Sodium binding site of factor Xa: role of sodium in the prothrombinase complex

The nature of residue 225 on a consensus loop in serine proteases determines whether a protease can bind Na(+). Serine proteases with a Pro at this position are unable to bind Na(+), but those with a Tyr or Phe can bind Na(+). Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na(+) is also known to stimulate the amidolytic activity of FXa toward cleavage of small synthetic substrates, but the role of Na(+) in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), respectively. In contrast, Na(+) minimally affected the cleavage rate of these substrates by the mutant, and no K(d(app)) for Na(+) binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na(+) and impaired approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor Va with a similar K(d(app)) of 1. 1-1.8 nM in the presence of Na(+), K(+), Li(+), Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na(+) in the reaction buffer. These results suggest that (1) the loop including residue 225 (225-loop) is a Na(+) binding site in FXa, (2) the Na(+)- and Ca(2+)-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na(+)-bound and Na(+)-free forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.     

Prothrombinase assembly and S1 site occupation restore the catalytic activity of FXa impaired by mutation at the sodium-binding site

Two loop segments (183-189 and 221-225) in the protease domain of factor Xa contribute to the formation of a Na(+)-binding site. Studies with factor Xa indicate that binding of a single Na(+) ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr(225) with Pro diminishes sensitivity to Na(+). Using full-length factor Xa(Y225P), the allosteric relationship between the Na(+) site and other structural determinants in factor Xa and prothrombinase was investigated. Direct binding and kinetic measurements with probes that target the S1 specificity pocket indicate that assembly of the mutant in prothrombinase corrected the impaired binding of these probes observed with free factor Xa(Y225P). This appears to result from the apparent allosteric linkage between the factor Va, S1, and Na(+)-binding sites, since binding of the cofactor to membrane-bound factor Xa(Y225P) enhances binding at the S1 site and vice versa. Additional studies revealed that the internal salt bridge (Ile(16)-Asp(194)) of factor Xa(Y225P) is partially destabilized, a process that is reversible upon occupation of the S1 site. The data establish that alterations at the factor Xa Na(+)-binding site shift the zymogen-protease equilibrium to a more zymogen-like state, and as a consequence binding of S1-directed probes and factor Va are adversely affected. Therefore, the zymogen-like characteristics of factor Xa(Y225P) have allowed for the apparent allosteric linkage between the S1, factor Va, and Na(+) sites to become evident and has provided insight into the structural transitions which accompany the conversion of factor X to factor Xa.     

Role of the alpha-helix 163-170 in factor Xa catalytic activity

Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa alpha-helix 163-170 (h163-170), Arg(165) and Lys(169), participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH(2)-terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na(+) because using a higher Na(+) concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH(2)-terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na(+)-binding site of the protease and that residues Val(163) and Ser(167) play a key role in this interaction.     

Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human coagulation factor xa. Studies on catalytic efficiency and inhibitor binding

The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.     

Role of the N-terminal epidermal growth factor-like domain of factor X/Xa

The functional importance of the N-terminal epidermal growth factor-like domain (EGF-N) of factor X/Xa (FX/Xa) was investigated by constructing an FX mutant in which the exon coding for EGF-N was deleted from FX cDNA. Following expression and purification to homogeneity, the mutant was characterized with respect to its ability to function as a zymogen for either the factor VIIa-tissue factor complex or the factor IXa-factor VIIIa complex and then to function as an enzyme in the prothrombinase complex to catalyze the conversion of prothrombin to thrombin. It was discovered that EGF-N is essential for the recognition and efficient activation of FX by both activators in the presence of the cofactors. On the other hand, the FXa mutant interacted with factor Va with a normal apparent dissociation constant and activated prothrombin with approximately 3-fold lower catalytic efficiency in the prothrombinase complex. Surprisingly, the mutant activated prothrombin with approximately 12-fold better catalytic efficiency than wild-type FXa in the absence of factor Va. The mutant was inactive in both prothrombin time and activated partial thromboplastin time assays; however, it exhibited a similar specific activity in a one-stage FXa clotting assay. These results suggest that EGF-N of FX is required for the cofactor-dependent zymogen activation by both physiological activators, but it plays no apparent role in FXa recognition of the cofactor in the prothrombinase complex.     

