Structure and Function of the Factor VIII - von Willebrand Factor Complex

本应用程序的目标是了解通过调节因子VIII （fVIII）功能和致病抗fVIII抗体的机制来控制凝血机制。本项目中涉及的调节过程包括fVIII-血管性血友病因子（vWf）复合物的激活和内在途径激活因子功能的控制，包括通过A2亚基解离发生的活化fVIII （fVIII）活性的衰减。fVIII C2结构域在fVIII激活中的作用以及抗C2抗体（Abs）抑制fVIII的机制将被确定。C2结构域的大型Ab表位图已经开发出来。这导致鉴定出一类新的C2结构域抗体，它们可以抑制凝血酶或Xa因子对fVIII的激活。在a型血友病小鼠和人类中，这些抗体对fVIII的抑制免疫反应起主要作用。此外，它们在小鼠中产生出血性素质。这些抗体对fVIII活化的抑制是令人惊讶的，因为它们的表位远离参与fVIII活化的蛋白水解裂解位点。实验将确定凝血酶和Xa因子催化的fVIII蛋白水解裂解是否被这些抗体抑制。此外，还将研究抗体的抑制机制。在vWf存在和不存在的情况下，还将分析抗c2抗体与fVIII的结合动力学。这将允许测试fVIII抑制剂的行为是由所谓的抗体对其抗原的亲和力限制所控制的假设。此外，这些研究旨在了解许多抑制剂的复杂行为，其中包括对fVIII的部分抑制和复杂的非二级动力学。此外，将通过位点定向诱变进行抗c2表位的高分辨率定位。该建议具有潜在的临床意义，包括开发新型重组fVIII分子，改善治疗A型血友病患者的止血疗效，改善fVIII抑制剂患者的诊断。

The goals of this application are to understand the control of the coagulation mechanism that occurs through the regulation of factor VIII (fVIII) function and the mechanisms underlying pathogenic anti-fVIII Abs. The regulatory processes addressed in this project include the activation of the fVIII-von Willebrand factor (vWf) complex and the control of intrinsic pathway activator function, including the decay of activated fVIII (fVIIIa) activity that occurs through A2 subunit dissociation. The role of the fVIII C2 domain in the activation of fVIII and the mechanism of inhibition of fVIII by anti-C2 antibodies (Abs) will be determined. A large Ab epitope map of the C2 domain has been developed. This has led to the identification of a novel class of C2 domain Abs that inhibit the activation of fVIII by either thrombin or factor Xa. These Abs make a dominant contribution to the inhibitory immune response to fVIII in hemophilia A mice and in humans. Additionally, they produce a bleeding diathesis in mice. The inhibition of fVIII activation by these Abs is surprising because their epitopes are remote from proteolytic cleavage sites that are involved in the activation of fVIII. Experiments will be done to determine which, if any, proteolytic cleavages in fVIII catalyzed by thrombin and factor Xa are inhibited by these Abs. Additionally, the mechanism of inhibition by the Abs will be investigated. The kinetics of binding of anti-C2 Abs to fVIII in the presence and absence of vWf also will be analyzed. This will allow testing of the hypothesis that the behavior of fVIII inhibitors is governed by the so-called affinity limit of Abs for their antigens. Additionally, these studies are directed toward understanding the complex behavior of many inhibitors, which can include partial inhibition of fVIII and complex, non-second order kinetics. Additionally, high-resolution mapping of anti-C2 epitopes by site directed mutagenesis will be performed. This proposal has potential clinical implications, including the development of novel recombinant fVIII molecules with improved hemostatic efficacy in the treatment of patients with hemophilia A and improved diagnostics for fVIII inhibitor patients.