Insertion loop 256-268 in coagulation factor IX restricts enzymatic activity in the absence but not in the presence of factor VIII

Insertions in surface loops bordering the substrate-binding groove have been shown to play a major role in the interaction of serine proteases with their cognate inhibitors and substrates. In the present study, we investigated the functional role of factor IX insertion loop 256-268, and in particular of residues Asn(264) and Lys(265) therein. To this end, the purified and activated mutants des-(N264,K265)-FIX and FIX-K265A were compared to normal factor IXa with regard to a number of functional properties. The catalytic efficiency of des-(N264,K265)-FIXa and FIXa-K265A toward the amide substrate CH(3)SO(2)-Leu-Gly-Arg-pNA was 2-3-fold increased relative to that of normal factor IXa. Comparison of the activities of normal and mutant factor IXa toward a series of closely related amide substrates indicates that mutation of residues Asn(264)-Lys(265) influences the interactions in the S2-binding site. The mutations in loop 256-268 also increased the susceptibility of factor IXa to antithrombin inhibition by approximately 3-fold. Factor X activation experiments in the absence of factor VIIIa revealed that the catalytic efficiency of des-(N264,K265)-FIXa and FIXa-K265A was about 20 times higher than that of normal factor IXa. In the presence of factor VIIIa, however, the activity toward factor X was similar to that of normal factor IXa. The reduced sensitivity of the factor IXa mutants to factor VIIIa was neither due to an increase in factor IXa-dependent inactivation of factor VIIIa, nor to a lower affinity for this cofactor. Overall, these data demonstrate that loop 256-268 restricts the activity of factor IXa toward both synthetic and natural substrates. Complex formation with factor VIIIa alleviates the inhibitory effect of this insertion loop on the activation of FX.     

A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca(2+), an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an approximately 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an approximately 4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.     

Factor IXa:factor VIIIa interaction. helix 330-338 of factor ixa interacts with residues 558-565 and spatially adjacent regions of the a2 subunit of factor VIIIa

The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca(2+) and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558-565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558-565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa(R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330-338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K(d(A2)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 100 nm, dEGR-IXa(R333Q) approximately 1.8 micrometer, and dEGR-IXa(helixVII) >10 micrometer. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558-565 of the A2 subunit. Apparent K(d(peptide)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 4 micrometer, and dEGR-IXa(R333Q) approximately 62 micrometer. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558-565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558-565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.     

Surface loop 199-204 in blood coagulation factor IX is a cofactor-dependent site involved in macromolecular substrate interaction.

In factor IX residues 199-204 encompass one of six surface loops bordering its substrate-binding groove. To investigate the contribution of this loop to human factor IX function, a series of chimeric factor IX variants was constructed, in which residues 199-204 were replaced by the corresponding sequence of factor VII, factor X, or prothrombin. The immunopurified and activated chimeras were indistinguishable from normal factor IXa in hydrolyzing a small synthetic substrate, indicating that this region is not involved in the interaction with substrate residues on the N-terminal side of the scissile bond. In contrast, replacement of loop 199-204 resulted in a 5-25-fold reduction in reactivity toward the macromolecular substrate factor X. This reduction was due to a combination of increased K(m) and reduced k(cat). In the presence of factor VIIIa the impaired reactivity toward factor X was largely restored for all factor IXa variants, resulting in a more pronounced stimulation by factor VIIIa compared with normal factor IXa (3 to 5 x 10(4)-fold versus 5 x 10(3)-fold). Inhibition by antithrombin was only slightly affected for the factor IXa variant with the prothrombin loop sequence, whereas factor IXa variants containing the analogous residues of factor VII or factor X were virtually insensitive to antithrombin inhibition. In the presence of heparin, however, all chimeric factor IXa variants formed complexes with antithrombin. Thus the cofactors heparin and factor VIIIa have in common that they both alleviate the deleterious effects of mutations in the factor IX loop 199-204. Collectively, our data demonstrate that loop 199-204 plays an important role in the interaction of factor IXa with macromolecular substrates.     

