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PubMed1980

Standardization of Factor VIII. II. A British Standard for Factor VIII related antigen.

A collaborative study on factor VIII related antigen (VIII R:Ag) has been carried out, involving 11 laboratories in the U.K. Samples of two different freeze-dried plasmas were assayed against participants' own local standards by the Laurell electroimmunoassay method. There was reasonably good agreement on the relative potencies of the two freeze-dried plasmas, but there were considerable differences in the VIII R:Ag content of the plasma pools used as local standards, with values ranging from 83% to 129% of the mean. All participants agreed on the need for a standard for VIII R:Ag, and that the unit be defined by the mean of the local standards. Accordingly, freeze-dried plasma 66/355 was established as the 1st British Standard for factor VIII related antigen, with an assigned potency of 1.05 units per ampoule.

Antigens↗
PubMed1985

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.

Antibodies, Monoclonal↗
PubMed2006

Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg(593) to Cys substitution.

Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.

Amino Acid Substitution↗
PubMed1993

Associations of factor VIII and von Willebrand factor with age, race, sex, and risk factors for atherosclerosis. The Atherosclerosis Risk in Communities (ARIC) Study.

Several coagulation proteins have been implicated as possible risk factors for the development of atherosclerotic diseases, among which are factor VIII and von Willebrand factor. As part of the Atherosclerosis Risk in Communities (ARIC) Study, a prospective study designed to assess risk factors for the development of atherosclerotic diseases, baseline measurements of factor VIII and von Willebrand factor (vWF) were performed to determine their relationship to the development of atherosclerosis. We herein report the associations of factor VIII and vWF with constitutional, lifestyle, and biochemical factors. Factor VIII and vWF were strongly correlated with each other (r = 0.73), and, therefore, had similar associations with risk factors. Mean levels of both factors were higher in women than in men, in blacks than in whites, and increased with age. In univariate analysis, both were positively associated with diabetes, body mass index, waist-to-hip ratio, serum insulin, and plasma triglycerides. Both were negatively associated with alcohol intake, educational level, physical activity (with some exceptions), and HDL-cholesterol. No correlations were observed between factor VIII or vWF and plasma LDL-cholesterol or lipoprotein(a). Although factor VIII was negatively associated with smoking in both sexes, vWF was not associated with smoking status. Most of these associations were confirmed in multivariate analysis. The strongest associations observed were of factor VIII and vWF with race and diabetes. In multivariate analysis, blacks had factor VIII and vWF levels 15 to 18 percentage points higher than whites, and diabetics had factor VIII and vWF levels 11 to 18 percentage points higher than non-diabetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

cis-acting elements and transcription factors involved in the promoter activity of the human factor VIII gene.

Factor VIII is a glycoprotein that is essential for blood coagulation. Although factor VIII mRNA has been detected in a variety of human tissues, hepatocytes are considered to be the major source of plasma factor VIII. In this report we demonstrate that the 5'-flanking region of the factor VIII gene is able to transcribe a luciferase reporter gene in three human liver-derived cell lines: PLC/PRF/5, Chang, and HepG2. DNase I footprinting showed the presence of 19 protein binding sites (labeled A to S, proximal to distal) distributed along the region from nucleotide -1175 to -9 of the factor VIII promoter (+1 refers to the translation initiation codon, ATG). Functional analysis of 5' and 3' deletion mutants of the promoter region in PLC/PRF/5 cells revealed that the region from -279 to -64, including sites B to D, contains all the necessary elements for maximal promoter activity. By using electrophoretic mobility shift assays with nuclear extracts and purified transcription factors, and antibody supershift assays we were able to characterize four liver-enriched factors and one ubiquitous transcription factor interacting with the proximal promoter binding sites (sites A to E): hepatocyte nuclear factor (HNF) 1 (site A), NF kappa B (site B), C/EBP alpha and C/EBP beta (proximal and distal regions of site C, and site D), and HNF4 (site E). Additionally, mutation of the putative TATA box GATAAA (positions -201 to -196) to GACCGA resulted in less than 2-fold decrease in promoter activity, suggesting that the putative TATA box is not essential for factor VIII promoter activity. These results significantly contribute to the understanding of the control of the hepatic transcription of the factor VIII gene.

