50 microm membrane concentration, but factor V(a) extends the range of efficient channeling to much lower or higher membrane concentrations.     

Effects of activation peptide bond cleavage and fragment 2 interactions on the pathway of exosite I expression during activation of human prethrombin 1 to thrombin

Activation of prothrombin (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg271-Thr272 and Arg320-Ile321, resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates fragment 1 and generates the zymogen analog, prethrombin 1 (Pre 1). The properties of exosite I on Pre 1 and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin(54-65) ([5F]Hir(54-65)-(SO3-)). Prethrombin 2 (Pre 2), formed by factor Xa cleavage of Pre 1 at Arg271-Thr272, had the same affinity for hirudin(54-65) peptides as Pre 1 in the absence or presence of near-saturating fragment 2 (F2). Pre 2 and thrombin also had indistinguishable affinities for F2. By contrast, cleavage of Pre 1 at Arg320-Ile321, to form active meizothrombin des-fragment 1 MzT(-F1), showed a 11- to 20-fold increase in affinity for hirudin(54-65), indistinguishable from the 13- to 20-fold increase seen for conversion of Pre 2 to thrombin. Thus, factor Xa cleavage of Pre 1 at Arg271-Thr272 does not effect exosite I expression, whereas cleavage at Arg320-Ile321 results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre 1 activation intermediates is not modulated by F2, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre 1 activation intermediates and the previously demonstrated role of (pro)exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre 1 activation pathway.     

Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.     

Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site.

Hirugen, a synthetic dodecapeptide corresponding to the carboxyl-terminal amino acids 53-64 of hirudin, binds within a deep groove in thrombin that contains a cationic region referred to as the anion-binding exosite. This region is important in many of the binary interactions of thrombin with macromolecular substrates and cofactors. Fluorescein-labeled hirugen was used to probe which steps in the prothrombin activation process generate this anion-binding exosite. Two activation cleavage sites exist in bovine prothrombin. Cleavage at Arg274-Thr275 releases the activation fragments to generate the thrombin precursor, prethrombin 2. Cleavage of prothrombin within a disulfide loop at Arg323-Ile324 leads to formation of meizothrombin with no loss of peptide material but with formation of amidolytic activity. Cleavage of the same bond in prethrombin 2 generates thrombin. Hirugen, labeled at the amino terminus with fluorescein isothiocyanate, does not bind to prothrombin but does bind to thrombin (Kd = 9.6 +/- 1.2 x 10(-8) M), prethrombin 2 (Kd = 1.3 +/- 0.1 x 10(-7) M), thrombin-fragment-2 complex (Kd = 1.1 +/- 0.2 x 10(-6) M), and meizothrombin (Kd = 1.6 +/- 0.5 x 10(-8) M). Prothrombin fragment-2 and hirugen both bind independently to thrombin. A ternary complex can form with hirugen and fragment-2 and either thrombin or prethrombin 2, suggesting that fragment-2 and hirugen bind to discrete sites. Hirugen also alters the active site conformation of thrombin as detected by modulation of synthetic substrate hydrolytic activity. These studies suggest that conformational changes, rather than alleviating steric hindrance, are responsible for the formation of the hirugen-binding site during prothrombin activation. Furthermore, this conformational change can be effected by the cleavage of either of the two bonds required for activation of prothrombin.     

Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism

The kinetics of the activation of human prothrombin catalyzed by human prothrombinase was studied using the fluorescent alpha-thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Prothrombinase proteolytically activates prothrombin to alpha-thrombin by cleavages at Arg273-Thr274 (bond A) and Arg322-Ile323 (bond B). The differential fluorescence properties of DAPA complexed with the intermediates and products of human prothrombin activation were exploited to study the kinetics of the individual bond cleavages in the zymogen. When the catalyst was composed of prothrombinase (human factor Xa, human factor Va, synthetic phospholipid vesicles, and calcium ion), initial velocity studies of alpha-thrombin formation indicated that the kinetic constants for the cleavage of bonds A or B were similar to the constants that were obtained for the overall reaction (bonds A + B). The progress of the reaction was also monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results indicated that the activation of human prothrombin catalyzed by prothrombinase proceeded exclusively via the formation of meizothrombin (bond B-cleaved) as an intermediate. Kinetic studies of the cofactor dependence of the rates of cleavage of the individual bonds indicated that, in the absence of the cofactor, cleavage at bond B would constitute the rate-limiting step in prothrombin activation. Progress curves for prothrombin activation catalyzed by prothrombinase and monitored using the fluorophore DAPA were typified by the appearance of a transient maximum, indicating the formation of meizothrombin as an intermediate. When factor Xa alone was the catalyst, progress curves were characterized by an initial burst phase, suggesting the rapid production of prethrombin 2 (bond A-cleaved) followed by its slow conversion to alpha-thrombin. Gel electrophoresis followed by autoradiography was used to confirm these results. Collectively, the results indicate that the activation of human prothrombin via the formation of meizothrombin as an intermediate is a consequence of the association of the cofactor, human factor Va, with the enzyme, human factor Xa, on the phospholipid surface.     

