Phospholipid-binding properties of bovine factor V and factor Va.

Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.     

Differentiation of enzyme and substrate binding in the prothrombinase complex

The Ca2+ dependence of factor Xa binding to phospholipid vesicles was measured in the presence and absence of factor Va. The increase in polarization of a fluorescently labeled derivative of factor Xa, [5-(dimethylamino)-1-naphthalenesulfonyl] glutamylglycylarginyl factor Xa (Dns-EGR-Xa), was used as a probe to measure the interaction of factor Xa with phospholipid. The Ca2+ concentration required for half-maximal binding of Dns-EGR-Xa to phospholipid vesicles was 3.5 X 10(-4) M in the presence of factor Va and 9.5 X 10(-4) M in the absence of factor Va. At a Ca2+ concentration of 5 X 10(-4) M, the binding of Dns-EGR-Xa to phospholipid-bound factor Va was near maximal, whereas there was no detectable interaction of Dns-EGR-Xa with phospholipid alone at this Ca2+ concentration as detected by fluorescence polarization. These results were qualitatively confirmed by high-performance liquid chromatography. The rate of hydrolysis of the factor Xa synthetic substrate, benzoylisoleucylglutamylglycylarginine p-nitroanilide, by factor Xa in the presence of factor Va and phospholipid decreased in a Ca2+-dependent manner. These data were analyzed as fraction of factor Xa bound to the phospholipid. A Ca2+ concentration of 2.7 X 10(-4) M resulted in half-maximal binding by this technique. The relationship observed between rates of prothrombin activation and Ca2+ concentration could be predicted quantitatively from calculations of local enzyme and substrate concentrations.     

The contribution of bovine Factor V and Factor Va to the activity of prothrombinase.

The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.     

Comparison of coagulation factor Xa and des-(1-44)factor Xa in the assembly of prothrombinase

The gamma-carboxyglutamic acid (Gla)-domain region of factor X (residues 1-44 of the light chain) was selectively removed by limited proteolysis with alpha-chymotrypsin. The Gla-domainless factor X was then activated by the factor X coagulant protein of Russell's viper venom. Apparent dissociation constants Kd' values for the interaction of factor Va with either factor Xa or Gla-domainless factor Xa were determined kinetically using prothrombin as the substrate. In the absence of phospholipid, factor Va interacted with Gla-domainless factor Xa with lower affinity (Kd' 4 X 10(-6) M) than with factor Xa (Kd' = 5 X 10(-8) M). At saturating concentrations of factor Va, maximal rates of thrombin formation were similar for either enzyme. The addition of phospholipid increased the affinity of factor Va for factor Xa approximately 75-fold (Kd' = 3.3 X 10(-10) M). In contrast, phospholipid had no effect on the affinity of Gla-domainless factor Xa for factor Va (Kd' = 4 X 10(-6) M). The maximal rate of thrombin formation increased approximately 300-fold with the addition of phospholipid to the factor Xa-factor Va system. Under the same conditions, phospholipid had no effect on the rate of thrombin formation when Gla-domainless factor Xa was the enzymatic moiety. These results demonstrate phospholipid has little or no effect on factor Va function when factor Xa has lost its Gla-mediated Ca2+-binding sites.     

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.     

The kinetics of activation of normal and gamma-carboxyglutamic acid-deficient prothrombins

The kinetics of activation of normal and gamma-carboxyglutamic acid (Gla)-deficient prothrombins isolated from cattle maintained for extended periods on the vitamin K antagonist dicoumarol were studied. The catalyst was prothrombinase, comprising isolated Factor Xa, Factor Va, phospholipid vesicles, and calcium ion. The Km and kcat values for prothrombins with 0, 1, 2, 5, 7, and 10 Gla residues were determined both by initial rate analysis and by integrated Michaelis-Menten-Henri analysis. Each of the Gla-deficient prothrombins exhibited kcat values similar to that of normal 10-Gla prothrombin but Km values that were 8- to 20-fold greater than that of the normal molecule. The increased Km coincided with a loss of Ca2+- and phospholipid-binding properties of the Gla-deficient prothrombins. The magnitude of the defect in both the kinetics of activation and Ca2+ and phospholipid binding is not progressive with the loss of Gla residues but rather appears abruptly with the loss of as few as 3 of the 10 Gla residues present in the normal substrate. The theoretical relationship between Km(app) and the dissociation constant (Kd) of the prothrombin-phospholipid interactions was derived. According to the result, the increase in apparent Km observed with the Gla-deficient prothrombins corresponds to at least a 100- to 1000-fold decrease in affinity for phospholipid compared to the affinity of normal prothrombin. In addition, the products of the activation of 10-Gla prothrombin were found to inhibit the activation of the Gla-deficient prothrombins.     