Animals↗
PubMed1993

Structural study of the sugar chains of porcine factor VIII--tissue- and species-specific glycosylation of factor VIII.

The asparagine-linked sugar chains of blood coagulation factor VIII purified from porcine plasma were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and the sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns. Structural study of each oligosaccharide by sequential exoglycosidase digestion and by methylation analysis revealed that porcine factor VIII contains high mannose-type and bi-, tri-, and tetraantennary complex-type sugar chains. Sixty-seven percent of the complex-type sugar chains contained the Gal alpha 1-->3Gal group, and 23% of the biantennary complex-type sugar chains contained the bisecting N-acetylglucosamine residue. These structures were not detected in the sugar chains of human plasma factor VIII. An in vitro competition study of von Willebrand factor and anti-Gal antibody for binding to factor VIII revealed that von Willebrand factor prevented antibody binding to Gal alpha 1-->3Gal groups in porcine factor VIII sugar chains. This suggests that anti-Gal antibody present in human plasma may not interact with the sugar chains of therapeutic porcine factor VIII. Reverse-transcription polymerase chain reaction was used to identify porcine tissues producing FVIII mRNA. These studies revealed that the kidney is one of the major tissues expressing factor VIII which may contain the sugar chains with the bisecting N-acetylglucosamine residue.

Animals↗
PubMed1988

Induction of immune tolerance in patients with hemophilia and antibodies to factor VIII by combined treatment with intravenous IgG, cyclophosphamide, and factor VIII.

The development of antibodies to factor VIII is a serious complication of the treatment of patients with hemophilia A. We successfully induced immune tolerance in patients with such antibodies with a new treatment protocol, which combined factor VIII, cyclophosphamide, and high-dose intravenous IgG, followed by regular prophylactic treatment with factor VIII. This protocol has now been used in 11 patients with hemophilia A, of whom 9 had a strong antibody response. When the initial concentration of antibodies exceeded 3 Malmö inhibitor units (corresponding to about 10 Bethesda units) per milliliter, treatment was preceded by adsorption of antibody to protein A. After two to three weeks of the combined treatment, factor VIII coagulant antibodies had disappeared in 9 of the 11 patients; in 8 of these 9 patients the half-life of infused factor VIII had normalized. The tolerant state appears to be stable after a median of 30 months. Two patients did not respond to the treatment. Because earlier treatment with factor VIII and cyclophosphamide or with factor VIII and IgG had been ineffective in these patients, our experience suggests that all three components of the protocol are required for the successful induction of tolerance to factor VIII.

Adolescent↗
PubMed2002

New characteristics of anti-factor VIII inhibitor antibody epitopes and unusual immune responses to Factor VIII.

Treatment of individuals with severe hemophilia A by plasma-derived or recombinant factor VIII leads to the production of anti-factor VIII antibodies in approximately 30% of such patients. Because some of these antibodies inactivate factor VIII, they are considered a major factor in preventing optimal therapeutic treatment. Factor VIII is a cofactor that must bind to factors IX and X and phospholipids in order for normal blood coagulation to occur. The inhibition of factor VIII activity is due to binding by anti- factor VIII antibodies in the patient plasma to the same sites required for factors IX and X and phospholipid binding. Previously, inhibitor epitopes were localized to the A2, A3, and C2 domains and to a region of acidic amino acids between the A1 and A2 domains. Inhibitor binding to these domains prevented factor VIII binding to factor IXa (A2, A3), factor Xa (C2), and phospholipids (C2), and binding to the acidic region interfered with factor X binding. Antibody binding to a minor C2 domain epitope slowed activated factor VIII release from von Willebrand factor (vWF) and interfered with factor Xa binding to factor VIII.

Amino Acid Sequence↗
PubMed1988

A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X.

