Activation of normal and abnormal human factor IX with trypsin

Human factor IX is activated to factor IXa beta when factor XIa cleaves two peptide bonds, Arg 145-Ala 146 and Arg 180-Val 181, to release an activation peptide. In factor IX Chapel Hill (IXCH), isolated from a hemophilia B patient with a mild bleeding disorder, the arginine 145 residue has been replaced with a histidine. Thus factor IXCH is activated by factor XIa by cleaving only at the Arg 180-Val 181 bond, leaving the activation peptide attached, and resulting in an activated species, factor IXa alpha CH, that, like normal factor IXa alpha, is only 20% as active as factor IXa beta. It is reported that both factor IX and factor IXCH could be activated by trypsin to forms of factor IXa beta and factor IXa beta CH that had clotting activities identical to factor XIa-activated factor IX. Amino-terminal amino acid sequence analysis showed that trypsin cleaved factor IX at the same bonds as did factor XIa; factor IXCH was cleaved at the Arg 180-Val 181 bond, as normal, and was cleaved near the histidine 145, at the Lys 142-Leu 143 bond, releasing a slightly larger activation peptide than from normal factor IXa beta. Metal ions had no effect on the rate of activation of factor IX by trypsin; however, metal ions had a profound effect on the rate at which further incubation with trypsin inactivated factor IXa. Calcium and manganese protected factor IXa from inactivation by trypsin more effectively than magnesium, which was more effective than no metal ion. It is concluded that trypsin can activate normal factor IX and factor IXCH to fully active IXa beta forms.     

gamma-Carboxyglutamic acid (Gla)-domainless blood coagulation factor IXa species: preparation and properties.

To investigate the function of the gamma-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated "factor IXa beta'," and its corresponding Gla-domainless form, designated "Gla-domainless factor IXa beta'," were prepared under controlled conditions and characterized. First, bovine factor IXa alpha was converted by alpha-chymotrypsin in the presence of calcium ions to factor IXa beta' (Mr 47,000). Compared with factor IXa beta, factor IXa beta' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXa beta' by additional selective cleavage by alpha-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXa beta' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXa alpha, factor IXa beta', and Gla-domainless factor IXa beta', with factor IXa beta revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXa beta' was less than 0.5% of that of factor IXa beta'.(ABSTRACT TRUNCATED AT 250 WORDS)     

A monoclonal antibody to factor IX that inhibits the factor VIII:Ca potentiation of factor X activation

A murine monoclonal antibody (IgG1k, Kd approximately 10(-8) M) specific for an epitope located on the heavy chain of human factor IXa was used to study structure-function relationships of factor IX. The antibody inhibited factor IX clotting activity but did not impair activation of factor IX either by factor XIa/calcium or by factor VIIa/tissue factor/calcium. The antibody also did not impair the binding of factor IXa to antithrombin III. Moreover, the antibody did not prevent calcium and phospholipid (PL) from inhibiting the binding of factor IXa to antithrombin III. The antibody also failed to impair activation of factor VII by factor IXa/calcium/PL. Furthermore, the antibody did not interfere with the very slow activation of factor X by factor IXa/calcium/PL. In contrast, the antibody did interfere with factor X activation when reaction mixtures also contained factor VIII:Ca/von Willebrand factor. The marked acceleration of factor X activation observed in control mixtures was not observed in mixtures containing the antibody. Similar results were obtained in reaction mixtures containing the Fab portion of the antibody and factor VIII:Ca free of von Willebrand factor. In additional experiments, factor VIII:Ca/von Willebrand factor was found to inhibit the binding of the antibody to 125I-factor IXa as determined using an immunosorbent assay. Moreover, the antibody displaced factor VIII:Ca from the factor X activator complex (IXa/calcium/PL/VIII:Ca) as evidenced by an altered elution pattern on gel filtration chromatography. From these observations, we conclude that the antibody impairs the clotting activity of factor IXa through interference with its binding of factor VIII:Ca. This suggests a significant role for the heavy chain (residues of 181-415) of factor IXa in binding factor VIII:Ca.     

Effects of gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IX on factor X activation. Studies using proteolytic fragments of bovine factor IX.

Factor IX is a vitamin K-dependent zymogen of a serine protease. The NH2-terminal half of the molecule consists of a Ca(2+)-binding gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. To elucidate the role of these non-catalytic modules of factor IXa beta in factor X activation, we have isolated and characterized fragments of bovine factor IX, containing one or both of the EGF-like modules as well as these modules linked to the Gla module. The fragments were used as inhibitors of factor IXa beta-mediated factor X activation in a plasma clotting system and in systems with purified components of the Xase complex. Fragments consisting of either the two EGF-like modules of factor IX linked together or the NH2-terminal EGF-like module alone were found to inhibit factor Xa generation both in the presence and absence of the cofactor, factor VIIIa. Moreover, a fragment consisting of the corresponding modules of factor X had a similar effect. We therefore propose that factor IXa beta and factor X interact directly through their EGF-like modules on or in the vicinity of a phospholipid surface. We have also found that the isolated Gla module of factor IX inhibits the formation of factor Xa both in the presence and absence of phospholipid but not in the absence of factor VIIIa. Our results are compatible with a model of the Xase complex, in which both the serine protease part and the Gla module of factor IXa beta interact with factor VIIIa.     