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.     

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.     

Activation of prothrombin as measured by the conversion of Nalpha-benzoylarginine ethyl ester

The activation of prothrombin has been studied by using highly purified preparations of activated factor X1 and activated factor X2, factor V and prothrombin. The rate of prothrombin activation was followed using an esterase assay involving the conversion of N alpha-benzoylarginine ethyl ester (BAEE) by thrombin generated in the course of prothrombin activation. The rate of thrombin generation increased by about 26000-fold when factor V and phospholipid were added to prothrombin, factor Xa and calcium. A comparison of the rates of thrombin formation obtained with activated factor X1 and activated factor X2 showed that activated factor X1 had only 70% of the biological activity of activated factor X2. Attempts to explain the rate of prothrombin activation and the difference between the activity of activated factor X1 and activated factor X2 are discussed.     

Soluble phosphatidylserine binds to a single identified site in the C2 domain of human factor Va.

Factor V(a) (FV(a)) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a membrane surface. FV(a) is a heterodimer composed of one heavy chain (A1 and A2 domains) and one light chain (A3, C1, and C2 domains). We use fluorescence, circular dichroism, and equilibrium dialysis to demonstrate that (1) the FV C2 domain expressed in Sf9 cells binds one molecule of C6PS with a k(d) of approximately 2 microM, (2) stabilizing changes occur in the FV C2 domain upon C6PS binding, (3) the C6PS binding site in the FV C2 domain is located near residue Cys(2113), which reacts with DTNB, and (4) binding to a PS-containing membrane is an order of magnitude tighter than that to soluble C6PS. Coupled with a recently published crystal structure of the C2 domain, these results support a model for the mechanism of C2-membrane interaction.     

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.     

Efficient thrombin generation requires molecular phosphatidylserine, not a membrane surface

Activation of prothrombin to thrombin is catalyzed by a "prothrombinase" complex, traditionally viewed as factor X(a) (FX(a)) in complex with factor V(a) (FV(a)) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through "channeling" (activation without the release of an intermediate). We ask here whether the PS molecule itself and not the membrane surface is sufficient to produce the fully active human "prothrombinase" complex in solution. Both FX(a) and FV(a) bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FX(a) and FV(a2) (light isoform of FV(a)), these proteins form a tight (Kd = 0.6 +/- 0.09 nM at 37 degrees C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels shows that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FX(a) and one FV(a2), further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2 x 10(8) M(-1) s(-1)) and via the same reaction pathway (50-60% channeling, with the rest via the meizothrombin intermediate) as the activation catalyzed by a complex assembled on PS-containing membranes (4.4 x 10(8) M(-1) s(-1)). These results question the accepted role of PS membranes as providing "dimensionality reduction" and favor a regulatory role for platelet-membrane-exposed PS.     

Temperature dependence of the thrombin-catalyzed proteolysis of prothrombin

Measurement of the temperature-dependence of thrombin-catalyzed cleavage of the Arg(155)-Ser(156) and Arg(284)-Thr(285) peptide bonds in prothrombin and prothrombin-derived substrates has yielded Arrhenius parameters that are far too large for classical mechanistic interpretation in terms of a simple hydrolytic reaction. Such a difference from the kinetic behavior exhibited in trypsin- and chymotrypsin-catalyzed proteolysis of peptide bonds is attributed to contributions by enzyme exosite interactions as well as enzyme conformational equilibria to the magnitudes of the experimentally determined Arrhenius parameters. Although the pre-exponential factor and the energy of activation deduced from the temperature-dependence of rate constants for proteolysis by thrombin cannot be accorded the usual mechanistic significance, their evaluation serves a valuable role by highlighting the existence of contributions other than those emanating from simple peptide hydrolysis to the kinetics of proteolysis by thrombin and presumably other enzymes of the blood coagulation system.     