The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.     

A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We improve hemostasis in vivo using a conformationally pliant variant of coagulation factor Xa (FXa(I16L)) rendered partially inactive by a defect in the transition from zymogen to active protease. Using mouse models of hemophilia, we show that FXa(I16L) has a longer half-life than wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXa(I16L) is more efficacious than FVIIa, which is used to treat bleeding in hemophilia inhibitor patients. FXa(I16L) may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.     

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.     

Characterization of a factor Xa binding site on factor Va near the Arg-506 activated protein C cleavage site

Prothrombin is proteolytically activated by the prothrombinase complex comprising the serine protease Factor (F) Xa complexed with its cofactor, FVa. Based on inhibition of the prothrombinase complex by synthetic peptides, FVa residues 493-506 were proposed as a FXa binding site. FVa is homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-activating complex, and FVIIIa residues 555-561 (homologous to FVa residues 499-506) are recognized as a FIXa binding sequence. To test the hypothesis that FVa residues 499-505 contribute to FXa binding, we created the FVa loop swap mutant (designated 499-505(VIII) FV) with residues 499-505 replaced by residues 555-561 of FVIIIa, which differ at five of seven positions. Based on kinetic measurements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity for FXa. The fully formed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k(cat) and K(m)) for cleavage of prothrombin at Arg-320. However, small changes in both Arg-320 and Arg-271 cleavage rates result together in a moderate change in the pathway of prothrombin activation. Although residues 499-505 directly precede the Arg-506 cleavage site for activated protein C (APC), the 499-505(VIII) FVa mutant was inactivated entirely normally by APC. These results suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different specificity for binding FXa and FIXa while retaining compatibility as substrate for APC. In an updated three-dimensional model for the FVa structure, residues 499-505, along with Arg-506, Arg-306, and other previously suggested FXa binding sequences, delineate a continuous surface on the A2 domain that is strongly implicated as an extended FXa binding surface in the prothrombinase complex.     

Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ～55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ～5,000- and ～80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ～85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.     

The elusive role of the potential factor X cation-binding exosite-1 in substrate and inhibitor interactions

A number of studies suggest that blood-clotting factor X (FX) uses secondary site(s) to interact (as a substrate) with its activators. Numerous pieces of evidence also imply that, within prothrombinase (as an enzyme), activated FX (FXa) uses exosite(s) for cofactor Va and/or prothrombin recognition. Similarly, FXa exosite(s) seem to govern interaction with inhibitors. An obvious difference between FXa and thrombin resides within a region called exosite-1: positively charged in thrombin and clearly of opposite polarity in FXa. To investigate the role of this potential cation-binding exosite, we prepared a series of mutants within loops 34-40 and 70-80 of FX. Overall, the mutations induced relatively subtle, non-synergistic modulation. The potential exosite was dispensable for FX activation and is unlikely to constitute a critical region for factor Va binding, albeit it is clearly important for prothrombin activation. Our data also implicate loop 34-40 of FXa in the interaction with the tissue factor pathway inhibitor, in prevention of plasminogen activator inhibitor-1 binding, and in tempering inhibition by heparin-activated antithrombin. Compared with FX, mutants with reduced electrostatic potential potentiated thrombin production in FX-depleted plasma, whereas mutants with inverted electrostatic potential impeded clotting. Despite the definite consequences observed, disruption of the potential cation-binding exosite of FX had rather weak effects, far from what would be expected if this region was as crucial as in thrombin.     

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.     