Two aspects of the activation of factor X by the intrinsic clotting pathway have been studied in purified human systems, in the presence of either purified phosphatidylserine:phosphatidylcholine vesicles (PS:PC) or platelets activated with ionophore A23187: (1) the activation of factor VIII by factor Xa and by thrombin, and (2) the activation of factor X by the factor IXa/VIIIa complex. Factor VIII activation by thrombin was unaffected in either rate or extent by the presence of PS:PC or activated platelets. In contrast, factor VIII activation by factor Xa required either PS:PC or platelets. The products of optimal factor VIII activation by the two enzymes, designated factor VIIIa(T) and factor VIIIa(Xa), are kinetically different in the activation of factor X by factor IXa, factor VIIIa(T) being approximately twice as active (in factor X activation) as factor VIIIa(Xa) in the presence of PS:PC or platelets. Factor VIIIa(Xa) can be converted to the more active VIIIa(T) by thrombin treatment, but the activity of factor VIIIa(T) is unchanged by factor Xa treatment. Factor X activation was also studied with optimally activated factor VIIIa(T), in the presence of PS:PC or activated platelets, as a function of factor IXa concentration in order to determine the apparent dissociation constant for the factor IXa-VIIIa interaction in the two cases. Activated platelets increased the apparent affinity more than fivefold.

Blood Coagulation↗
PubMed1984

Heterogeneity of human procoagulant factor VIII (VIIIC) antibodies in their reaction with factor VIII related antigen (VIIIRAG).

Human inhibitors to procoagulant factor VIII (VIIIC) were tested for possible reactivity with factor VIII related antigen (VIIIRAg) by a modified VIIIRAg immunoradiometric assay (IRMA). Inhibitor IgG's were screened for anti-VIIRAg by competition with I125 labelled VIIIRAg antibodies for common antigenic determinants using either an VIIIRAg concentrate from which factor VIII coagulant antigen (VIIICAg) had been dissociated, or normal plasma (containing VIIIRAg and VIIICAg) as an antigen source. There was no evidence of VIIIRAg antibodies in the five haemophilic VIIIC inhibitor IgG's tested but low titre VIIIRAg antibodies were detected in one spontaneous VIIIC inhibitor IgG.

Antibodies↗
PubMed1978

Survival of 125iodine-labeled Factor VIII in normals and patients with classic hemophilia. Observations on the heterogeneity of human Factor VIII.

Radiolabeled human Factor VIII was used to study its survival in normals and patients with classic hemophilia, and to study the heterogeneity of Factor VIII; Purified Factor VIII was radiolabeled with 125iodine (125I-VIII) without loss of its structural integrity. The survival of 125I-VIII was studied in six normals and six hemophiliacs of whom four of the hemophiliacs had received transfusions with normal cryoprecipitate before the 125I-VIII infusion. No significant difference was observed between the disappearance of Factor VIII coagulant activity and radioactivity in these hemophiliacs. 125I-VIII in plasma showed a biphasic disappearance with an average t1/2 of 2.9 +/- 0.4 h (SEM) for the first phase and 18.6 +/- 0.7 h (SEM) for the second phase, respectively. The survival of 125I-VIII was similar comparing normals and hemophiliacs. The highest molecular weight forms of Factor VIII disappear more rapidly than the lower molecular weight ones. This was established by analysis of the fractions obtained by gel chromatography of plasma collected at several times after infusion and by analysis of the in vivo disappearance of three subfractions of Factor VIII. The fraction of 125I-VIII binding to platelets in the presence of ristocetin (containing the highest molecular weight forms of Factor VIII including the ristocetin cofactor) represented about 50% of the radioactivity present in plasma after infusion and showed a t 1/2 of 11.7 +/- 0.9 h (SEM) for the second phase. The fraction, which was recovered in cryoprecipitate of the recipient's plasma, represented about 90% of the initial radioactivity and showed a t 1/2 of 16.3 +/- 0.8 h (SEM) for the second phase. The fraction of 125I-VIII remaining in the cryosupernatant plasma (containing low molecular weight forms of Factor (VIII) showed a t 1/2 of 27.2 +/- 1.1 h (SEM). The first phase of the disappearance of 125I-VIII is caused in part by the disappearance of the highest molecular weight forms, which are possibly removed by the reticuloendothelial system.

