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PubMed1994

Baculovirus-mediated expression of the epidermal growth factor-like modules of human factor IX fused to the factor XIIIa transamidation site in fibronectin. Evidence for a direct interaction between the NH2-terminal epidermal growth factor-like module of factor IXa beta and factor X.

Factor IX is a vitamin K-dependent procoagulant zymogen of a serine protease. In the presence of Ca2+ the active form of factor IX (factor IXa beta) forms a complex with factor VIIIa on suitable phospholipid surfaces such as aggregated platelets. This macromolecular complex rapidly activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-like module of factor IXa beta and the substrate factor X. In an alternative approach to study this protein-protein interaction, we have expressed three recombinant baculovirus constructs encoding the EGF-like modules of human factor IX and a truncated form of fibronectin in a system based on the infection of insect cells (Spodoptera frugiperda 21). This strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gelatin-binding part derived from fibronectin by proteolytic cleavage. The fusion proteins were isolated at yields of 20-50 micrograms/ml culture medium. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro-beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount found in factor IX from human plasma, and appeared to be glycosylated at Ser-53. The NH2-terminal EGF-like module, which contained a transamidation acceptor site derived from fibronectin, was cross-linked by factor XIIIa in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fragments containing only the gamma-carboxyglutamic acid module and the two EGF-like modules. The cross-linking results suggest a specific interaction between the NH2-terminal EGF-like module of factor IXa beta and the heavy chain of unactivated factor X. This interaction, albeit weak as judged by competition experiments, may be important for the targeting of factor X to the factor IXa beta-factor VIIIa complex on biological membranes and for the subsequent dissociation of factor Xa from the complex after activation.

Amino Acid Sequence↗
PubMed1992

Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa.

Factor IXa was shown to inactivate both factor VIII and factor VIIIa in a phospholipid-dependent reaction that could be blocked by an antifactor IX antibody. Factor IXa-catalyzed inactivation correlated with proteolytic cleavages within the A1 subunit of factor VIIIa and within the heavy chain (contiguous A1-A2-B domains) of factor VIII. Furthermore, a relatively slow conversion of factor VIII light chain to a 68-Kd fragment was observed after prolonged incubation. Sites of cleavage were identified within the A1 domain at Arg336-Met337 and within the factor VIII light chain at Arg1719-Asn1720. Factor IXa failed to cleave isolated factor VIII heavy chains, yet cleaved isolated factor VIII light chain. In addition, the purified A1/A3-C1-C2 dimer derived from factor VIIIa was a substrate for factor IXa; however, cleavage of the A1 subunit occurred at less than 30% the rate of cleavage of A1 in trimeric factor VIIIa. These data suggest that factor VIII light chain contributes to the binding site for factor IXa and also support a role for a heavy chain determinant located within the A2 subunit in the association of factor VIIIa with factor IXa. Furthermore, the capacity of factor IXa to proteolytically inactivate its cofactor, factor VIIIa, suggests a mode of regulation within the intrinsic tenase complex.

Amino Acid Sequence↗
PubMed1998

A human alloantibody interferes with binding of factor IXa to the factor VIII light chain.

Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa-factor VIIIa complex assembly.

Binding Sites↗
PubMed1983

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.

Animals↗
PubMed1999

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.

Binding Sites↗
PubMed1990

Factor IXa and von Willebrand factor modify the inactivation of factor VIII by activated protein C.

Activated protein C inactivates factor VIII by proteolytic cleavage of the heavy chain of factor VIII. Protein S and calcium ions are cofactors in this reaction. We have examined the effects of several potential modulators of this reaction, including phospholipids, von Willebrand factor, and factor IXa, all of which bind factor VIII. Our results indicate that neither resting nor stimulated platelets nor phospholipid vesicles protect factor VIII from inactivation by activated protein C in either the presence or the absence of protein S. However, the addition of von Willebrand factor decreases the inactivation of factor VIII by activated protein C by 20% to 30%, and factor IXa, which is known to protect factor VIII from inactivation by activated protein C, confers additional protection with von Willebrand factor. The active site of factor IXa is necessary for the protective effect, because native factor IX and active site-inhibited factor IXa do not protect factor VIII from inactivation. Thus there is an additive protective effect when von Willebrand factor and factor IXa are present with factor VIII, leading to a decrease in the inactivation by activated protein C. These factors may be particularly important in stabilizing factor VIII in the circulation and during the early stages of coagulation.