Directed glycosylation of human coagulation factor X at residue 333. Insight into factor Va-dependent prothrombin catalysis

Based on homology, amino acids 326-336 (143-154 in chymotrypsin numbering) of factor X (fX) comprise a flexible surface loop, which is susceptible to self-proteolysis and influences substrate catalysis. To investigate the role of this autolysis loop in fX function, a recombinant variant with a new site for asparagine-linked glycosylation has been produced by changing glutamine 333 to asparagine. Q333N fX is activated normally by factor VIIa and tissue factor, factors IXa and VIIIa, and Russell's viper venom. Proteolysis of the loop is prevented by the mutation. Reactivity of the free enzyme toward substrates and inhibitors is attenuated 4-20-fold; relative to wild type fXa, Spectrozyme Xa(TM) hydrolysis is 25%, inhibition by antithrombin III and the tissue factor pathway inhibitor is approximately 20%, and prothrombin activation in the absence of the cofactor Va is only 5%. Surprisingly, activities of the variant and wild type enzymes are equivalent when part of the prothrombinase complex. N-Glycanase cleaves the new oligosaccharide from Q333N fXa leaving aspartic acid. Q333D fXa is approximately 1.6-fold more reactive with Spectrozyme Xa(TM), antithrombin III and tissue factor pathway inhibitor, and prothrombin than its glycosylated counterpart, Q333N fXa, but still quite abnormal relative to wild type fXa. Like Q333N fXa, Q333D fXa is fully functional as part of the prothrombinase complex. We conclude that Gln-333 is geographically close to a site of proteolytic degradation but not to activator, cofactor, or membrane binding sites. Mutation of Gln-333 impairs catalytic function, but given normal prothrombin activation by the complexed enzyme, the importance of Gln-333 for catalysis is not manifest in the prothrombinase assembly, suggesting a conformational change in complexed fXa.     

Identification of a basic region on tissue factor that interacts with the first epidermal growth factor-like domain of factor X

Tissue factor (TF) facilitates the recognition and rapid activation of factor X (fX) by factor VIIa (fVIIa) in the extrinsic Xase pathway. TF makes extensive interactions with both light and heavy chains of fVIIa; however, with the exception of a basic recognition site for the Gla domain of fX, no other interactive site on TF for the substrate has been identified. Structural and modeling data have predicted that a basic region of TF comprised of residues Asn-199, Arg-200, and Lys-201 is located at a proper height on the membrane surface to interact with either the C-terminus of the Gla domain or the EGF-1 domain of fX. To investigate this possibility, we prepared the Ala substitution mutants of these residues and evaluated their ability to function as cofactors for fVIIa in the activation of wild-type fX and its two mutants which lack either the Gla domain (GD-fX) or both the Gla and EGF-1 domains (E2-fX). All three TF mutants exhibited normal cofactor activity in the amidolytic activity assays, but the cofactor activity of Arg-200 and Lys-201 mutants in fVIIa activation of both fX and GD-fX, but not E2-fX, was impaired approximately 3-fold. Further kinetic analysis revealed that kcat values with both TF mutants are impaired with no change in Km. These results suggest that both Arg-200 and Lys-201 of TF interact with EGF-1 of fX to facilitate the optimal docking of the substrate into the catalytic groove of the protease in the activation complex.     

Protease and EGF1 domains of factor IXa play distinct roles in binding to factor VIIIa. Importance of helix 330 (helix 162 in chymotrypsin) of protease domain of factor IXa in its interaction with factor VIIIa.

Previous studies revealed that cleavage at Arg-318-Ser-319 in the protease domain autolysis loop of factor IXa results in its diminished binding to factor VIIIa. Now, we have investigated the importance of adjacent surface-exposed helix 330-338 (162-170 in chymotrypsin numbering) of IXa in its interaction with VIIIa. IXWT, eight point mutants mostly based on hemophilia B patients, and a replacement mutant (IXhelixVII in which helix 330-338 is replaced by that of factor VII) were expressed, purified, and characterized. Each mutant was activated normally by VIIa-tissue factor-Ca2+ or XIa-Ca2+. However, in both the presence and absence of phospholipid, interaction of each activated mutant with VIIIa was impaired. The role of IXa EGF1 domain in binding to VIIIa was also examined. Two mutants (IXQ50P and IXPCEGF1, in which EGF1 domain is replaced by that of protein C) were used. Strikingly, interactions of the activated EGF1 mutants with VIIIa were impaired only in the presence of phospholipid. We conclude that helix 330 in IXa provides a critical binding site for VIIIa and that the EGF1 domain in this context primarily serves to correctly position the protease domain above the phospholipid surface for optimal interaction with VIIIa.     