Prothrombinase complex assembly. Contributions of protein-protein and protein-membrane interactions toward complex formation

Equilibrium binding studies of prothrombinase complex formation were undertaken using phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS), factor Va, and factor Xa modified with dansyl glutamylglycinylarginyl chloromethyl ketone (DEGR.Xa). The interaction between the Va.PCPS and DEGR.Xa.PCPS binary complexes was experimentally isolated using saturating concentrations of PCPS. Fluorescence titrations indicated that the membrane-bound proteins interact tightly (Kd approximately 10(-9) M) with a stoichiometry of 1 mol of Va bound/mol of DEGR.Xa at saturation. Complex formation was also investigated by kinetic studies of prothrombin activation using unmodified factor Xa. The kinetic studies yielded a Kd approximately 10(-9) M, which was independent of the concentration of prothrombin in the range of 0.5-5.0 microM. Fluorescence studies of complex assembly at limiting PCPS concentrations provided evidence for an altered DEGR.Xa-PCPS interaction when the enzyme was assembled into the complex. The data suggest that although both proteins are associated with PCPS when complexed with each other, the presence of factor Va on the membrane surface increases the affinity for the Xa-PCPS interaction by an estimated 100-fold. Prothrombinase complex assembly therefore proceeds independently of the availability of substrate and is stabilized by protein-protein and protein-phospholipid interactions. Linkage between the two protein-membrane combination events leads to the further stabilization of the complex on the vesicle surface.     

The binding of activated protein C to factors V and Va

Activated protein C has been derivatized with the active site-directed fluorophore 2-(dimethylamino)-6-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (2,6-DEGR-APC). Covalently modified activated protein C has been used to investigate the binding interactions of the protein to factors V and Va in the presence of phospholipid vesicles. The fluorescence polarization of the 6-dimethylaminonaphthalene-2-sulfonyl moiety increased saturably with increasing phospholipid concentrations in the presence or absence of factor V or Va. Differences in the limiting polarization values indicated distinguishable differences in the interactions between 2,6-DEGR-APC and phospholipid in the presence of factor V or Va. The dissociation constant calculated for the 2,6-DEGR-APC/phospholipid interaction (7.3 X 10(-8) M) was not significantly altered by factor V but was decreased to 7 X 10(-9) M in the presence of factor Va. The interaction between 2,6-DEGR-APC and factor V or Va was characterized by a 1:1 stoichiometry. The binding of 2,6-DEGR-APC to factor V or Va in the presence of phospholipid could be reduced in a competitive manner by diisopropylphosphofluoridate-treated activated protein C. An analysis of the displacement curves indicated that the binding of 2,6-DEGR-APC was indistinguishable from the binding of diisopropylphosphofluoridate-treated activated protein C. The interaction between 2,6-DEGR-APC and phospholipid-bound factor Va was further examined using the isolated subunits of factor Va. Fluorescence polarization changes observed with component E of Va (light chain) closely corresponded with the changes observed with factor Va, whereas isolated component D (heavy chain) had little influence on the binding of 2,6-DEGR-APC to phospholipid vesicles. The data presented are consistent with the interpretation that component E of factor Va contains a binding site for activated protein C.     

Membrane-mediated assembly of the prothrombinase complex

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was Kd = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to Kd = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of Kd = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin. Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of kon = 2.8 x 10(13) (mol/cm2)-1 s-1. In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of kon should be proportional to vesicle diameter. This was verified with a method for estimation of kon values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.     