Amino Acid Chloromethyl Ketones↗
PubMed1976

Purification and properties of human factor IXa.

Human factor IXa was purified 5,000-fold from serum by ion exchange chromatography. The preparation was free from other clotting factors. Both pH sensitivity and heat stability of purified factor IXa appeared to be different from those of factor IX in the plasma. The molecular weight of human factor IXa is 80,000 as estimated from gel-filtration experiments. Modification of seryl or histidyl side chains abolished the activity of factor IXa.

Chromatography, Ion Exchange↗
PubMed2004

SAR and factor IXa crystal structure of a dual inhibitor of factors IXa and Xa.

Modifications to the P4 moiety and pyrazole C3 substituent of factor Xa inhibitor SN-429 provided several new compounds, which are 5-10nM inhibitors of factor IXa. An X-ray crystal structure of one example complexed to factor IXa shows that these compounds adopt a similar binding mode to that previously observed with pyrazole inhibitors in the factor Xa active site both with regard to how the inhibitor binds and the position of Tyr99.

Benzimidazoles↗
PubMed1992

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.

Amino Acid Chloromethyl Ketones↗
PubMed2004

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.

1-Carboxyglutamic Acid↗
PubMed1999

Region of factor IXa protease domain that interacts with factor VIIIa: analysis of select hemophilia B mutants.

Essential to hemostasis is the interaction of factor IXa with factor VIIIa. Recent studies indicate that helix-330 in the protease domain of factor IXa provides a critical binding site for factor VIIIa. Although weaker interactions cannot be ruled out, a primary role of the EGF1 domain of factor IXa in this context may be to serve as a spacer in properly positioning the factor IXa protease domain for optimal interaction with factor VIIIa. The role of the Gla domain, as well as of the EGF2 domain of factor IXa, in binding to factor VIIIa is not clear. The region of factor VIIIa that interacts with the protease domain of factor IXa is quite possibly located in the A2 domain. Furthermore, it should be noted (Table 1) that the corresponding helix residues in factor VIIa bind to tissue factor, and, in factor Xa, they are involved in binding to factor Va. Thus, a common function of this helix (162 in chymotrypsin numbering) in several blood coagulation proteases may be to serve as an anchoring point for the respective cofactor.

Animals↗
PubMed1994

Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.

The interaction between human factor IXa and factor VIII or its constituent units was investigated. Equilibrium binding studies were performed employing factor VIII light chain that was immobilized on a monoclonal antibody. Factor VIII light chain was observed to bind factor IXa with high affinity (Kd = 14.8 +/- 3.2 nM) and approximately 1:1 stoichiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution effectively inhibited binding of factor IXa to the immobilized light chain (Ki = 10.9 +/- 1.9 nM). The isolated factor VIII light chain and the factor VIII heterodimer were equally effective in factor IXa binding, demonstrating that this interaction did not require the factor VIII heavy chain. Factor Xa and activated Protein C were found to be inefficient (Ki > or = 1.2 microM) in competing with factor IXa, indicating that the high affinity for factor VIII light chain was unique for factor IXa. The factor IXa-factor VIII light chain interaction was inhibited by von Willebrand factor, but this effect was abolished by cleavage of the factor VIII light chain by thrombin. An antibody that inhibits von Willebrand factor-factor VIII complex formation did not compete for factor IXa binding. In contrast, association of factor IXa with the factor VIII light chain was inhibited by an antibody directed against the factor VIII region Gln1778-Asp1840. We propose that this sequence provides a factor IXa binding site and that its exposure requires dissociation of the factor VIII-von Willebrand factor complex.

Antibodies, Monoclonal↗
PubMed2000

Amphipathic helices support function of blood coagulation factor IXa.