Adult↗
PubMed1994

Continuous infusion of monoclonal antibody-purified factor VIII: rational approach to serious hemorrhage in patients with allo-/autoantibodies to factor VIII.

Hemorrhage in a patient with factor VIII inhibitor is associated with increased morbidity and mortality. Treatment with factor IX complex concentrates or recombinant factor VIIa (rVIIa) may not control bleeding and may induce thrombosis. In this study, continuous infusion of a monoclonal antibody-purified factor VIII [correction of factor VII] concentrate (Monoclate-P) was used successfully in two hemophilic patients with factor VIII alloantibodies and one nonhemophilic patient with a factor VIII autoantibody. In two patients, hemorrhage was life-threatening, and, in one, bleeding did not stop with repeated infusions of activated factor IX complex concentrates. The patients' ages ranged from 4 to 15 years, and the inhibitor levels from 6 to 300 Bethesda units/ml. Clinical hemostasis was excellent, and in vivo recovery of infused factor VIII was achieved. When an excess of monoclonal factor VIII was added to the inhibitor plasma in vitro, a stable level of residual factor VIII activity was noted after an initial rapid loss. This second-order reaction occurs in plasmas of patients with type I factor VIII inhibitors. In one patient, we showed that the saturation dose of the factor VIII inhibitor predicted in vivo recovery of factor VIII:C. These data emphasize the importance of characterizing the kinetic reactions of the factor VIII inhibitor. Furthermore, we confirm previous reports that continuous infusion of monoclonal factor VIII is a safe and effective treatment of patients with factor VIII inhibitors in whom hemorrhage is either life-threatening or refractory to standard treatment.

Adolescent↗
PubMed1996

Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the factor VR506Q mutation.

Factor V and protein S are cofactors of activated protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effects of factor V and protein S were quantitated in a reaction system in which plasma factor VIII was inactivated by APC and the loss of factor VIII activity was monitored in a factor X-activating system in which a chromogenic substrate was used to probe factor Xa formation. Factor V increased the rate of APC-mediated factor VIII inactivation in a dose-dependent manner in representative plasma samples with protein S or factor V deficiency, abnormal factor V (heterozygous or homozygous for factor VR506Q), or a combination of heterozygous protein S deficiency and heterozygous factor VR506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response in these plasmas was corrected by addition of protein S, indicating that both factor V and protein S are required for optimal inactivation of factor VIII by APC. The effects of factor V and protein S were also studied in a reaction system with purified proteins. APC-catalysed factor VIII inactivation was enhanced 3.7-fold in the presence of 1.1 nM factor V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was 11-fold. Factor V is a more potent cofactor than protein S, as can be concluded from the fact that 0.04 nM factor V gave the same stimulation as 2.4 nM protein S. Protein S lost its cofactor function after complexation with C4b binding protein, which indicates that it is free protein S that acts as a cofactor. To investigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lost when factor V was activated with thrombin or with the factor V activator from Russell's viper venom. These data indicate that optimal inactivation of factor VIII by APC requires the presence of an intact factor V molecule and free protein S.

Arginine↗
PubMed2006

High plasma levels of factor VIII: an important risk factor for isolated pulmonary embolism.

OBJECTIVE: The aim of the study was to investigate whether factor V Leiden and prothrombin G20210A mutations, elevated levels of factor VIII and factor IX are associated with pulmonary embolism (PE). METHODS: Sixty-four patients with objectively documented PE and 64 control subjects were included in this study. The authors divided the 64 subjects with PE into those with PE and deep vein thrombosis (combined form of venous thromboembolism, n = 26) and those with PE without deep vein thrombosis (isolated PE n = 38). RESULTS: There was no significant difference between the PE groups and the control subjects with regard to the presence of factor V Leiden and prothrombin mutations and elevated levels of factor IX. Using the 90th percentile measured in control subjects (P(90) = 168 U/dL) as a cut-off point for factor VIII levels, the authors found an 11-fold increased risk for both isolated PE patients and patients with a combined form of venous thromboembolism who have factor VIII levels >168 U/dL compared with individuals having factor VIII levels below this cut-off point. The risk was not affected by adjustments for other possible risk factors. CONCLUSIONS: Elevated plasma factor VIII levels were found to be a significant, independent risk factor for PE.