Kinetics of factor Xa inhibition by recombinant tick anticoagulant peptide: both active site and exosite interactions are required for a slow- and tight-binding inhibition mechanism

Recombinant tick anticoagulant peptide (rTAP) is a competitive slow- and tight-binding inhibitor of factor Xa (FXa) with a reported equilibrium dissociation constant (K(I)) of approximately 0.2 nM. The inhibitory characteristics and the high selectivity of rTAP for FXa are believed to arise from the ability of the inhibitor to specifically interact with the residues of both the active site as well as those remote from the active site pocket of the protease. To localize the rTAP-interactive sites on FXa, the kinetics of inhibition of wild-type and 18 different mutants of recombinant FXa by the inhibitor were studied by either a discontinuous assay method employing the tight-binding quadratic equation or a continuous assay method employing the slow-binding kinetic approach. It was discovered that K(I) values for the interaction of rTAP with four FXa mutants (Tyr(99) --> Thr, Phe(174) --> Asn, Arg(143) --> Ala, and a Na(+)-binding loop mutant in which residues 220-225 of FXa were replaced with the corresponding residues of thrombin) were elevated by 2-3 orders of magnitude for each mutant. Further studies revealed that the characteristic slow type of inhibition by rTAP was also eliminated for the mutants. These findings suggest that the interaction of rTAP with the P2-binding pocket, the autolysis loop, and the Na(+)-binding loop is primarily responsible for its high specificity of FXa inhibition by a slow- and tight-binding mechanism.     

Removal of B-domain sequences from factor V rather than specific proteolysis underlies the mechanism by which cofactor function is realized

Factor V, the precursor of factor Va, circulates in plasma with little or no procoagulant activity. Activity is generated following limited proteolysis indicating that the conversion of factor V to factor Va results in appropriate structural changes, which impart cofactor function. We have produced recombinant partial B-domain-truncated derivatives of factor V (FV(des811-1491) and FV(des811-1491) with Arg(709) and Arg(1545) mutated to Gln) to investigate whether discrete proteolysis within the B-domain followed by a conformational transition is responsible for activation. Direct binding fluorescence measurements as well as steady-state kinetic assays were employed to assess the ability of these factor V derivatives to assemble and function in prothrombinase. In contrast to human factor V, single-chain B-domain-truncated factor V bound to FXa membranes with an affinity that was identical to factor Va. Additionally, it was found that, once this modified derivative was assembled in prothrombinase, it functioned in an equivalent manner to factor Va. Taken together these data support the hypothesis that proteolysis within the B-domain of factor V, although necessary, is incidental to the mechanism by which cofactor function is realized. Instead, our results are more consistent with the interpretation that proteolytic activation of factor V simply eliminates steric and/or conformational constraints contributed by the B-domain that otherwise interfere with discrete binding interactions that govern the eventual function of factor Va.     

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.     

The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex

The location of the active site of membrane-bound factor Xa relative to the phospholipid surface was determined both in the presence and absence of factor Va using fluorescence energy transfer. Factor Xa was reacted with 5-(dimethylamino)-1-naphthalenesulfonyl- glutamylglycylarginyl(DEGR) chloromethyl ketone to yield DEGR-Xa, an analogue of factor Xa with a fluorescent dye attached covalently to the active site. When DEGR-Xa was titrated with phosphatidylcholine/phosphatidylserine vesicles containing octadecylrhodamine, fluorescence energy transfer was observed between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the outer surface of the phospholipid bilayer. Based on the dependence of the efficiency of singlet-singlet energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between the active site probe and the surface of the phospholipid bilayer averaged 61 A in the absence of factor Va and 69 A in the presence of factor Va. These direct measurements show that the active site of factor Xa is located far above the membrane surface. Also, association of factor Xa with factor Va on the membrane surface to form the prothrombinase complex results in a substantial movement of the active site of the enzyme relative to the membrane surface. The 5-(dimethylamino)-1-naphthalenesulfonyl emission in the complete prothrombinase complex was distinct from that in any other combination of components. It therefore appears that the optimum conformation of the prothrombinase active site is achieved only when factor Va, Ca2+, and a membrane surface interact simultaneously with factor Xa. Thus, in addition to its previously demonstrated ability to stimulate factor Xa binding to membranes, factor Va, upon association with factor Xa on a phospholipid surface, allosterically induces a particular active site conformation in factor Xa and also positions the active site at the correct distance above the membrane for prothrombin activation.     

