Antifactor VIII antibody inhibiting allogeneic but not autologous factor VIII in patients with mild hemophilia A.
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Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF. (+info)
Cleavage of factor V at Arg 506 by activated protein C and the expression of anticoagulant activity of factor V.
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Activated protein C (APC) inhibits coagulation by cleaving and inactivating procoagulant factor Va (FVa) and factor VIIIa (FVIIIa). FV, in addition to being the precursor of FVa, has anticoagulant properties; functioning in synergy with protein S as a cofactor of APC in the inhibition of the FVIIIa-factor IXa (FIXa) complex. FV:Q506 isolated from an individual homozygous for APC-resistance is less efficient as an APC-cofactor than normal FV (FV:R506). To investigate the importance of the three APC cleavage sites in FV (Arg-306, Arg-506, and Arg-679) for expression of its APC-cofactor activity, four recombinant FV mutants (FV:Q306, FV:Q306/Q506, FV:Q506, and FV:Q679) were tested. FV mutants with Gln (Q) at position 506 instead of Arg (R) were found to be poor APC-cofactors, whereas Arg to Gln mutations at positions 306 or 679 had no negative effect on the APC-cofactor activity of FV. The loss of APC-cofactor activity as a result of the Arg-506 to Gln mutation suggested that APC-cleavage at Arg-506 in FV is important for the ability of FV to function as an APC-cofactor. Using Western blotting, it was shown that both wild-type FV and mutant FV was cleaved by APC during the FVIIIa inhibition. At optimum concentrations of wild-type FV (11 nmol/L) and protein S (100 nmol/L), FVIIIa was found to be highly sensitive to APC with maximum inhibition occurring at less than 1 nmol/L APC. FV:Q506 was inactive as an APC-cofactor at APC-concentrations +info)
The A1 and A2 subunits of factor VIIIa synergistically stimulate factor IXa catalytic activity.
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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. (+info)
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.
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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. (+info)
Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.
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Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary. (+info)
Surface loop 199-204 in blood coagulation factor IX is a cofactor-dependent site involved in macromolecular substrate interaction.
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In factor IX residues 199-204 encompass one of six surface loops bordering its substrate-binding groove. To investigate the contribution of this loop to human factor IX function, a series of chimeric factor IX variants was constructed, in which residues 199-204 were replaced by the corresponding sequence of factor VII, factor X, or prothrombin. The immunopurified and activated chimeras were indistinguishable from normal factor IXa in hydrolyzing a small synthetic substrate, indicating that this region is not involved in the interaction with substrate residues on the N-terminal side of the scissile bond. In contrast, replacement of loop 199-204 resulted in a 5-25-fold reduction in reactivity toward the macromolecular substrate factor X. This reduction was due to a combination of increased K(m) and reduced k(cat). In the presence of factor VIIIa the impaired reactivity toward factor X was largely restored for all factor IXa variants, resulting in a more pronounced stimulation by factor VIIIa compared with normal factor IXa (3 to 5 x 10(4)-fold versus 5 x 10(3)-fold). Inhibition by antithrombin was only slightly affected for the factor IXa variant with the prothrombin loop sequence, whereas factor IXa variants containing the analogous residues of factor VII or factor X were virtually insensitive to antithrombin inhibition. In the presence of heparin, however, all chimeric factor IXa variants formed complexes with antithrombin. Thus the cofactors heparin and factor VIIIa have in common that they both alleviate the deleterious effects of mutations in the factor IX loop 199-204. Collectively, our data demonstrate that loop 199-204 plays an important role in the interaction of factor IXa with macromolecular substrates. (+info)
Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity.
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The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor IXa to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A., Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains. Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII. Activated factor IX/factor X chimeras were indistinguishable from normal factor IXa with respect to factor IXa enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed approximately 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX(106-108)/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor IXa enzymatic activity requires hydrophobic contact between the two EGF-like domains. (+info)
Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase.
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Factor VIIIa is a trimer of A1, A2, and A3-C1-C2 subunits. Inactivation of the cofactor by human activated protein C (APC) results from preferential cleavage at Arg336 within the A1 subunit, followed by cleavage at Arg562 bisecting the A2 subunit. In the presence of human protein S, the rate of APC-dependent factor VIIIa inactivation increased several-fold and correlated with an increased rate of cleavage at Arg562. (Active site-modified) factor IXa, blocked cleavage at the A2 site. However, APC-catalyzed inactivation of factor VIIIa proceeded at a similar rate independent of factor IXa, consistent with the location of the preferential cleavage site within the A1 subunit. Addition of protein S failed to increase the rate of cleavage at the A2 site when factor IXa was present. In the presence of factor X, cofactor inactivation was inhibited, due to a reduced rate of cleavage at Arg336. However, inclusion of protein S restored near original rates of factor VIIIa inactivation and cleavage at the A1 site, thus overcoming the factor X-dependent protective effect. These results suggest that in the human system, protein S stimulates APC-catalyzed factor VIIIa inactivation by facilitating cleavage of A2 subunit (an effect retarded in the presence of factor IXa), as well as abrogating protective interactions of the cofactor with factor X. (Blood. 2000;95:1714-1720) (+info)