Blood coagulation factor IXa gains proteolytic efficiency upon binding to a phospholipid membrane. We have found that an amphipathic, membrane-binding peptide from the C2 domain of factor VIII, fVIII(2303)(-23), enhances proteolytic efficiency of factor IXa in the absence of phospholipid membranes. This enhancement is the result of a reduction in the K(M) for the substrate, factor X, with little effect on the k(cat). Enhanced function requires interaction of the gamma-carboxyglutamic acid (Gla) domains of factor IXa and factor X since (i) a synthetic peptide comprising the Gla domain of factor IXa and antibodies directed to the Gla domain of factor IXa inhibit this acceleration, (ii) the acceleration is Ca(II) dependent, and (iii) conversion of Gla-domainless factor X is not affected by the presence of fVIII(2303)(-23). The effect of fVIII(2303)(-23) on factor IXa parallels the enhanced function produced by phosphatidylserine-containing bilayers, and fVIII(2303)(-23) does not further enhance function of factor IXa when phospholipid vesicles are present. The critical feature of fVIII(2303)(-23) is apparently its amphipathic helix-forming structure [Gilbert, G. E., and Baleja, J. D. (1995) Biochemistry 34, 3022-3031] because other alpha-helical peptides such as a homologous peptide from the C2 domain of factor V and melittin have similar effects. Diastereomeric analogues of fVIII(2303)(-23) and melittin, which have reduced helical content, do not support factor IXa activity. A truncated peptide of fVIII(2303)(-23) with three C-terminal residues deleted retains alpha-helical content but loses capacity to enhance factor X cleavage, suggesting that a minimum length of alpha-helix is required. Although these results probably do not illuminate the physiologic function of the factor VIII peptide corresponding to fVIII(2303)(-23), they demonstrate a novel, membrane-mimetic role of amphipathic helical peptides in supporting function of factor IXa.

1-Carboxyglutamic Acid↗
PubMed1992

Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation.

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

1-Carboxyglutamic Acid↗
PubMed1994

The role of amino-terminal residues of the heavy chain of factor IXa in the binding of its cofactor, factor VIIIa.

The purpose of this study is to determine which residues of the factor IXa heavy chain are important for interaction with the cofactor of factor IXa, factor VIIIa. Because the monoclonal antibody (MoAb) FXC008 inhibits interaction between factors IXa and VIIIa, and because it also reacts with residues 181-310 of the factor IXa heavy chain, we used the computer-modelled structure of the factor IXa heavy chain to select charged surface residues likely to interact with FXC008 and/or factor VIIIa. We made mutations in the region of residues 181-310 of the heavy chain of factor IX, and replaced these amino acids individually with those located at the same position in factor X. The mutated factor IX retained complete clotting activity and thus interacted normally with factor VIIIa. Five mutant proteins (factor IXK214F, factor IXK228R, factor IXE240Q, factor IXK247V, and factor IXN260K) reacted with heavy chain-specific MoAbs FXC008 and A-5. Neither factor IXD276K nor factor IXR248H bound to FXC008. Factor IXR252V had reduced affinity to FXC008. Our results suggest the following: (1) factor IXa residues 214, 228, 240, 247, 248, 252, 260, and 276 are not involved in specific interaction with factor VIIIa; and (2) the FXC008 and factor VIIIa binding sites may not share critical residues.

Antibodies, Monoclonal↗
PubMed1982

Measurement of human factor IXa activity in an isolated factor X activation system.

To determine the functional properties of factor IX isolated from the plasma of CRM+ hemophilia B patients, an assay system using proteins isolated from human plasma had to be developed which would be amenable to kinetic studies under a variety of experimental conditions. The present study describes the activation of factor X by factor IXa, isolated from normal human plasma, in an assay system which allows manipulation of calcium, phospholipid and factor VIIIa concentrations. Initial rate measurements with factor VIIIa present in the system were made by incubating factor VIII with factor Xa immediately before factor IXa assay. With this approach, the initial rate of factor X activation was constant, with little evidence for a lag period. Within the framework of the assay system described in the present study, it should be possible to examine not only genetic variants of factor IX, but also variants of factor VIII, as well as providing a means of routine factor IXa and factor VIII(a) assays.

Blood Coagulation Tests↗