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At least 19 recordsLinked to original sources

PubMed1997

Synergistic cofactor function of factor V and protein S to activated protein C in the inactivation of the factor VIIIa - factor IXa complex -- species specific interactions of components of the protein C anticoagulant system.

Human factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC. For this purpose, the effects of protein S and factor V on APC-mediated inactivation of factor VIIIa were studied using purified APC, protein S and factor V of human and bovine origin. The factor VIIIa investigated here was part of a Xase complex (i.e. factor IXa, factor VIIIa, phospholipid and calcium) and the APC-mediated inhibition of factor VIIIa was monitored by the ability of the Xase complex to activate factor X. Synergistic APC-cofactor function of factor V and protein S was demonstrated in the bovine system. The effect of bovine APC was potentiated by bovine protein S but not by human protein S, whereas both human or bovine protein S stimulated the function of human APC. Factor V did not express species specificity in its APC-cofactor activity even though bovine factor V was more potent than its human counterpart. Recombinant human/bovine protein S chimeras were used to demonstrate that the thrombin sensitive region and first epidermal growth factor-like module of protein S determine the species specificity of the APC-protein S interaction. In conclusion, both human and bovine factor V were found to express APC-cofactor activity which depends on the presence of protein S. The species specificity of APC was shown to be caused by the interaction between APC and protein S.

Animals↗

PubMed1997

Partial activation of the factor VIIIa-factor IXa enzyme complex by dihexanoic phosphatidylserine at submicellar concentrations.

Phosphatidylserine (PS)-containing membranes increase the kcat of the factor VIIIa-factor IXa enzyme complex by more than 1000-fold. While PS supports specific, high-affinity membrane binding of factor VIIIa and factor IXa, it is not known whether PS is the lipid that activates the membrane-bound complex. It is also not known whether PS or other activating lipids must reside in the two-dimensional membrane matrix for efficacy. We have found that submicellar concentrations of dihexanoic phosphatidylserine (C6PS) increase the activity of the factor VIIIa-factor IXa complex in a biphasic manner with half-maximal concentrations of 0.2 and 1.6 mM while the micelle-forming concentration is 4.0 mM. Increased cleavage of factor X at 0.25 mM C6PS was due to a 25-fold enhancement of the kcat and a 30-fold increase in the affinity of factor VIIIa for factor IXa. C6 phosphatidylethanolamine and C6 phosphatidic acid, but not C6 phosphatidylcholine, also accelerated the Xase complex, indicating that kcat enhancement has less structural specificity than membrane binding. Submicellar C6PS enhanced activity of factor IXa in the absence of factor VIIIa, but the effect was due to a decreased KM rather than an increased kcat. These results suggest that activation of the factor VIIIa-factor IXa complex can result from binding of individual C6PS molecules or small aggregates in the absence of a membrane bilayer. They provide a model system in which the phospholipid-induced activation may be distinguished from membrane-binding of the enzyme complex.

Calcium↗

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↗

PubMed1992

Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa.

Factor VIIIa is a heterotrimer of the factor VIII heavy chain-derived A1 and A2 subunits plus the factor VIII light chain-derived A3-C1-C2 subunit. While the A1 and A3-C1-C2 subunits can be isolated as a stable dimer, the A2 subunit is weakly associated with the dimer. In the human protein, the association of A2 with dimer is reversible and governed by a pH-dependent dissociation constant. Using the specific activity of factor VIIIa as an indicator of trimer concentration, the Kd (pH 6.0) was determined to be 28 nM whereas at the more physiologic pH (pH 7.4) this value was approximately 260 nM. Results from pH shift experiments confirmed the reversible binding of A2 to dimer as did the capacity for high levels of exogenous A2 subunit to inhibit the spontaneous decay of factor VIIIa activity. A2 subunit associated with the A1 subunit in the A1/A3-C1-C2 dimer based upon the capacity for free A1 subunit to inhibit the reconstitution of factor VIIIa from A2 subunit and dimer. These results indicate that the primary mechanism for the spontaneous decay of human factor VIIIa is the reversible dissociation of A2 subunit from the A1 subunit of the A1/A3-C1-C2 dimer.

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa.

