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PubMed1989

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.

Blood Platelets↗
PubMed1996

Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis.

The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit. At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min(-1)) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate of proteolysis of Al subunit. However, the rate of decay of FXase containing the FVIIIaR336I was reduced (0.048 min(-1)) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values.

Amino Acid Sequence↗
PubMed1985

The role of factor VIII in the activation of human blood coagulation factor X by activated factor IX.

The role of factor VIII in the activation of human factor X by factor IXa, Ca2+ and phospholipid has been investigated. Factor VIII stimulated the factor Xa formation after activation by factor Xa or thrombin; the activity of thrombin-activated factor VIII was about 4-fold that of factor Xa-activated factor VIII. The isolated procoagulant moiety of the factor VIII complex behaved identically to the complete complex, whereas the von Willebrand factor moiety did not participate in the factor Xa formation. Thrombin-activated factor VIII complex (factor VIIIa) was used to study the effect of factor VIIIa in kinetic experiments. The results revealed a complex kinetic behaviour, including substrate inhibition and non-linearity of the reaction rate with the enzyme concentration. Using previously obtained insight into the kinetics of factor X activation in the absence of factor VIII, the results were found to support the hypothesis that factor VIIIa participates in the factor Xa formation in a complex with phospholipid-bound factor IXa; the formation of the factor VIIIa-factor IXa complex then increases the catalytic efficiency of the factor IXa by 500-fold.

Blood Coagulation↗
PubMed2004

Activation of factor VIII and mechanisms of cofactor action.

The factor VIII procofactor circulates as a metal ion-dependent heterodimer of a heavy chain and light chain. Activation of factor VIII results from limited proteolysis catalyzed by thrombin or factor Xa, which binds the factor VIII substrate over extended interactive surfaces. The proteases efficiently cleave factor VIII at three sites, two within the heavy and one within the light chain resulting in alteration of its covalent structure and conformation and yielding the active cofactor, factor VIIIa. The role of factor VIIIa is to markedly increase the catalytic efficiency of factor IXa in the activation of factor X. This effect is manifested in a dramatic increase in the catalytic rate constant, k(cat), by mechanisms that remain poorly understood.

Cysteine Endopeptidases↗
PubMed1995

The role of the second growth-factor domain of human factor IXa in binding to platelets and in factor-X activation.

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX(Xegf2), and the wild-type, factor IXwt, produced in embryonic kidney cells 293 were radiolabelled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXawt in the presence of factor VIIIa (2 units/ml) and factor X (1.5 microM). However, under similar experimental conditions, factor IXa(Xegf2) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (Kd) of 1.4 nM, compared with normal factor IXa, factor IXaN (558 sites/platelet; Kd 0.67 nM), or factor IXawt (590 sites/platelet; Kd 0.61 nM). The concentrations of factor IXaN and factor IXawt required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a close correspondence of the Kd,app. for binding of factor IXawt to the factor-X activating complex on activated platelets to the Kd obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa(Xegf2) showed a decreased affinity (Kd 1.5 nM), in agreement with results of binding studies. These studies with factor IX(Xegf2) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.

Binding Sites↗
PubMed2006

Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism.

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties and inhibits factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The mechanism and molecular target for intrinsic tenase inhibition were determined and compared with inhibition by low-molecular-weight heparin (LMWH). DHG inhibited factor X activation in a noncompetitive manner (reduced V(max(app))), with 50-fold higher apparent affinity than LMWH. DHG did not affect factor VIIIa half-life or chromogenic substrate cleavage by factor IXa-phospholipid but reduced the affinity of factor IXa for factor VIIIa. DHG competed factor IXa binding to immobilized LMWH with an EC(50) 35-fold lower than soluble LWMH. Analysis of intrinsic tenase inhibition, employing factor IXa with mutations in the heparin-binding exosite, demonstrated that relative affinity (K(i)) for DHG was as follows: wild type > K241A > H92A > R170A > > R233A, with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity and the potency of LMWH for intrinsic tenase. DHG also accelerated decay of the intact intrinsic tenase complex. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interactions.

Antithrombins↗
PubMed1985

The functional defect of factor VIII Leiden, a genetic variant of coagulation factor VIII.