Identification of functionally important residues of the epidermal growth factor-2 domain of factor IX by alanine-scanning mutagenesis. Residues Asn(89)-Gly(93) are critical for binding factor VIIIa

This paper describes the consequences of alanine-scanning mutagenesis on 28 positions of the second epidermal growth factor (EGF-2) domain of factor IX. We identified four positions of Gln(97), Phe(98), Tyr(115), and Leu(117) that are critical for secretion of factor IX. Of the remaining mutations, 4 mutants (V86A, E113A, K122A, and S123A) are as active as wild-type factor IX (IXwt); 16 (D85A, K100A, N101A, D104A, N105A, R116A, E119A, T87A, I90A, K91A, R94A, E96A, S102A, K106A, T112A, and N120A) retain reduced but detectable activity, and 4 (N89A, N92A, G93A, and V107A) are nearly inert in the clotting assay. Both factor XIa and the factor VIIa-tissue factor complex effectively catalyzed the activation of these mutants except N89A. The mutant V107A failed to form the factor tenase complex with factor VIIIa because of a 35-fold increase in K(d). The mutants N89A and N92A did not compete with factor IXwt for factor VIIIa binding, and G93A exhibited a 6-fold increase in K(i) values in the competitive binding assay. It appears that mutations at these positions have significantly affected the interaction between factor IX and factor VIIIa, although other mutations had little effect on the binding of factor IX to factor VIIIa. Mutations in two regions, Thr(87)-Gly(93) and Asn(101)-Val(107), significantly increased the K(m) value of factor IXa (2-10-fold) in cleavage of factor X in the absence of factor VIIIa. In the presence of factor VIIIa, the catalytic efficiency of each mutant toward factor X paralleled its clotting activity. Briefly, we propose two relatively distinctive functions of factor IX for two adjacent regions in the EGF-2 domain; the first loop region (residues 89-94) is involved with the binding of its cofactor, factor VIIIa, and the third loop with connected beta-sheets (residues 102-108) is involved in the proper binding to the substrate, factor X.     

Contribution of basic residues of the autolysis loop to the substrate and inhibitor specificity of factor IXa

The autolysis loop (residues 143-154 in chymotrypsinogen numbering) plays a pivotal role in determining the macromolecular substrate and inhibitor specificity of coagulation proteases. This loop in factor IXa (FIXa) has 3 basic residues (Arg143, Lys147, and Arg150) whose contribution to the protease specificity of factor IXa has not been studied. Here, we substituted these residues individually with Ala in Gla-domainless forms of recombinant factor IX expressed in mammalian cells. All mutants exhibited normal amidolytic activities toward a FIXa-specific chromogenic substrate. However, Arg143 and Lys147 mutants showed a approximately 3- to 6-fold impairment in FX activation, whereas the Arg150 mutant activated factor X normally both in the absence and presence of factor VIIIa. By contrast, Arg143 and Lys147 mutants reacted normally with antithrombin (AT) in both the absence and presence of the cofactor, heparin. However, the reactivity of the Arg150 mutant with AT was impaired 6.6-fold in the absence of heparin and 33- to 70-fold in the presence of pentasaccharide and full-length heparins. These results suggest that Arg143 and Lys147 of the autolysis loop are recognition sites for FX independent of factor VIIIa, and Arg150 is a specific recognition site for AT that can effectively interact with AT only if the serpin is in the heparin-activated conformation.     