Factor VIIIa is a heterotrimer of A1,A2 and A3-C1-C2 subunits which is labile due to a relatively weak affinity interaction between the A2 subunit and the Me(2+)-linked A1/A3-C1-C2 dimer. Previously we speculated that the acidic region at the COOH terminus of the A1 subunit was involved with the A2 subunit retention. This region, delineated by factor VIII residues 337-372, was chemically synthesized. Both the peptide, designated FVIII337-372, and an IgG fraction prepared from rabbit anti-FVIII337-372 antiserum inhibited the reconstitution of factor VIIIa from A1/A3-C1-C2 dimer plus A2 subunit. A primary component of the inhibitory activity of the peptide was attributed to its acidic nature based upon similar inhibition of factor VIIIa reconstitution using a synthetic polymer of aspartic acid. Trypsin cleaved the peptide at Arg359 and the resultant two fragments were isolated. Inhibitory activity was associated with the NH2-terminal fragment which contained 10 of the 13 acidic residues present in the original peptide. The fluorescence of a dansylated FVIII337-372 was enhanced 2-fold by A2 subunit and this effect was reversed by addition of excess unmodified peptide. The inhibitory activity of FVIII337-372 was attenuated by the presence of Ca2+. Ca2+ also inhibited the reconstitution of factor VIIIa in the absence of peptide and increased the rate and extent of factor VIIIa decay, suggesting that Ca2+ effectively shielded charges important for the intersubunit interactions. The above results support a role for this acidic region in the association of A2 subunit with A1/A3-C1-C2 dimer.

Amino Acid Sequence↗

PubMed1996

Activation of the factor VIIIa-factor IXa enzyme complex of blood coagulation by membranes containing phosphatidyl-L-serine.

Factor IXa, a serine protease of blood coagulation, functions at least 100,000 times more efficiently when bound to factor VIIIa on a phospholipid membrane than when free in solution. We have utilized the catalytic activity of the factor VIIIa-factor IXa complex to report the effect of phospholipid membranes on binding of factor IXa to factor VIIIa and on enzymatic cleavage of the product. The apparent affinity of factor IXa for factor VIIIa was 10-fold lower in the absence of phospholipid membranes with a KD of 46 nM versus 4.3 nM with phospholipid membranes. The Km for activation of factor X by the factor VIIIa-factor IXa complex was 1700 nM in solution, 70-fold higher than the value of 28 nM when bound to membranes containing phosphatidyl-L-serine, phosphatidylethanolamine, and phosphatidylcholine at a ratio of 4:20:76. The largest effect of phosphatidyl-L-serine-containing membranes on the factor VIIIa-factor IXa complex was the accelerated rate of peptide bond cleavage, with the k(cat) increased by 1,500-fold from 0.022 to 33 min-1. Membranes in which phosphatidyl-L-serine was replaced by phosphatidyl-D-serine, phosphatidic acid, or phosphatidylglycerol were at least 10-fold less effective for enhancing the k(cat). Thus, while membranes containing phosphatidyl-L-serine enhance condensation of the enzyme with its cofactor and substrate, their largest effect is activation of the assembled factor VIIIa-factor IXa enzyme complex.

Animals↗

PubMed1994

Factor IXa protects factor VIIIa from activated protein C. Factor IXa inhibits activated protein C-catalyzed cleavage of factor VIIIa at Arg562.

Factor VIIIa is inactivated by both factor IXa and activated protein C. The latter protease rapidly attacked a site at Arg562 (A2 subunit), whereas both proteases slowly cleaved factor VIIIa at Arg336 (A1 subunit). Cofactor inactivation catalyzed by activated protein C was 8-fold faster than that catalyzed by factor IXa. Simultaneous reaction of factor VIIIa with the two enzymes resulted in a rate of inactivation intermediate to that observed for the individual proteases. Under these conditions, the activated protein C-catalyzed cleavage at Arg562 was inhibited such that cofactor inactivation resulted primarily from cleavage at Arg336. Substitution of factor IXa modified in its active site with 6-(dimethylamino)-2-naphthalenesulfonyl-glutamylglycylarginyl++ + chloromethyl ketone (DEGR-IXa) for the native enzyme yielded a similar rate of activated protein C-catalyzed cleavage at the A1 site, whereas cleavage at the A2 site was virtually eliminated. However, the inclusion of protein S resulted in a marked increase in cleavage at the A2 site that correlated with an increased rate of cofactor inactivation. Active site-modified activated protein C inhibited the factor IXa-dependent enhancement of factor VIIIa reconstitution from isolated subunits. In addition, the factor VIIIa-dependent fluorescence enhancement of DEGR-activated protein C was inhibited by EGR-IXa. These results indicate that factor IXa can reduce the rate of activated protein C-catalyzed cofactor inactivation by selectively blocking cleavage at the A2 domainal site, an effect reversed by protein S. One mechanism consistent with the reciprocal inhibitory effects of the proteases is that activated protein C and factor IXa occupy overlapping sites on the cofactor. Thus, factor IXa may protect factor VIIIa by preventing activated protein C binding.