Calcium binding to a human factor IXa derivative lacking gamma-carboxyglutamic acid: evidence for two high-affinity sites that do not involve beta-hydroxyaspartic acid

A derivative of human blood clotting factor IXa beta lacking gamma-carboxyglutamic acid (Gla) residues was prepared by limited proteolysis with chymotrypsin, and subsequently examined for its ability to bind calcium ions. By amino acid analysis, Gla-domainless human factor IXa beta contained 0.3-0.4 moles of beta-hydroxyaspartic acid per mole of protein. Equilibrium dialysis experiments demonstrated that Gla-domainless human factor IXa beta retained two high-affinity calcium binding sites (Kd=52 microM), a finding essentially identical to that observed for Gla-domainless bovine factor IX that contains 0.8-0.9 moles of beta-hydroxyaspartic acid per mole of protein. These data strongly suggest that the beta-hydroxyaspartic acid residue in these proteins does not participate in their high affinity calcium sites.     

The interaction of bovine factor IX, its activation intermediate, factor IX alpha, and its activation products, factor IXa alpha and factor IXa beta, with acidic phospholipid vesicles of various compositions.

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues

Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.     

Zymogenic and enzymatic properties of the 70-80 loop mutants of factor X/Xa

The Ca(2+) binding 70-80 loop of factor X (fX) contains one basic (Arg(71)) and three acidic (Glu(74), Glu(76), and Glu(77)) residues whose contributions to the zymogenic and enzymatic properties of the protein have not been evaluated. We prepared four Ala substitution mutants of fX (R71A, E74A, E76A, and E77A) and characterized their activation kinetics by the factor VIIa and factor IXa in both the absence and presence of cofactors. Factor VIIa exhibited normal activity toward E74A and E76A and less than a twofold impaired activity toward R71A and E77A in both the absence and presence of tissue factor. Similarly, factor IXa in the absence of factor VIIIa exhibited normal activity toward both E74A and E76A; however, its activity toward R71A and E77A was impaired approximately two- to threefold. In the presence of factor VIIIa, factor IX activated all mutants with approximately two- to fivefold impaired catalytic efficiency. In contrast to changes in their zymogenic properties, all mutant enzymes exhibited normal affinities for factor Va, and catalyzed the conversion of prothrombin to thrombin with normal catalytic efficiencies. However, further studies revealed that the affinity of mutant enzymes for interaction with metal ions Na(+) and Ca(2+) was impaired. These results suggest that although charged residues of the 70-80 loop play an insignificant role in fX recognition by the factor VIIa-tissue factor complex, they are critical for the substrate recognition by factor IXa in the intrinsic Xase complex. The results further suggest that mutant residues do not play a specific role in the catalytic function of fXa in the prothrombinase complex.     

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

beta-Hydroxyaspartic acid is a post-translationally modified amino acid found in a number of plasma proteins in a domain homologous to epidermal growth factor. Its presence can be correlated with a high affinity Ca2+ binding site, with a dissociation constant of 10-100 microM. We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post-translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo. This system has been used to express eight different point mutations of human factor IX in the first epidermal growth factor domain in order to study the role of beta-hydroxyaspartate at residue 64, and the adjacent carboxylate residues at positions 47, 49 and 78. We conclude that this domain is essential for factor IX function and suggest that Ca2+ binds to carboxylate ions in this domain and stabilizes a conformation necessary for the interaction of factor IXa with factor X, factor VIII and phospholipid in the next step of the clotting cascade.     

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 IX Amagasaki: a new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. We identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate factor X in the presence of factor VIII, phospholipids, and Ca2+, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311----Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.     

Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity

The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor IXa to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A., Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains. Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII. Activated factor IX/factor X chimeras were indistinguishable from normal factor IXa with respect to factor IXa enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed approximately 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX(106-108)/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor IXa enzymatic activity requires hydrophobic contact between the two EGF-like domains.     

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.     

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.     

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.     

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 binding of calcium to the activation products of bovine factor IX.

Binding isotherms of Ca2+ to the bovine Factor IX activation intermediates and products, i.e. Factor IXalpha, Factor IXa alpha, and Factor IXa beta have been examined. At pH 7.4, Factor IX alpha possesses at least two strong Ca2+ sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 3.7 mM. Bovine Factor IXa alpha also contains at least two Ca2+ binding sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 1.3 mM. Factor IXa beta, the ultimate activation product of Factor IX, in the intrinsic system, likewise contains at least two strong Ca2+ sites, of average KD 0.1 mM, as well as seven additional weaker sites, possessing an average KD of 1.0 mM. The Ca2+-binding properties of the above proteins are similar to those of their precursor molecule, Factor IX, which we have earlier shown to possess at least two strong Ca2+ sites, with an average KD of 0.1 mM, and 11 weaker sites, of average KD 1.3 mM (Amphlett, G.W., Byrne, R., and Castellino, F.J. (1978) J. Biol. Chem. 253, 6774-6779). Circular dichroism analysis of all of the above proteins was consistent with the molecules possessing a low alpha-helical content, and a high quantity of beta structure and random coil conformations.     

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.