Activation of prothrombin by two subtilisin-like serine proteases from Acremonium sp

Two novel subtilisin-like serine proteases (AS-E1 and -E2) that activate prothrombin have been identified in a culture of the fungus Acremonium sp. The enzymes were purified through repeated hydrophobic interaction chromatography. The N-terminal sequences of AS-E1 (34.4 kDa) and AS-E2 (32 kDa) showed high similarity to the internal sequences of two distinct subtilisin-like hypothetical proteins from Chaetomium globosum. Both enzymes proteolytically activated prothrombin to meizothrombin(desF1)-like molecules, while the activation cleavage seemed to occur at a site (Tyr(316)-Ile(317)) that is four residues proximal to the canonical Xa cleavage site (Arg(320)-Ile(321)). Both enzymes inhibited plasma clotting, possibly due to extensive degradation of fibrinogen and production of meizothrombin(desF1)-like molecule.     

Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin

Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin.F1.2 complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.     

Phosphatidylserine binding alters the conformation and specifically enhances the cofactor activity of bovine factor Va

Factor V(a) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a platelet membrane surface. A platelet membrane lipid, phosphatidylserine (PS), regulates the proteolytic activity of factor X(a) as well as the structure of prothrombin. Here we ask whether PS also regulates the structure and cofactor activity of factor V(a), which is a heterodimer composed of one heavy chain (A1-A2 domains) and one light chain (A3-C1-C2 domains). We use fluorescence, circular dichroism, equilibrium dialysis, and activity measurements to demonstrate the following: (1) Factor V(a) has four sites for dicaproyl-sn-glycero-3-phospho-L-serine (C(6)PS, a soluble form of PS); the heavy and light chains each bind two C(6)PS molecules. (2) In the absence of Ca(2+), only two sites remain, one in the heavy chain and another in the light chain. (3) Binding to these sites causes conformational changes evidenced by changes in intrinsic fluorescence and in CD spectra and changes in cofactor activity. (4) At least some of the four lipid binding sites are nonspecific with respect to soluble lipid species, but the site(s) that regulate(s) cofactor activity is (are) specific for C(6)PS, phosphatidic acid, or phosphatidyl(homo)serine and produce a response comparable to that seen with a PS-containing membrane. (5) Like Ca(2+), C(6)PS also mediates the interaction between factor V(a) heavy (V(a)-HC) and light (V(a)-LC) chains. We conclude that PS regulates both the cofactor and the enzyme of the prothrombin-activating complex.     

Kinetic intermediates in prothrombin activation. Bovine prethrombin 1 conversion to thrombin by factor X

Two pathways are possible during the proteolytic formation of alpha-thrombin (alpha-IIa) from prothrombin (II) or prethrombin 1 (P1). One of the pathways, with prethrombin 2 or prethrombin 2 associated with fragment 2 (P2F2) as intermediates, has long been known to exist when activation is catalyzed by Factor Xa (Xa) alone. The second pathway, with meizothrombin or meizothrombin (des fragment 1) (MzIIa(-F1)) as intermediate, has been shown to exist when Factor Va and phospholipids are present with Xa. Until now, MzIIa(-F1) has not been detected in reactions catalyzed by Xa alone. In this study, we demonstrate that P1 activation by Xa alone occurs via both pathways, and we provide rate constants and kinetic equations for calculating the relative contributions of each of the pathways to the formation of alpha-IIa by Xa. Investigation of the initial rates of proteolytic cleavage of P2F2 and P1 by Xa alone indicated first-order dependence on substrate concentration with no evidence of saturation of Xa with either substrate at concentrations as high as 200 microM. Apparent second-order rate constants (kc/Km) of 113 +/- 9 M-1 s-1 for the formation of thrombin from P2F2 and 1,410 +/- 19 M-1 s-1 for the disappearance of P1 were determined at pH 7.5, 25 degrees C, 10 mM CaCl2, 0.15 M ionic strength. A two-step sequential first-order pathway employing these rate constants for thrombin activity production from P1 via P2F2 could not, however, account for the quantity of thrombin that was produced during the early stages of P1 activation. Addition of a parallel first-order reaction to produce thrombin activity from P1 independently of P2F2, tentatively identified as the formation of MzIIa(-F1), yielded progress curves in quantitative agreement with the experimental data. kc/Km for the parallel reaction was estimated to be 98 +/- 10 M-1 s-1. Independent determination of the second-order rate constant for the cleavage of isolated MzIIa (-F1), 15,000 +/- 420 M-1 s-1, indicated that MzIIa(-F1) could meet the kinetic requirements for an intermediate in the parallel activation pathway. The transient formation of MzIIa (-F1), as well as the generation of alpha-IIa, was directly demonstrated during activation of P1 by active site-affinity labeling of the reaction products with a biotin derivative of D-Phe-Pro-Arg chloromethyl ketone and visualization by semiquantitative Western blotting.(ABSTRACT TRUNCATED AT 400 WORDS)     