Amino Acid Chloromethyl Ketones↗

PubMed1991

Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit.

Heterodimeric human factor VIII was proteolytically activated by catalytic levels of thrombin to yield the (labile) active cofactor factor VIIIa possessing an initial specific activity of approximately 80 units/microgram. Activation paralleled the generation of fragments A1 and A2 derived from the heavy chain and A3-C1-C2 derived from the light chain. Chromatography of factor VIIIa, on Mono-S buffered at pH 6.0 resulted in separation of the bulk of the A2 fragment from a fraction composed predominantly of A1/A3-C1-C2 dimer plus low levels of A2 fragment. Only the latter fraction contained clotting activity (approximately 20 units/microgram) which was stable and represented a less than 10% yield when compared with the peak activity of unfractionated factor VIIIa. Further depletion of A2 fragment from Mono-S-purified factor VIIIA, achieved using an immobilized monoclonal antibody to the A2 domain, yielded a relatively inactive A1/A3-C1-C2 dimer (less than 0.4 unit/microgram). Factor VIIIa (greater than 40 units/microgram) was reconstituted from the A1/A3-C1-C2 dimer plus the A2 fragment in a reaction that was Me(2+)-independent and inhibited by moderate ionic strength. Reassociation of A2 required the A1 subunit in that the A2 subunit associated weakly if at all to A3-C1-C2 in the absence of A1. These results indicated that human factor VIIIa is a trimer represented by the subunits A1/A2/A3-C1-C2 and that the A2 subunit is required for expression of factor VIIIa activity.

Blotting, Western↗

PubMed1992

Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.

The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs

Animals↗

PubMed1999

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

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

Antibodies↗

PubMed1996

Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase.

Factor VIIIa, the cofactor for the factor IXa-dependent conversion of factor X to factor Xa, is proteolytically inactivated by activated protein C (APC). APC cleaves at two sites in factor VIIIa, Arg336, near the C terminus of the A1 subunit; and Arg562, bisecting the A2 subunit (Fay, P., Smudzin, T., and Walker, F. (1991) J. Biol. Chem. 266, 20139-20145). Factor VIIIa increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa (Fl-FFR-FIXa; Kd = 42.4 nM), whereas cleavage of factor VIIIa by APC eliminated this property. Isolation of the APC-cleaved A1/A3-C1-C2 dimer (A1336/A3-C1-C2), and the fragments derived from cleaved A2 subunit (A2N/A2C), permitted dissection of the roles of individual cleavages in cofactor inactivation. Intact A1/A3-C1-C2 dimer increased Fl-FFR-FIXa anisotropy and bound factor X in a solid phase assay, while these activities were absent in the A1336/A3-C1-C2. However, the residues removed by this cleavage, Met337 Arg372, did not directly participate in these functions since neither a synthetic peptide to this sequence nor an anti-peptide polyclonal antibody blocked these activities using intact dimer. CD spectral analysis of the intact and truncated dimers indicated reduced alpha and/or beta content in the latter. The A1/A3-C1-C2 dimer plus A2 subunit reconstitutes cofactor activity and produced a factor VIIIa-like effect on the anisotropy of Fl-FFR-FIXa. However, when A2 was replaced by the A2N/A2C fragments, the resulting fluorescence signal was equivalent to that observed with the dimer alone. These results indicate that APC inactivates the cofactor at two levels within the intrinsic factor Xase complex. Cleavage of either subunit modulates the factor IXa active site, suggesting an essential synergy of interactive sites in factor VIIIa. Furthermore, cleavage of the A1 site alters the conformation of a factor X binding site within that subunit, thereby reducing the affinity of cofactor for substrate.