Factor VIII Leiden is a genetic variant of coagulation factor VIII which has been detected in the plasma of a patient with mild haemophilia A. In this patient's plasma factor VIII procoagulant antigen was in 5-fold excess over factor VIII procoagulant activity, indicating the presence of an abnormal factor VIII molecule. The variant factor VIII was isolated from the patient's plasma, and its functional properties were studied in a factor X-activating system consisting of purified components. The isolated factor VIII Leiden was normally activated by factor Xa and by thrombin, but the activity of the factor VIIIa was about 3% of normal. The defect of factor VIIIa Leiden was studied by comparison with normal factor VIIIa in kinetic experiments of factor Xa formation. The results support the hypothesis that factor VIIIa Leiden has a reduced affinity for phospholipid-bound factor IXa in the intrinsic factor X-activating complex.

Calcium↗
PubMed2003

Heparin inhibits the intrinsic tenase complex by interacting with an exosite on factor IXa.

The specific molecular target for direct heparin inhibition of factor X activation by intrinsic tenase (factor IXa-factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa-factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa-PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa-PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa-PL complex. LMWH induced a modest decrease in factor IXa-factor VIIIa affinity [K(D(app))] on PL vesicles that did not account for the inhibition. In contrast, LMWH caused a substantial reduction in factor IXa-factor VIIIa affinity in the presence of C6PS that fully accounted for the inhibition. Factor IXa bound LMWH with significantly higher affinity than factor X by competition solution affinity analysis, and the K(D(app)) for the factor IXa-LMWH complex agreed with the K(I) for inhibition of the factor IXa-PL complex by LMWH. Thus, LMWH binds to an exosite on factor IXa that antagonizes cofactor activity without disrupting factor IXa-factor VIIIa assembly on the PL surface. This exosite may contribute to the clinical efficacy of heparin and represents a novel target for antithrombotic therapy.

Binding Sites↗
PubMed2005

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.

Alanine↗
PubMed1998

Blood coagulation factor IX residues Glu78 and Arg94 provide a link between both epidermal growth factor-like domains that is crucial in the interaction with factor VIII light chain.

Recently, we established that mutations at calcium-binding sites within the first epidermal growth factor (EGF)-like domain of activated factor IX affect its interaction with factor VIIIa (Lenting, P. J., Christophe, O. D., ter Maat, H., Rees, D. J. G., and Mertens, K. (1996) J. Biol. Chem. 271, 25332-25337). In the present study, we have investigated the functional role of residue Glu78, which is not involved in calcium binding. Glu78 is also located in the first EGF-like domain and, when mutated to Lys, is associated with severe hemophilia B. Because Glu78 is conserved in related vitamin K-dependent proteins, it is difficult to understand how a mutation at this position is associated with factor IX-specific function. In this study, we addressed the hypothesis that Glu78 exerts its biological activity by interacting with another residue. One candidate was found to be the second EGF-like domain residue, Arg94, which is also associated with severe hemophilia B when mutated. We constructed a series of mutants that included mutations at position 78 alone (Glu78 to Lys/Glu78 to Asp) or at both positions 78 and 94 (Glu78 to Lys and Arg94 to Asp). The functional parameters of immunopurified and activated mutants were compared with normal activated factor IX. Mutants were indistinguishable from normal factor IXa in cleaving the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide or activating factor X in the absence of factor VIIIa. In contrast, in the presence of factor VIIIa, factor IXa Glu78 to Asp and factor IXa Glu78 to Lys/Arg94 to Asp were stimulated to the same extent as normal factor IXa, whereas factor IXa Glu78 to Lys was markedly less stimulated (140-fold versus 2,000-fold). This suggests that residues 78 and 94 should carry an opposite charge for a normal interaction of factor IXa to factor VIIIa. This hypothesis was confirmed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy (Ser558-Gln565) or light chain (Glu1811-Lys1818) and in direct binding studies. We propose that residues 78 and 94 link both EGF-like domains and thereby maintain the integrity of the factor VIII light chain binding site.

Animals↗
PubMed1995

Anticoagulant actions of tissue factor pathway inhibitor on tissue-factor-dependent plasma coagulation.

The initiation and propagation of in vivo coagulation are thought to be catalyzed by factor VIIa-tissue factor (an activator of factor X and factor IX) and factor IXa-factor VIIIa (an activator of factor IX), respectively. The enzymatic activity of factor VIIa-tissue factor is abrogated by tissue factor pathway inhibitor (TFPI), which anchors a quaternary complex consisting of equimolar TFPI, factor Xa, factor VIIa, and tissue factor in which both factor Xa and factor VIIa are inactive. This study compared the anticoagulant effectiveness of TFPI (which also inactivates prothrombinase-bound factor Xa), hirudin (which inactivates thrombin), and heparin (which catalyzes the inactivation of factor Xa and thrombin by antithrombin III). Factor X and prothrombin activation were initiated by adding 5 pM r-tissue factor in a suspension of coagulant phospholipids and CaCl2 to defibrinated plasma. Compared on the basis of their ability to delay the initiation of and inhibit factor X and prothrombin activation, the anticoagulant effectiveness of 0.5 nM TFPI was greater than those of 10 nM hirudin and approximately 100 nM (0.1 unit/mL) heparin. However, a 100-fold molar excess of TFPI over tissue factor could not abrogate factor X and prothrombin activation in plasma. These results suggest that propagation of tissue-factor dependent coagulation is catalyzed by factor IXa-factor VIIIa, which unlike factor VIIa-tissue factor, is not inactivated by TFPI.