Thrombin generation and inactivation in the presence of antithrombin III and heparin

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex

The kinetic parameters of bovine prothrombin activation by factor Xa were determined in the absence and presence of factor Va as a function of the phospholipid concentration and composition. In the absence of factor Va, the Km for prothrombin increases proportionally with the phospholipid concentration and correlates well with the affinity of prothrombin for the different membranes. Phospholipid vesicles with a high affinity for prothrombin yield low Km values compared to membranes with less favorable binding parameters. At limited phospholipid concentrations, the Vmax of prothrombin activation correlates with the binding affinity of factor Xa for the various phospholipid vesicles. Membranes with a high affinity for factor Xa have high Vmax values, while for membranes with a low affinity a low Vmax is observed. Extrapolation of double-reciprocal plots of 1/Vmax vs. 1/[phospholipid] to infinite phospholipid concentrations, a condition at which all factor Xa would participate in prothrombin activation, yields a kcat of 2-4 min-1 independent of the type and amount of acidic phospholipid present in the vesicles. Also, in the presence of factor Va the Km for prothrombin varies proportionally with the phospholipid concentration. There is, however, no correlation between the binding parameters and the Km. Factor Va drastically lowers the Km for prothrombin for vesicles that have a low affinity for prothrombin. Vesicles composed of 20 mol % phosphatidylglycerol and 80 mol % phosphatidylcholine have a Km of 0.04 microM when factor Va is present, compared to 2.2 microM determined in the absence of factor Va.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation

Regulatory exosite I of thrombin is present on prothrombin in a precursor state (proexosite I) that specifically binds the Tyr(63)-sulfated peptide, hirudin(54-65) (Hir(54-65)(SO(3)(-))) and the nonsulfated analog. The role of proexosite I in the mechanism of factor Va acceleration of prothrombin activation was investigated in kinetic studies of the effects of peptide binding. The initial rate of human prothrombin activation by factor Xa was inhibited by the peptides in the presence of factor Va but not in the absence of the cofactor. Factor Xa and factor Va did not bind the peptide with significant affinity compared with prothrombin. Maximum inhibition reduced the factor Va-accelerated rate to a level indistinguishable from the rate in the absence of the cofactor. The effect of Hir(54-65)(SO(3)(-)) on the kinetics of prothrombin activation obeyed a model in which binding of the peptide to proexosite I prevented productive prothrombin interactions with the factor Xa-factor Va complex. Comparison of human and bovine prothrombin as substrates demonstrated a similar correlation between peptide binding and inhibition of factor Va acceleration. Inhibition of prothrombin activation by hirudin peptides was opposed by assembly on phospholipid vesicles of the membrane-bound factor Xa-factor-Va-prothrombin complex. Factor Va interactions of human and bovine prothrombin activation are concluded to share a common mechanism in which proexosite I participates in productive interactions of prothrombin as the substrate of the factor Xa-factor Va complex, possibly by directly mediating productive prothrombin-factor Va binding.     

Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents

Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of an amphitropic protein (factor Xa) with membrane models in a complex system

Phosphatidylserine (PS) plays a crucial role, in the conversion of prothrombin into thrombin by the protease, factor Xa. Physiologically, the conversion occurs in the prothrombinase complex. The question of how water-soluble proteins that normally circulate in plasma bind remains to be unambiguously determined. We previously found that the amphitropic proteins (prothrombin, factors V and Va) penetrate into phospholipid layers. AC polarography has allowed the detection for the first time of insertion of factor Xa into condensed monolayers containing phosphatidylserine (PS) and phosphatidylcholine (PC) either 100% PS or 25% PS in the presence of Ca2+. This observation demonstrates that part of factor Xa can cross the phospholipid polar headgroup/hydrocarbon chain interface. In parallel experiments, radioactive surface measurements permitted measuring binding of tritium-labeled factor Xa onto a PS monolayer and calculate an association constant, 6x10(6) M(-1). Penetration of factor Xa into PS-containing vesicles was investigated also using photoactivable 5-[125I]iodonaphthalene-1-azide, which binds selectively to the lipid embedded domains of the protein. These experiments suggest that Factor Xa penetrates preferentially by its heavy chain, an alternative mode of binding to the commonly accepted binding via its Gla domain. Interaction of factor Xa with PS vesicles also changes its apparent K(m) for S 2222.     