Amino Acid Sequence↗

PubMed1998

The A2 subunit of factor VIIIa modulates the active site of factor IXa.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Isolated subunits of factor VIIIa were examined for their ability to accelerate the factor IXa-catalyzed activation of factor X. The A2 subunit enhanced the kcat for this conversion by 100-fold whereas the Km for factor X was unaffected. The apparent Kd for the interaction of A2 subunit with factor IXa was approximately 300 nM. Similar results were obtained using purified A2 expressed as the isolated domain in Chinese hamster ovary cells, although this material was less stable than the factor VIIIa-derived material. Isolated A1 and A3-C1-C2 subunits showed no effect on the rate of factor X conversion. A2 subunit increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa (Deltar = 0.015) and markedly increased anisotropy in the presence of factor X (Deltar = 0.057), suggesting that it contributes to the orientation of the factor IXa active site and its relation to substrate. A synthetic peptide to A2 residues 558-565 inhibited the A2-dependent enhancement of factor X activation with an IC50 = 40 microM, a value similar to its Ki for inhibition of the intrinsic factor Xase (105 microM). These results indicate that the isolated A2 subunit modulates the active site of factor IXa and identifies a functional role for this subunit in factor VIIIa.

Animals↗

PubMed1994

Characterization of chemically crosslinked human factor VIIIa.

The activity of factor VIIIa was enhanced and stabilized by treatment of factor VIII with the crosslinker disuccinimidyl suberate. The activity was > 200-fold higher compared with that of native factor VIIIa and was stable for at least 15 days at 4 degrees C and pH 7.2. The crosslinked factor VIIIa was purified by immunoaffinity chromatography and gel filtration. Electrophoretic analysis revealed high-molecular-mass (approximately 150 kDa) molecules as well as the three bands characteristic of native factor VIIIa. Thus crosslinking appeared to yield molecules stabilized by intra- and/or inter-subunit crosslinks. The material was further fractionated using immobilized von Willebrand factor and the factor VIIIa activity could be ascribed to trimers containing only intra-subunit crosslinks. Moreover, reduction of crosslinked factor VIIIa produced using dithiobis(succinimidylpropionate) suggested that the molecules containing intersubunit crosslinks had not been cleaved by thrombin at arginine 372.

Chromatography, Affinity↗

PubMed1981

The role of phospholipid and factor VIIIa in the activation of bovine factor X.

The kinetic parameters of bovine factor X activation by bovine factor IXa have been determined in the absence and presence of Ca2+, thrombin-activated bovine factor VIII (VIIIa), and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 75/25; mol/mol). Factor IXa in the absence of Ca2+, factor VIIIa, and phospholipid is able to catalyze factor X activation. The Km for factor X is 299 microM which is well above its concentration in bovine plasma, about 0.2 microM. The Vmax of factor Xa formation is 0.0022 mol of Xa . min-1 . mol of IXa-1 under these conditions. Addition of Ca2+ has little effect on the kinetic constants of factor X activation by factor IXa. In the presence of 10 mM CaCl2 the Km for factor X is 181 microM, and the Vmax is 0.0105 mol of Xa . min-1 . mol of IXa-1. The presence of 10 microM phospholipid dramatically decreases the Km for factor X to 0.058 microM, and the Vmax becomes 0.0025 mol of Xa . min-1 . mol of IXa-1. The Vmax of factor Xa formation slightly increases when more phospholipid is present in our experiments, and there is a considerable increase of the Km for factor X at higher phospholipid concentrations. Therefore, the Km measured in the presence of phospholipid has to be regarded as an apparent Km. The possible explanations for this phenomenon are discussed. For the complete factor X-activating complex (i.e. factor IXa, factor VIIIa, Ca2+, and 10 microM phospholipid) the Km for factor X is 0.0063 microM, and the Vmax is raised 200,000-fold to 500 mol of Xa . min-1 . mol of IXa-1. In order to exert its stimulating effect on factor X activation factor VIII has to be activated with thrombin. Our results show that factor IXa is an enzyme which can activate factor X at a very low rate. The stimulating effect of phospholipid in factor X activation is mainly due to an effect on the Km for factor X, bringing it within the range of the plasma concentration. The stimulatory effect of factor VIIIa is explained by its 200,000-fold increase of the Vmax of factor Xa formation.

Animals↗

PubMed1994

Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site.