Animals↗
PubMed1997

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate.

The decay of human coagulation factor VIIIa has been studied by kinetic methods that ensure no interference through proteolytic feedback. The rate of decay of factor VIIIa activity was found to vary with the activator used to activate factor VIII. Thrombin-activated factor VIII-von Willebrand factor complex (fVIII-vWf) decayed at a rate of 0.31 min-1, whereas factor Xa-activated fVIII-vWf decayed at 0.11 min-1 under the same conditions. Factor VIII free of von Willebrand factor (factor VIII: C), although decaying at a generally slower rate after activation, still showed a dependence of decay rate on activator: thrombin-activated factor VIII:C decaying at a rate of 0.06 min-1, and factor Xa-activated factor VIII: C at 0.01 min-1. Readdition of von Willebrand factor (18 micrograms/ml) to factor VIII:C did not alter the observed activity or decay rate. The decay of the two species of factor VIIIa was studied, using the fVIIIa-vWf complex, in the presence of varying levels of factor IXa. Plots of reciprocal decay rates vs factor IXa concentration were linear, and nearly parallel for the two factor VIIIa species, with a mean slope of 0.56 min.nM-1. In addition to these studies, we have confirmed previous studies showing that the two forms of factor VIIIa differ in cofactor activity, but they do so in the same ratio as in their decay rates. We suggest that this difference and that observed in decay rate have a common cause, and incorporate this into a potential kinetic model of factor VIII activation and decay.

Animals↗
PubMed2000

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

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

Amino Acid Sequence↗
PubMed1992

Factor IXa-factor VIIIa-cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo.

We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.

Adolescent↗
PubMed2004

Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain.

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys36 cleavage site, generating the A137-372 intermediate with approximately 20% the catalytic efficiency of wild type. This defect likely resulted from an approximately 4-fold increase in Km for the A1 substrate because kcat values for the wild type and mutant were equivalent. Cleavage of the A1-A2 domain junction by factor Xa R240A was not blocked by the 337-372 peptide. Studies using mutant factor VIII where clustered acidic residues in the 337-372 segment were replaced by Ala showed that a factor VIIIa D361A/D362A/D363A mutant possessed a approximately 1.6-fold increase in Km for factor X compared with wild type. However, similar Km values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase.

Alanine↗
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↗
PubMed1996

The role of electrostatic interactions in the assembly of the factor X activating complex on both activated platelets and negatively-charged phospholipid vesicles.

Factor X was activated by factor IXa on the surface of either activated platelets or artificial lipid vesicles in the presence of different NaCl concentrations. The Vmax of reactions using platelets was optimal at physiologic [NaCl] both in the absence and in the presence of factor VIIIa. In contrast, the Vmax of reactions using vesicles decreased with increasing [NaCl] in the absence of factor VIIIa, and increased with increasing [NaCl] when cofactor was present. In the absence of factor VIIIa, the EC50FIXa' although stable to changes in [NaCl] in platelet-supported reactions, was found to increase significantly as [NaCl] increased in vesicle-supported reactions and correlated with the decreased Vmax. Thus, in contrast to platelet-supported reactions, enzyme interaction with negatively-charged vesicles was highly dependent upon electrostatic interactions. In the presence of factor VIIIa, the EC50FIXa of vesicle-supported reactions decreased with increasing [NaCl], indicating that interactions between FIXa and FVIIIa can increase enzyme affinity when fewer ionic interactions are favored. The EC50FVIIIa was insensitive to changes in [NaCl] on both surfaces. The K(m)app derived from platelet-supported titrations of factor X was lowest just above physiological [NaCl], whereas on vesicles K(m)app was minimal at the lowest [NaCl] tested. Thus, the direct interaction of factor X and factor IXa with the artificial lipid surface is highly dependent upon ionic interactions with the negatively-charged polar heads of phospholipids. However, the interaction of factor IXa and factor X with the activated platelet surface must rely both on electrostatic interactions with lipid and on other interactions provided by surface proteins.

Animals↗