The heparin-binding exosite is critical to allosteric activation of factor IXa in the intrinsic tenase complex: the role of arginine 165 and factor X

Heparin inhibits the intrinsic tenase complex (factor IXa-factor VIIIa) via interaction with a factor IXa exosite. To define the role of this exosite, human factor IXa with alanine substituted for conserved surface residues (R126, N129, K132, R165, N178) was characterized. Chromogenic substrate hydrolysis by the mutant proteases was reduced 20-30% relative to factor IXa wild type. Coagulant activity was moderately (N129A, K132A, K126A) or dramatically (R165A) reduced relative to factor IXa wild type. Kinetic analysis demonstrated a marked reduction in apparent cofactor affinity (23-fold) for factor IXa R165, and an inability to stabilize cofactor activity. Factor IXa K126A, N129A, and K132A demonstrated modest reductions ( approximately 2-fold) in apparent cofactor affinity, and accelerated decay of intrinsic tenase activity. In the absence of factor VIIIa, factor IXa N178A and R165A demonstrated a defective Vmax(app) for factor X activation. In the presence of factor VIIIa, Vmax(app) varied in proportion to the predicted factor IXa-factor VIIIa concentration. However, factor IXa R165A had a 65% reduction in the kcat for factor X, suggesting an additional effect on catalysis. The ability of factor IXa to compete for physical assembly into the intrinsic tenase complex was enhanced by EGR-chloromethylketone bound to the factor IXa active site or addition of factor X, and reduced by selected mutations in the heparin-binding exosite (N178A, K126A, R165A). These results suggest that the factor IXa heparin-binding exosite participates in both cofactor binding and protease activation, and cofactor affinity is linked to active site conformation and factor X interaction during enzyme assembly.     

The factor IXa heparin-binding exosite is a cofactor interactive site: mechanism for antithrombin-independent inhibition of intrinsic tenase by heparin

Therapeutic heparin concentrations selectively inhibit the intrinsic tenase complex in an antithrombin-independent manner. To define the molecular target and mechanism for this inhibition, recombinant human factor IXa with alanine substituted for solvent-exposed basic residues (H92, R170, R233, K241) in the protease domain was characterized with regard to enzymatic activity, heparin affinity, and inhibition by low molecular weight heparin (LMWH). These mutations only had modest effects on chromogenic substrate hydrolysis and the kinetics of factor X activation by factor IXa. Likewise, factor IXa H92A and K241A showed factor IXa-factor VIIIa affinity similar to factor IXa wild type (WT). In contrast, factor IXa R170A demonstrated a 4-fold increase in apparent factor IXa-factor VIIIa affinity and dramatically increased coagulant activity relative to factor IXa WT. Factor IXa R233A demonstrated a 2.5-fold decrease in cofactor affinity and reduced ability to stabilize cofactor half-life relative to wild type, suggesting that interaction with the factor VIIIa A2 domain was disrupted. Markedly (R233A) or moderately (H92A, R170A, K241A) reduced binding to immobilized LMWH was observed for the mutant proteases. Solution competition demonstrated that the EC(50) for LMWH was increased less than 2-fold for factor IXa H92A and K241A but over 3.5-fold for factor IXa R170A, indicating that relative heparin affinity was WT > H92A/K241A > R170A > R233A. Kinetic analysis of intrinsic tenase inhibition demonstrated that relative affinity for LMWH was WT > K241A > H92A > R170A > R233A, correlating with heparin affinity. Thus, LMWH inhibits intrinsic tenase by interacting with the heparin-binding exosite in the factor IXa protease domain, which disrupts interaction with the factor VIIIa A2 domain.     

Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop

Coagulation factor IXa (fIXa) plays a central role in the coagulation cascade. Enzymatically, fIXa is characterized by its very low amidolytic activity that is not improved in the presence of cofactor, factor VIIIa (fVIIIa), distinguishing fIXa from all other coagulation factors. Activation of the fIXa-fVIIIa complex requires its macromolecular substrate, factor X (fX). The 99-loop positioned near the active site partly accounts for the poor activity of fIXa because it adopts a conformation that interferes with canonical substrate binding in S2-S4. Here we show that residues Lys-98 and Tyr-99 are critically linked to the amidolytic properties of fIXa. Exchange of Tyr-99 with smaller residues resulted not only in an overall decreased activity but also in impaired binding in S1. Replacement of Lys-98 with smaller and uncharged residues increased activity. Simultaneous mutagenesis of Lys-98, Tyr-177, and Tyr-94 produced an enzyme with 7000-fold increased activity and altered specificity. This triple mutant probably mimics the conformational changes that are physiologically induced by cofactor and substrate binding. It therefore provides a cooperative two-step activation model for fIXa. Tyr-177 locks the 99-loop in an inactive conformation which, in the physiologic complex, is released by cofactor fVIIIa. FX is then able to rearrange the unlocked 99-loop and subsequently binds to the active site cleft.     