Factor VIIIa is a non-covalent heterotrimer of A1, A2, and A3-C1-C2 subunits. Previously, we speculated that the central portion of the A2 subunit, in and around the activated protein C-sensitive bond at Arg562-Gly (Fay, P. J., Smudzin, T.M., and Walker, F.J. (1991) J. Biol. Chem. 266, 20139-20145), is important for macromolecular interactions within the factor Xase enzyme complex. A peptide corresponding to factor VIII residues 558-565, SVDQRGNQ and designated FVIII558-565, was chemically synthesized and inhibited factor Xa generation in a purified system with an apparent KI of 105 microM. Tryptic cleavage of FVIII558-565 eliminated its inhibitory activity, whereas a scrambled sequence version of the peptide possessed < 30% the inhibitory activity of the native version. Overlapping peptides FVIII556-564 and FVIII561-569 were also inhibitory and confirmed the importance of residues in and around the scissile bond for functional factor Xase. Kinetic analysis revealed that peptide-mediated inhibition was non-competitive with respect to factor X. However, increasing factor IXa concentration overcame the observed inhibition. Furthermore, the peptide inhibited the factor IXa-dependent enhancement of factor VIIIa reconstituted from isolated A1/A3-C1-C2 dimer plus A2 subunit. Isolated factor VIII heavy chain (contiguous A1-A2 domains) was cleaved at Arg336 by an equimolar concentration of factor IXa in a reaction that was phospholipid-independent. No proteolysis of the isolated A1 subunit was observed in a similar reaction. These results indicate that the A2 subunit sequence delineated by residues 558-565 contributes to the interaction of cofactor with protease and that this interaction is essential for intrinsic factor Xase activity. Furthermore, that this peptide blocks both factor Xase activity and the capacity of factor IXa to stabilize the labile factor VIIIa heterotrimer suggest that this latter property is of physiologic significance.

Amino Acid Sequence↗

PubMed1985

Assembly of the intrinsic factor X activating complex--interactions between factor IXa, factor VIIIa and phospholipid.

The activation of blood coagulation factor X by factor IXa is strongly stimulated by the non-enzymatic cofactors phospholipid, Ca2+ and activated factor VIII. In this paper we present a method by which we were able to determine binding affinities of factor IXa for phospholipids (either in the absence or presence of factor VIIIa) from kinetic measurements of factor X activation. It is shown that rates of factor X activation in the presence of phospholipids can be saturated with an excess factor VIIIa at limiting amounts of factor IXa and vice versa. Our data indicate that the enzymatic unit in the intrinsic factor X activator is a 1:1 stoichiometrical complex of factor IXa and factor VIIIa bound to phospholipid. Titrations with factor IXa at fixed concentrations of phospholipid and factor X show that the apparent dissociation constant of factor IXa for phospholipid is lowered from 10(-6) M to 10(-8) M by the presence of factor VIIIa. We conclude, that in analogy with the role of factor Va in prothrombin activation, phospholipid-bound factor VIIIa functions as a high-affinity binding site ("receptor") for factor IXa in the intrinsic factor X activating complex. Therefore, factor VIIIa increases the observed Vmax of factor X activation by 1) enhancing the kcat of the reaction and 2) increasing the amount of phospholipid-bound factor IXa that participates in factor X activation.

Blood Coagulation Factors↗

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↗

PubMed1988

Subunit structure of thrombin-activated human factor VIIIa.

Proteolytic activation of human Factor VIII (VIIIa) by thrombin was correlated with the generation of a light-chain-derived 73(71) kDa polypeptide plus polypeptides of 51 and 43 kDa derived from the heavy chain(s). Factor VIIIa activity was unstable and decayed to an inactive form (VIIIi) in the absence of additional proteolysis. The subunit structure of Factor VIIIa was studied using two rapid chromatographic methods. Gel filtration of Factor VIIIa showed that coagulant activity was correlated with the 73 and 51 kDa polypeptides which co-eluted with a Stokes radius of 46 A and was separated from the 43 kDa fragment. A similar polypeptide elution pattern was obtained for Factor VIIIi following prolonged incubation with thrombin. Gel filtration of EDTA-inactivated Factor VIIIa showed that the 73 and 51 kDa polypeptides eluted separately with Stokes radii of 32 and 38 A, respectively. Anion-exchange HPLC of Factor VIIIa resolved the coagulant-active 73/51 kDa dimer from the inactive dimer. The labile activity of Factor VIIIa was stabilized by chemical crosslinking reagents, presumably by formation of intra-chain crosslinks. A native Mr of 136,000 for Factor VIIIa, calculated from its Stokes radius (46 A) and sedimentation coefficient (7.1 S), was compatible with a non-covalent dimer composed of 73 and 51 kDa subunits.

Enzyme Activation↗