Exposure of platelet membrane phosphatidylserine regulates blood coagulation

This article addresses the role of platelet membrane phosphatidylserine (PS) in regulating the production of thrombin, the central regulatory molecule of blood coagulation. PS is normally located on the cytoplasmic face of the resting platelet membrane but appears on the plasma-oriented surface of discrete membrane vesicles that derive from activated platelets. Thrombin, the central molecule of coagulation, is produced from prothrombin by a complex ("prothrombinase") between factor Xa and its protein cofactor (factor V(a)) that forms on platelet-derived membranes. This complex enhances the rate of activation of prothrombin to thrombin by roughly 150,000 fold relative to factor X(a) in solution. It is widely accepted that the negatively charged surface of PS-containing platelet-derived membranes is at least partly responsible for this rate enhancement, although there is not universal agreement on mechanism by which this occurs. Our efforts have led to an alternative view, namely that PS molecules bind to discrete regulatory sites on both factors X(a) and V(a) and allosterically alter their proteolytic and cofactor activities. In this view, exposure of PS on the surface of activated platelet vesicles is a key regulatory event in blood coagulation, and PS serves as a second messenger in this regulatory process. This article reviews our knowledge of the prothrombinase reaction and summarizes recent evidence leading to this alternative viewpoint. This viewpoint suggests a key role for PS both in normal hemostasis and in thrombotic disease.     

The use of prothrombin(S525C) labeled with fluorescein to directly study the inhibition of prothrombinase by antithrombin during prothrombin activation

Serine 525 of human prothrombin was mutated to cysteine and covalently labeled with fluorescein to make II(S525C)-fluorescein. Kinetics of cleavage of this derivative by prothrombinase are identical to those of wild-type prothrombin. Cleavage is coincident with a 50% increase in fluorescence intensity and the product is catalytically inactive. Thus, it allows convenient monitoring of prothrombin activation without generating active thrombin. The kinetics of inhibition of factor Xa (FXa) by antithrombin (AT) and AT-heparin were measured by monitoring activation of II(S525C)-fluorescein and the hydrolysis of the chromogenic substrate S2222 in the presence of AT. With S2222 as the substrate the rate constant for inhibition of FXa, Ca(2+), and unilamellar vesicles of phosphatidylcholine and phosphatidylserine (75:25) (PCPS) vesicles by AT was 3.51 x 10(3) m(-1) s(-1); when factor Va (FVa) was included the rate constant was 1.55 x 10(3) m(-1) s(-1). In the absence of FVa, II(S525C)-fluorescein had no effect on inhibition. When II(S525C)-fluorescein was the substrate, however, FVa at saturating concentrations profoundly protected FXa from inhibition by AT, increasing the half-life from 3 min with FXa, Ca(2+), PCPS, and II(S525C)-fluorescein, to greater than 69 min when FVa was included. Thus, both FVa and prothrombin are necessary for this level of protection. In the absence of prothrombin, FVa decreased the second order rate constant for inhibition by the AT-heparin complex from 1.58 x 10(7) m(-1) s(-1), for FXa, Ca(2+), and PCPS, to 7.72 x 10(6) m(-1) s(-1). II(S525C)-fluorescein and factor Va together reduced the rate constant to less than 1% of that for FXa, Ca(2+), and PCPS. At a heparin concentration of 0.2 unit/ml, this corresponds to a half-life increase from 1 s to 136 s.     

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.