Nogami Keiji, Shima Midori, Nishiya Katsumi, Sakurai Yoshihiko, Tanaka Ichiro, Giddings John C, Saenko Evgueni L, Yoshioka Akira
Department of Pediatrics, Nara Medical University, Kashihara, Japan.
Thromb Haemost. 2002 Mar;87(3):459-65.
Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in one-stage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)'2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.
凝血因子VIII（FVIII）抑制性同种抗体与FVIII的A2、C2或A3-C1结构域发生反应，并使FVIII活性失活。我们最近证明，一种具有Val2248-Gly2285表位的抗C2单克隆抗体可抑制因子Xa（FXa）催化的FVIII活化，并且FVIII的FXa结合位点位于Thr2253-Gln2270残基内。在本研究中，我们调查了抗C2同种抗体是否抑制FXa催化的FVIII活化。来自4名患者的抗C2同种抗体在一期凝血试验中抑制了FXa对FVIII的活化。此外，SDS-PAGE分析表明，所有同种抗体均能独立于磷脂抑制FXa对FVIII的蛋白水解切割。为了证实对FVIII和FXa相互作用的直接抑制，我们在ELISA中检测了同种抗体对FVIII与无活性FXa脱水FXa结合的影响。所有同种抗体和C2亲和纯化的F(ab)'2制剂均剂量依赖性地抑制FVIII与脱水FXa的结合。我们的结果揭示了一种新的FVIII抑制机制，即在存在抗C2同种抗体的情况下通过抑制FXa介导。