The gamma-carboxyglutamic acid and epidermal growth factor-like domains of factor X. Effect of isolated domains on prothrombin activation and endothelial cell binding of factor X

Factor Xa is the enzymatically active constituent of the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin. We have isolated fragments, from tryptic digests of factor X, that consists of the gamma-carboxyglutamic acid (Gla) region linked to one or two epidermal growth factor (EGF)-like domains. Calcium ion binding measurements indicated that these fragments have a native conformation. The factor X-GlaEGF fragments inhibit factor Xa-induced blood clotting in a manner suggesting that they compete with factor Xa for phospholipid binding sites. The same conclusion was reached when thrombin generation was studied in a system of purified components (factor Xa, factor Va, prothrombin, phospholipid, and Ca2+). There was no evidence for a strong interaction between the EGF-like domains of factor Xa and factor Va in either system. However, experiments in the purified system without phospholipid indicated a direct, albeit weak, interaction between the Gla region of factor Xa and factor Va and between the COOH-terminal EGF-like domain of factor Xa and factor Va. Using domain-specific Fab fragments, we have confirmed that the conformation of the serine protease region alters dramatically upon activation of factor X. Furthermore, we have demonstrated that the conformation of the Gla region is affected by the activation, whereas the EGF-like domains appear to be unaltered. The association constant for factor X binding to endothelial cells was two orders of magnitude lower than that for binding of factor IX to these cells. Binding of the Gla and GlaEGF fragments suggested Gla-mediated binding to phospholipid rather than binding to a specific receptor.     

Inhibition of prothrombinase complex by plasma proteinase inhibitors

The rate of inactivation of human coagulation factor Xa by the plasma proteinase inhibitors antithrombin III and alpha 1-antitrypsin has been studied in the presence of the accessory components which constitute the prothrombinase complex. The rate of inactivation of factor Xa by antithrombin III was found to be decreased in the presence of phospholipid vesicles with high affinity for factor Xa. The second-order rate constant for the reaction fell from 6.21 X 10(4) to 3.40 X 10(4) M-1 min-1 in the presence of 20 microM phospholipid. Purified factor Va had no effect on the rate of inactivation of factor Xa in the absence of phospholipid. In the presence of phospholipid, factor Va increased the protective effect displayed by phospholipid, further reducing the rate constant to 2.20 X 10(4) M-1 min-1. The rate of inactivation of factor Xa by alpha 1-antitrypsin was unaffected under these conditions. Platelet-bound prothrombinase complex was formed by incubation of factor Xa with washed human platelets activated by a mixture of collagen and thrombin. The prothrombinase activity was inhibited by antithrombin III was a second-order rate constant of 0.85 X 10(4) M-1 min-1. This rate was obtained in both the presence and absence of exogenous factor Va. Platelet factor 3 vesicles, isolated from platelet aggregation supernatants, also formed prothrombinase complex in the presence of factor Va, and this was inhibited by antithrombin III at the same rate as the platelet-bound complex. There was no protection of the platelet-bound prothrombinase complex from inhibition by alpha 1-antitrypsin.     

The factor V activation paradox

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.     

A monoclonal antibody which inhibits the factor Va:factor Xa interaction

An immunoprecipitation technique has been used to determine the subunit specificity of two of the monoclonal antibodies to bovine Factor V(Va) developed by this laboratory. One of the antibodies is specific for the 74,000-dalton subunit (the E chain) of Factor Va, and the other antibody is specific for the 94,000-dalton subunit (the D chain). The binding of Factor Va to phospholipid was studied by light scattering, and the interaction of Factor Xa with phospholipid-bound Factor Va was examined using 5-dimethylaminonaphthalene-1-sulfonyl-glutamyl-glycyl-arginyl-Xa (Dns-EGR-Xa). Neither the antibody specific for the E chain nor the antibody specific for the D chain inhibit the binding of Factor Va to phospholipid vesicles. The antibody specific for the E chain blocks the increase in fluorescence polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid vesicles and calcium. This antibody also inhibits the association of Dns-EGR-Xa with phospholipid-bound Factor Va as determined by gel-exclusion high pressure liquid chromatography. The antibody specific for the D chain of Factor Va does not block the increase in polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid, and calcium. It was concluded that the antibody specific for the E chain of Factor Va binds at or near the Factor Xa-binding site on the E chain and that the Factor Va E chain plays a significant role in binding Factor Xa.     

