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.     

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.     

The second epidermal growth factor-like domain of human factor IXa mediates factor IXa binding to platelets and assembly of the factor X activating complex.

Factor IXa binding to the activated platelet surface is required for efficient catalysis of factor X activation. Platelets possess a specific binding site for factor IXa, occupancy of which has been correlated with rates of factor X activation. However, the specific regions of the factor IXa molecule that are critical to this interaction have not yet been fully elucidated. To assess the importance of the second epidermal growth factor (EGF2) domain of factor IXa for platelet binding and catalysis, a chimeric protein (factor IXa(Xegf2)) was created by replacement of the EGF2 domain of factor IX with that of factor X. Competition binding experiments showed 2 different binding sites on activated platelets (approximately 250 each/platelet): (1) a specific factor IXa binding site requiring the intact EGF2 domain; and (2) a shared factor IX/IXa binding site mediated by residues G(4)-Q(11) within the Gla domain. In kinetic studies, the decreased V(max) of factor IXa(Xegf2) activation of factor X on the platelet surface (V(max) 2. 90 +/- 0.37 pM/min) versus normal factor IXa (37.6 +/- 0.15 pM/min) was due to its decreased affinity for the platelet surface (K(d) 64.7 +/- 3.9 nM) versus normal factor IXa (K(d) 1.21 +/- 0.07 nM), resulting in less bound enzyme (functional complex) under experimental conditions. The hypothesis that the binding defects of factor IXa(Xegf2) are the cause of the kinetic perturbations is further supported by the normal k(cat) of bound factor IXa(Xegf2) (1701 min(-)(1)) indicating (1) an intact catalytic site and (2) the normal behavior of bound factor IXa(Xegf2). The EGF2 domain is not a cofactor binding site since the mutant shows a normal rate enhancement upon the addition of cofactor. Thus, the intact EGF2 domain of factor IXa is critical for the formation of the factor X activating complex on the surface of activated platelets.     

Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding

During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na+ and Ca2+ with increasing concentrations of a synthetic substrate (CH3-SO2-d-Leu-Gly-Arg-p-nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca2+ decreased this affinity but increased k(cat) dramatically. Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa. FIXa(Y225P), a Na+ site mutant, was severely impaired in Na+ potentiation of its catalytic activity and in binding to p-aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na+ binding. Moreover, the rate of carbamylation of NH2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXa(Y225P) and addition of Ca2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis. In the presence of Ca2+ and phospholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but threefold reduced k(cat). Further, interaction of FVIIIa:FIXa(Y225P) was impaired fourfold. Our previous data revealed that Ca2+ binding to the protease domain increases the affinity of FIXa for FVIIIa approximately 15-fold. The present data indicate that occupancy of the Na+ site further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold. Thus, in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by approximately 12-fold, implicating its role in physiologic coagulation.     

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.     

Characterization of the functional defect in factor IX Alabama. Evidence for a conformational change due to high affinity calcium binding in the first epidermal growth factor domain

Factor IX Alabama is a factor IX variant in which a glycine has been substituted for Asp47 in the first epidermal growth factor (EGF) domain. The structural defect in factor IX Alabama results in a molecule with 10% of normal coagulant activity. The interactions of immunoaffinity-purified factor IX Alabama with its activator, cofactors, and substrate have been investigated to determine the functional defect in the variant. Factor IX Alabama is activated by factor XIa/calcium at near normal rates. Calcium fluorescence-quenching experiments indicate that high affinity calcium binding in the first EGF domain is not altered in factor IX Alabama. The active site of factor IXa Alabama is fully competent to activate factor X in the absence of calcium when using polylysine as a surface to catalyze the reaction. Factor IXa Alabama has only 64% of normal factor IXa activity in the presence of 300 microM CaCl2 in the polylysine-catalyzed system although apparent high affinity calcium binding constants are similar. Factor IXa Alabama has 52-60% of normal activity in a calcium/phospholipid vesicle system. The addition of factor VIIIa to the phospholipid vesicle system decreases the relative rate of factor IXa Alabama to 18-19% of normal. Three-dimensional computer-aided models of the first EGF domain of normal factor IX and factor IX Alabama indicate no major structural alterations resulting from the glycine substitution for Asp47. The model of the first EGF domain of normal factor IX predicts a calcium-binding site involving Asp47, Asp49, Asp64, and Asp65. Our binding data, however, indicate that Asp47 is not necessary to form the high affinity binding site. We conclude that Asp47 in normal factor IX coordinates to the bound calcium, inducing a conformational change in the molecule essential for proper interaction with factor X and factor VIIIa.     

Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer.

Heterotrimeric factor VIIIa was reconstituted from isolated A2 subunit and A1/A3-C1-C2 dimer of thrombin-activated human factor VIII in a reaction that was sensitive to pH. Maximal levels of reconstituted factor VIIIa at pH 6.0 were as much as 20-fold greater than were values observed at pH 7.5. The presence of factor IXa and phospholipid resulted in a marked increase in factor VIIIa reconstituted at physiologic pH. However, the resultant factor VIIIa was unstable due to slow proteolysis of the A1 subunit. Factor IXa modified by the active site-specific reagent dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone (DEGR-IXa) increased the level of factor VIIIa reconstituted from subunits to a similar extent as was observed for unmodified factor IXa and yielded stable factor VIIIa. This enhancement was saturated above a 1:1 molar ratio of DEGR-IXa to factor VIIIa subunits and could be blocked by an anti-factor IX antibody, suggesting that the DEGR-IXa-dependent increase in factor VIIIa reconstitution correlated with assembly of the factor X-ase complex. At a saturating amount of DEGR-IXa, the level of factor VIIIa reconstitution at pH 7.5 approached values obtained at pH 6.0. Fluorescence polarization measurements indicated that factor VIIIa altered binding of DEGR-IXa to phospholipid. However, neither the A2 subunit nor the A1/A3-C1-C2 dimer alone produced this effect. This result suggested that both A2 and A1/A3-C1-C2 were necessary for association of the cofactor with factor IXa. These results suggest a model in which assembly of the intrinsic factor X-ase complex stabilizes factor VIIIa through inhibition of subunit dissociation.     

Platelet receptor occupancy with factor IXa promotes factor X activation

To investigate the activated platelet surface as a locus for factor X activation, the functional consequences of factor IXa binding to platelets were studied. The concentration of factor IXa required for half-maximal rates of factor X activation in the presence of factor VIIIa and thrombin-activated platelets was 0.53 nM, which is close to the Kd (0.56 nM) for factor IXa binding to platelets under identical conditions, determined from equilibrium binding studies. In direct comparative experiments, there was a close correspondence between equilibrium binding of factor IXa to thrombin-activated platelets in the presence of factor VIIIa and kinetic determinations of factor X activation rates. Analysis by polyacrylamide gel electrophoresis revealed that 125I-labeled factor IXa bound to platelets was structurally intact and did not form covalent complexes with platelet proteins. Factor IXa active site-inhibited by 5-dimethylaminonaphthalene-1-sulfonyl glutamyl-glycylarginyl chloromethyl ketone was shown to be a competitive inhibitor of factor IXa binding in the absence (Ki = 2.3 nM) and presence (Ki = 0.43 nM) of factor VIIIa and factor X and of factor X activation (Ki = 0.4 nM) by factor IXa in the presence of factor VIIIa, indicating that the generation of factor Xa is not required for factor IXa binding and that factor IXa bound to activated platelets in the presence of factor VIIIa is closely coupled with rates of factor X activation. We conclude that factor IXa bound tightly to a platelet receptor in the presence of factor VIIIa is the enzyme active in factor X activation.     

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 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.     