Comparative platelet binding and kinetic studies with normal and variant factor IXa molecules

We have recently shown that thrombin-stimulated human platelets have specific, saturable receptors for factor IXa, occupancy of which promotes factor X activation (Ahmad, S. S., Rawala-Sheikh, R., and Walsh, P. N. (1989) J. Biol. Chem. 264: 3244-3251, 20012-20016; Rawala-Sheikh, R., Ahmad, S. S., and Walsh, P. N. (1990) Biochemistry 29, 2606-2611). To study the structural requirements for factor IXa binding to platelets, equilibrium binding studies and kinetic studies of factor X activation were carried out with normal factor IXa and with two variant proteins: factor IXaAlabama (FIXaAL; Asp47----Gly substitution) and factor IXaChapel Hill (FIXaCH; Arg145----His substitution). In the absence of factors VIIIa and X, there were 331 binding sites/platelet for FIXaCH (Kdapp = 2.8 nM), and 540 sites/platelet for FIXaAL (Kdapp = 3.2 nM), compared with 540 sites/platelet (Kdapp = 2.3 nM) for normal factor IXa. The addition of factors VIIIa and X, both at saturating concentrations, had no effect on the number of binding sites for either normal or variant factor IXa, resulted in a decrease in the Kd for normal factor IXa to 0.67 nM, resulted in a suboptimal decrease in Kd for FIXaAL (1.4 nM), and had no effect on the Kd for FIXaCH. Kinetic studies of factor X activation at variable factor IXa concentration confirmed these values of Kd in the presence of factors VIIIa and X. Determination of rates of factor X activation at variable substrate concentrations yielded normal values of catalytic efficiency (kcat/Km) for the variant proteins, thereby indicating that the abnormally low rates of factor X activation obtained were a consequence of the low affinity binding of FIXaAL and FIXaCH to thrombin-activated platelets in the presence of factors VIIIa and X. These studies suggest that the presence of Asp47 and the cleavage of factor IX at Arg145-Ala146 are important structural features required for specific, high affinity factor IXa binding to platelets in the presence of factors VIIIa and X.     

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.     

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with ∼40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31–41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ∼5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ∼4-fold decreased k_cat and ∼2-fold decreased K_m, although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.     

Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa.

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A2 subunit to stimulate factor IXa activity, we show that a panel of these inhibitors block this activity. Inhibition of activity parallels the antibody potency as measured in the Bethesda assay. These antibodies also block the A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa. Similar to the IgG fractions, a peptide representing the sequence of the inhibitor epitope (A2 residues 484-509) blocked the A2-dependent stimulation of factor IXa. These results indicate that antibodies possessing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. Furthermore, these results also suggest that factor VIII residues 484-509 contribute to a factor IXa-interactive site.     

The tissue factor/factor VIIa/factor Xa complex: a model built by docking and site-directed mutagenesis

Factor X is activated to factor Xa (fXa) in the extrinsic coagulation pathway by the tissue factor (TF)/factor VIIa (fVIIa) complex. Upon activation, the fXa molecule remains associated with the TF/fVIIa complex, and this ternary complex is known to activate protease-activated receptors (PARs) 1 and 2. Activation of fVII in the TF complex by fXa is also seen at physiologic concentrations. The ternary complexes TF/fVII/fXa, TF/fVIIa/fX, and TF/fVIIa/fXa are therefore all physiologically relevant and of interest as targets for inhibition of both coagulation and cell-signaling pathways that are important in cardiovascular disease and inflammation. We therefore present a model of the TF/fVIIa/fXa complex, built with the use of the available structures of the TF/fVIIa complex and fXa by protein-protein docking calculations with the program Surfdock. The fXa model has an extended conformation, similar to that of fVIIa in the TF/fVIIa complex, with extensive interactions with TF and the protease domain of fVIIa. All four domains of fXa are involved in the interaction. The gamma-carboxyglutamate (Gla) and epithelial growth factor (EGF1 and EGF2) domains of fVIIa are not significantly involved in the interaction. Docking of the Gla domain of fXa to TF/fVIIa has been reported previously. The docking results identify potential interface residues, allowing rational selection of target residues for site-directed mutagenesis. This combination of docking and mutagenesis confirms that residues Glu51 and Asn57 in the EGF1 domain, Asp92 and Asp95 in the EGF2 domain, and Asp 185a, Lys 186, and Lys134 in the protease domain of factor Xa are involved in the interaction with TF/fVIIa. Other fX protease domain residues predicted to be involved in the interaction come from the 160s loop and the N-terminus of the fX protease domain, which is oriented in such a way that activation of both fVII by fXa, and the reciprocal fX activation by fVIIa, is possible.     