The interaction of bovine factor V and factor V-derived peptides with phospholipid vesicles

The binding of bovine Factor V, isolated Factor Va, and isolated activation intermediates to single bilayer phospholipid vesicles was studied by light scattering. The vesicles composed of 25% phosphatidylserine and 75% phosphatidylcholine had a mean radius of approximately 163 A as determined by quasi-elastic light scattering. When these vesicles were saturated with Factor V, the radii increased by approximately 120 A in both 0.15 and 1 M NaCl. At saturation, about 35 molecules of Factor V and 141 molecules of Factor Va were bound to each vesicle. Studies of the binding of Factor V and Factor Va at various ionic strengths showed little change in either Kd or n, suggesting that the binding is not electrostatic. The dissociation constants (Kd) and the lipid to protein ratios at saturation, moles/mol (n), obtained by relative light scattering intensities were: Factor V (Kd = 4.3 X 10(-8) M, n = 214); isolated Factor Va (Kd = 1.7 X 10(-7) M, n = 57); component B, Mr = 205,000 (Kd = 1.8 X 10(-7) M, n = 140); component C, Mr = 150,000 (Kd = 7.0 X 10(-7) M, n = 136); component D, Mr = 94,000 (no binding could be demonstrated); component E, Mr = 74,000 (Kd = 3.8 X 10(-7) M, n = 42). The results presented here indicate that the lower Kd exhibited by Factor V compared to Factor Va (components D and E) is primarily due to the interaction present within the component C portion of the molecule which is destroyed when component C is further cleaved to give component D. The interactions responsible for the binding of Factor Va are expressed in component E as well as in its precursor peptide component B. Dissociation of components D and E by the addition of EDTA indicate that component E alone is responsible for the interaction of bovine Factor Va with phospholipid.     

Human prothrombinase complex assembly and function on isolated peripheral blood cell populations

A membrane-bound Ca2+-dependent complex of the cofactor Factor Va and the enzyme Factor Xa comprises the prothrombinase coagulation complex which catalyzes the proteolytic conversion of prothrombin to thrombin. Analyses of the kinetics of prothrombin activation permit calculation of the stoichiometry and binding parameters governing the functional interactions of Factor Va and Factor Xa with isolated thrombin-activated human platelets and isolated leukocyte subpopulations. Our kinetic approach indicates that Factor Xa binds to approximately 2700 +/- 1000 (n = 8) functional sites on the surface of thrombin-activated platelets with an apparent dissociation constant (Kd) equal to 1.18 +/- 0.53 X 10(-10) M and kcat equal to 19 +/- 7 mol of thrombin/s/mol of Factor Xa bound. The store of Factor V in normal platelets prevents an analogous determination of the functional Factor Va platelet binding sites. Factor Va and Factor Xa titrations performed using platelets from a Factor V antigen-deficient individual indicate that Factor Va and Factor Xa form a 1:1 stoichiometric complex on the surface of thrombin-activated platelets. Both binding isotherms are governed by the same apparent Kd (approximately equal to 10(-10) M) and expressed the same kcat/site (14-17 s-1. Factor Xa-platelet binding parameters are not altered by the use of different platelet agonists, the choice of anticoagulant, or platelet washing procedure. Kinetics of prothrombin activation indicate also that monocytes, lymphocytes, and neutrophils possess, respectively, 16,000, 45,000, and 8,000 Factor Va-Factor Xa receptor sites/cell, which are all governed by apparent KdS approximately equal to 10(-10) M. Enzymatic complexes bound to monocytes or neutrophils exhibit kcat values similar to the platelet-bound complex. Complexes bound to lymphocytes are only 25% as active.