The regulation of factor IXa by supersulfated low molecular weight heparin

Supersulfated low molecular weight heparin (ssLMWH) inhibits the intrinsic tenase (factor IXa-factor VIIIa) complex in an antithrombin-independent manner. Recombinant factor IXa with alanine substitutions in the protease domain (K126A, N129A, K132A, R165A, R170A, N178A, R233A) was assessed with regard to heparin affinity in solution and ability to regulate protease activity within the factor IXa-phospholipid (PL) and intrinsic tenase complexes. In a soluble binding assay, factor IXa K126A, K132A, and R233A dramatically (10-20-fold) reduced ssLMWH affinity, while factor IXa N129A and R165A moderately (5-fold) reduced affinity relative to wild type. In the factor IXa-PL complex, binding affinity for ssLMWH was increased 4-fold, and factor X activation was inhibited with a potency 7-fold higher than predicted for wild-type protease-ssLMWH affinity in solution. In the intrinsic tenase complex, ssLMWH inhibited factor X activation with a 4-fold decrease in potency relative to wild-type factor IXa-PL. The mutations increased resistance to inhibition by ssLMWH in a similar fashion for both enzyme complexes (R233A > K126A > K132A/R165A > N129A/N178A/wild type) except for factor IXa R170A. This protease had ssLMWH affinity and potency for the factor IXa-PL complex similar to wild-type protease but was moderately resistant (6-fold) to inhibition in the intrinsic tenase complex based on increased cofactor affinity. These results are consistent with conformational regulation of the heparin-binding exosite and macromolecular substrate catalysis by factor IXa. An extensive overlap exists between the heparin and factor VIIIa binding sites on the protease domain, with residues K126 and R233 dominating the heparin interaction and R165 dominating the cofactor interaction.     

Kinetic comparison of bovine blood coagulation factors IXa alpha and IXa beta toward bovine factor X

The Vmax/Km (microM -1 min -1.) for bovine factor X activation by bovine factor IXa alpha, in the presence of sufficient [Ca2+] to saturate the initial reaction rate, was 0.007. When factor IXa beta was substituted for factor IXa alpha in this reaction, the Vmax/Km decreased to 0.001, suggesting that factor IXa alpha was a more potent catalyst under these conditions. When phospholipid (PL) vesicles (egg phosphatidylcholine/bovine brain phosphatidylserine, 4:1 w/w) were added to these same systems, at levels sufficient to saturate their effects, little change in the Vmax/Km occurred when factor IXa alpha was the enzyme. However, when factor IXa beta was employed, the Vmax/Km dramatically increased to 0.023, demonstrating that factor IXa beta responded to PL addition to a much greater extent than did factor IXa alpha. Upon addition of thrombin-activated factor VIII (factor VIIIa,t), at a suboptimal level, to the above systems, the Vmax/Km for factor X activation by factor IXa alpha/Ca2+/PL/factor VIIIa,t was increased to 1.0, whereas this parameter for factor X activation by factor IXa beta/Ca2+/PL/factor VIIIa,t under the same conditions was found to be 27.3. During these studies, it was discovered that the factor X which became activated to factor Xa during the course of reaction participated in several feedback reactions: activation of factor X, activation of factor VIII, and conversion of factor IXa alpha to factor IXa beta. All feedback reactions, which are capable of complicating the kinetic interpretation, were inhibited by performing the studies in a system which contained a rapid factor Xa inhibitor, Glu-Gly-Arg-CH2Cl, thus allowing kinetic constants to be accurately determined. The results show that while factor IXa alpha is a more efficient enzyme than factor IXa beta toward factor X activation in the absence of cofactors, the response of factor IXa beta to the reaction cofactors, PL and factor VIIIa,t, is much greater than that of factor IXa alpha.     

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 epidermal growth factor-like domains of factor IX. Effect on blood clotting and endothelial cell binding of a fragment containing the epidermal growth factor-like domains linked to the gamma-carboxyglutamic acid region

The binding of factor IX to cultured bovine endothelial cells was characterized using isolated domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like domain(s) also contributes to the interaction of the enzyme with its cofactor.     

The A1 and A2 subunits of factor VIIIa synergistically stimulate factor IXa catalytic activity.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Recently, we showed that isolated A2 subunit enhanced the kcat for factor IXa-catalyzed activation of factor X by approximately 100-fold ( approximately 1 min-1), whereas isolated A1 or A3-C1-C2 subunits showed no effect on this rate (Fay, P. J., and Koshibu, K. J. (1998) J. Biol. Chem. 273, 19049-19054). However, A1 subunit increased the A2-dependent stimulation by approximately 10-fold. The Km for factor X in the presence of A2 subunit was unaffected by A1 subunit, whereas the kcat observed in the presence of saturating A1 and A2 subunits ( approximately 15 min-1) represented 5-10% of the value observed for native factor VIIIa (approximately 200 min-1). An anti-A1 subunit antibody that blocks the association of A2 eliminated the A1-dependent contribution to factor IXa activity. Inclusion of both A1 and A2 subunits resulted in greater increases in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa than that observed for A2 subunit alone and approached values obtained with factor VIIIa. These results indicate that A1 subunit alters the A2 subunit-dependent modulation of the active site of factor IXa to synergistically increase cofactor activity, yielding an overall increase in kcat of over 1000-fold compared with factor IXa alone.     