Factor Va alters the conformation of the Na+-binding loop of factor Xa in the prothrombinase complex

Structural and mutagenesis data have indicated that the 220-loop of thrombin is stabilized by a salt-bridge between Glu-217 and Lys-224, thereby facilitating the octahedral coordination of Na (+) with contributions from two carbonyl O atoms of Arg-221a and Lys-224. All three residues are also conserved in fXa and the X-ray crystal structure of fXa indicates that both Glu-217 and Lys-224 are within hydrogen-bonding distance from one another. To investigate the role of these three residues in the catalytic function of fXa and their contribution to interaction with Na (+), we substituted them with Ala and characterized their properties in both amidolytic and proteolytic activity assays. The results indicate that the affinity of all three mutants for interaction with Na (+) has been impaired. The mutant with the greatest loss of affinity for Na (+) (E217A or E217Q) also exhibited a dramatic impairment ( approximately 3-4 orders of magnitude) in its activity toward both synthetic and natural substrates. Interestingly, factor Va (fVa) restored most of the catalytic defect with prothrombin, but not with the synthetic substrate. Both Glu-217 mutants exhibited a near normal affinity for fVa in the prothrombinase assay, but a markedly lower affinity for the cofactor in a direct-binding assay. These results suggest that, similar to thrombin, an ionic interaction between Glu-217 and Lys-224 stabilizes the 220-loop of fXa for binding Na (+). They further support the hypothesis that the Na (+) and fVa-binding sites of fXa are energetically linked and that a cofactor function for fVa in the prothrombinase complex involves inducing a conformational change in the 220-loop of fXa that appears to stabilize this loop in the Na (+)-bound active conformation.     

Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323-331 of factor V represent an interactive site for activated factor X

We have recently shown that amino acid region 307-348 of factor Va heavy chain (42 amino acids, N42R) is critical for cofactor activity and may contain a binding site for factor Xa and/or prothrombin [(2001) J. Biol. Chem. 276, 18614-18623]. To ascertain the importance of this region for factor Va cofactor activity, we have synthesized eight overlapping peptides (10 amino acid each) spanning amino acid region 307-351 of the heavy chain of factor Va and tested them for inhibition of prothrombinase activity. The peptides were also tested for the inhibition of the binding of factor Va to membrane-bound active site fluorescent labeled Glu-Gly-Arg human factor Xa ([OG488]-EGR-hXa). Factor Va binds specifically to membrane-bound [OG488]-EGR-hXa (10nM) with half-maximum saturation reached at approximately 6 nM. N42R was also found to interact with [OG488]-EGR-hXa with half-maximal saturation observed at approximately 230 nM peptide. N42R was found to inhibit prothrombinase activity with an IC50 of approximately 250 nM. A nonapeptide containing amino acid region 323-331 of factor Va (AP4') was found to be a potent inhibitor of prothrombinase. Kinetic analyses revealed that AP4' is a noncompetitive inhibitor of prothrombinase with respect to prothrombin, with a K(i) of 5.7 microM. Thus, the peptide interferes with the factor Va-factor Xa interaction. Displacement experiments revealed that the nonapeptide inhibits the direct interaction of factor Va with [OG488]-EGR-hXa (IC50 approximately 7.5 microM). The nonapeptide was also found to bind directly to [OG488]-EGR-hXa and to increase the catalytic efficiency of factor Xa toward prothrombin in the absence of factor Va. In contrast, a peptadecapeptide from N42R encompassing amino acid region 337-351 of factor Va (P15H) had no effect on either prothrombinase activity or the ability of the cofactor to interact with [OG488]-EGR-hXa. Our data demonstrate that amino acid sequence 323-331 of factor Va heavy chain contains a binding site for factor Xa.     

Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va.

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481-525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493-506), with 50% inhibition at 2.5 microM. VP493 also inhibited FXa in plasma in FXa-1-stage clotting assays by 50% at 3 microM. When the C-terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity-purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa-1-stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd approximately 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493-506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.     

Alboserpin, a factor Xa inhibitor from the mosquito vector of yellow fever, binds heparin and membrane phospholipids and exhibits antithrombotic activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) ~ 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.