Molecular defect (Gla+14----Lys) and its functional consequences in a hereditary factor X deficiency (factor X "Vorarlberg")

Factor X (FX) "Vorarlberg" is a congenital FX deficiency characterized clinically by a mild bleeding tendency. Homozygous individuals have a FX activity of less than 10% in the extrinsic system and 25% in the intrinsic system. FX antigen is 20%. Using molecular techniques, two point mutations were detected in the coding sequence of the FX Vorarlberg gene: a G----A at base pair 160 in exon II resulting in a change of Gla14 (GAA) to Lys (AAA); a G----A at base pair 424 in exon V resulting in a change from Glu102 (GAG) to Lys (AAG). The mutations abolished a TaqI restriction site in exon II and an MnlI site in exon V. To determine whether these mutations are present on one or on both alleles, restriction analyses of amplified exon II and exon V fragments were performed. Analysis of the pedigree showed that the genotype for the mutation on exon II (homozygous versus heterozygous) correlates with the severity of the phenotypic coagulation defect. We therefore conclude that the mutation in exon II is responsible for the functional defect in FX Vorarlberg. We have also purified the mutant FX protein from patient plasma. Purified FX Vorarlberg is indistinguishable from normal FX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its activity is 15% of normal FX upon activation with factor VIIa/tissue factor, 75% upon activation with factor IXa/factor VIIIa, and 100% upon activation with RVV. Activation at varying Ca2+ concentrations shows that the affinity of FX Vorarlberg for Ca2+ is decreased. Factor Xa Vorarlberg is able to convert prothrombin at a normal rate but also shows decreased affinity for Ca2+ in this interaction. Upon addition of Ca2+, FX Vorarlberg does not undergo the same conformational change as normal FX. Our data show that FX Vorarlberg has a decreased affinity for Ca2+ which impedes a normal conformational change. This leads to a decreased rate of activation by factor VIIa/tissue factor and by factor IXa. The decrease is much more marked for the extrinsic than for the intrinsic pathway.     

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.     

Identification of residues Asn89, Ile90, and Val107 of the factor IXa second epidermal growth factor domain that are essential for the assembly of the factor X-activating complex on activated platelets

Activated platelets promote intrinsic factor X-activating complex assembly by presenting high affinity, saturable binding sites for factor IXa mediated by two disulfide-constrained loop structures (loop 1, Cys88-Cys99; loop 2, Cys95-Cys109) within the second epidermal growth factor (EGF2) domain. To identify amino acids essential for factor X activation complex assembly, recombinant factor IXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared. All seven mutants were similar to the native factor IXa by SDS-PAGE, active site titration, and content of gamma-carboxyglutamic acid residues. Kinetic constants obtained by either titrating factor X or factor VIIIa on SFLLRN-activated platelets or phospholipid vesicles revealed near normal values of Km(app) and Kd(app)FVIIIa for all mutants, indicating normal substrate and cofactor binding. In a factor Xa generation assay in the presence of activated platelets and cofactor factor VIIIa, compared with native factor IXa (Kd(app)FIXa approximately 1.1 nm, Vmax approximately 12 nm min(-1)), N89A displayed an increase of approximately 20-fold in Kd(app)FIXa and a decrease of approximately 20-fold in Vmax; I90A had an increase of approximately 5-fold in Kd(app)FIXa and approximately 10-fold decrease in Vmax; and V107A had an increase of approximately 3-fold in Kd(app)FIXa and approximately 4-fold decrease in Vmax. We conclude that residues Asn89, Ile90, and Val107 within loops 1 and 2 (Cys88-Cys109) of the EGF2 domain of factor IXa are essential for normal interactions with the platelet surface and for the assembly of the factor X-activating complex on activated platelets.