Adult↗
PubMed1976

Antibodies to factor VIII. V. Patterns of immune response to factor VIII in hemophilia A.

The natural history of factor VIII antibodies was studied in 20 severe, multitransfused hemophiliacs. Two patterns of humoral immune reactivity were observed. In one group of ten, who developed antibodies after an average of 22 cumulative exposure days to factor VIII, the antibody titers increased after each antigenic stimulation or persisted for years in the absence of transfusion. These patients were designated as high-responding hemophiliacs. In the second group of ten patients, the factor VIII neutralizing activity appeared after a longer exposure period (48 days). Antibody titers remained low, and there was no significant difference in individual titers before and 8--20 days following transfusion. Antibody affinity did not increase after renewed antigenic challenge. This pattern characterized low-responding hemophiliacs. The latter group of patients benefited from repeated placement therapy required by the clinical situation.

Antibodies↗
PubMed2005

Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A.

Inhibitor development is the major treatment complication in children with severe hemophilia A. It is not clear whether the risk of inhibitors is higher with recombinant factor VIII or with plasma-derived factor VIII. We used multivariate analysis to compare 2 cohorts of previously untreated patients (PUPs) with severe hemophilia A: 62 patients treated with the same brand of high-purity plasma-derived FVIII (pFVIII) containing von Willebrand factor (VWF) and 86 patients treated with full-length recombinant FVIII (rFVIII). In addition to the usual end points (all inhibitors, high inhibitors), we also examined a third end point (high inhibitors and/or immune tolerance induction). The risk of inhibitor development was higher in patients treated with rFVIII than in patients treated with pFVIII, regardless of other risk factors (F8 genotype; nonwhite origin; history of inhibitors in patients with a family history of hemophilia; age at first FVIII infusion). The adjusted relative risk (RRa) for inhibitor development with rFVIII versus pFVIII was 2.4 (all inhibitors), 2.6 (high inhibitors), and 3.2 (high inhibitors and/or immune tolerance induction), respectively, depending on the end point (above). The pathophysiology of this large effect must be understood in order to improve the characteristics of recombinant products and to reduce the incidence of inhibitors to FVIII.

Autoantibodies↗
PubMed1995

The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor.

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid-phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.

Adult↗
PubMed2006

The interface between the EGF2 domain and the protease domain in blood coagulation factor IX contributes to factor VIII binding and factor X activation.

The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the protease domain. The role thereof has been addressed in this study. Recombinant FIX variants FIX-N92A, FIX-N92H, FIX-Y295A, and FIX-F299A were produced in 293 cells. After activation, the purified mutants were analyzed for a variety of functional parameters. None of these substitutions had a major effect on the interaction with antithrombin or the cleavage of the chromogenic substrate CH(3)SO(2)-d-CHG-Gly-Arg-p-nitroanilide. All FIXa mutants, however, exhibited a reduced level of factor X (FX) activation. Defective proteolytic activity occurred both in the absence and in the presence of activated factor VIII (FVIIIa). All mutants also exhibited a reduced level of FX activation in the absence of phospholipids. This suggests that putative interdomain contacts involving residues Asn(92), Tyr(295), and Phe(299) affect reactivity toward FX. Detailed kinetic studies in the presence of phospholipids and FVIIIa revealed substrate inhibition, particularly for mutants FIXa-N92A and FIXa-N92H. Surface plasmon resonance demonstrated that the same replacements weaken the association with the isolated factor VIII (FVIII) A2 domain and the FVIII light chain. This implies a defect in the formation of the FX-activating complex that is membrane-independent. We conclude that contacts between EGF2 and the protease domain of FIXa are crucial for FIXa enzymatic activity and for the assembly of the FX-activating complex.

Aminophenols↗