Kinetics of coagulation factor X activation by platelet-bound factor IXa

Thrombin-activated human platelets, in the presence of factors VIIIa and X, have specific, high-affinity (Kd approximately 0.5 nM), saturable binding sites for factor IXa that are involved in factor X activation [Ahmad, S.S., Rawala-Sheikh, R., & Walsh, P.N. (1989) J. Biol. Chem. 264, 3244-3251]. To determine the functional consequences of factor IXa binding to platelets, a detailed kinetic analysis of the effects of platelets, phospholipids, and factor VIII on factor IXa catalyzed factor X activation was done. In the absence of platelets, phospholipids, or factor VIII, the Michaelis constant (Km = 81 microM) was greater than 500-fold higher than the factor X concentration in human plasma. Unactivated platelets and thrombin-activated factor VIII, alone or in combination, had no effect on the kinetic parameters, whereas thrombin-activated platelets caused a major decrease in Km (0.39 microM) with no significant effect on kcat (0.052 min-1) and allowed factor VIIIa to decrease the Km further to a concentration (0.16 microM) near that of factor X in plasma and to increase the kcat 24,000-fold to 1240 min-1. Sonicated mixed phosphatidylserine/phosphatidylcholine vesicles (25/75, mol/mol) had kinetic effects similar to those of activated platelets. When factor IXa binding to thrombin-activated platelets and rates of factor X activation were measured simultaneously at saturating concentrations of factor X and factor VIIIa, the kcat was independent of factor IXa concentration, and the mean kcat value was 2391 min-1. The increase in catalytic efficiency (kcat/Km) in the presence of thrombin-activated platelets and factor VIIIa was (17.4 x 10(6))-fold.     

Experimental and theoretical evidence supporting the role of Gly363 in blood coagulation factor IXa (Gly193 in chymotrypsin) for proper activation of the proenzyme

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.     

Factor VIIIa cofactor activity shows enhanced ionic strength sensitivity in the absence of phospholipid.

Factor VIIIa, a cofactor for the protease factor IXa, is a trimer of A1, A2 and A3-C1-C2 subunits. In the absence of phospholipid (PL), the k(cat) for factor VIIIa-dependent, factor IXa-catalyzed conversion of factor X was markedly less than that observed in the presence of PL (approx. 150 min(-1)) and decreased as the ionic strength of the reaction increased. At low salt concentration, the k(cat) (5.5 min(-1)) was approx. 8-fold greater than observed at near physiologic ionic strength (0.7 min(-1)). However, this level of salt showed minimal effects on the intermolecular affinities of factor VIIIa (or isolated A2 subunit) for factor IXa or on the K(m) for factor X. Alternatively, the association of A2 subunit with A1 subunit was sensitive to increases in salt and paralleled the reduction in k(cat) observed with factor VIIIa. This instability was not observed in PL-containing reactions. Fluorescence energy transfer between acrylodan-A2 and fluorescein-A1/A3-C1-C2 dimer showed a requirement for both PL and factor IXa for maximal association of A2 with dimer. These results indicate that in the presence of factor IXa, the salt-dependent dissociation of factor VIIIa subunits is significantly enhanced in the absence of PL, promoting a reduced k(cat) for the cofactor-dependent generation of factor Xa.     

Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase

Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed in the absence of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa). However, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phospholipid was poor with a K(i) values (approximately 400 nM) that were approximately 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM). These results indicated that a significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain (Bajaj et al. (2001) J. Biol. Chem. 276, 16302-16309). Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII/Asp712Ala exhibited an 8-fold increased K(d) (35 +/- 1.5 nM) relative to wild-type suggesting a contribution by this residue of approximately 10% of the FVIIIa-FIXa binding energy. Thus both A2 and A3-C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.     

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.