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

这项应用的目的是了解通过调节因子VIII(FVIII)功能而发生的凝血机制的控制，以及致病性抗FVIII抗体的潜在机制。本项目涉及的调控过程包括FVIII-von Willebrand因子(VWF)复合体的激活和内在途径激活功能的控制，包括通过A2亚基解离而发生的激活的FVIII(FVIIIa)活性的衰退。FVIII C2结构域在激活FVIII中的作用以及抗C2抗体抑制FVIII的机制将被确定。已开发出C2结构域的大容量抗体表位图。这导致了一类新的C2结构域抗体的鉴定，这些抗体可以抑制凝血酶或Xa因子激活FVIII。这些抗体在血友病A小鼠和人类对FVIII的抑制性免疫反应中起主导作用。此外，它们还会在小鼠身上产生出血的素质。这些抗体对FVIII激活的抑制是令人惊讶的，因为它们的表位远离参与FVIII激活的蛋白水解性切割位点。将进行实验，以确定这些抗体是否抑制了凝血酶和Xa因子催化的FVIII中的哪些蛋白水解性切割。此外，还将探讨单抗的抑制作用机制。还将分析在VWF存在和不存在的情况下抗C2抗体与FVIII结合的动力学。这将允许对FVIII抑制剂的行为受所谓的抗体对其抗原的亲和力限制的假设进行检验。此外，这些研究旨在了解许多抑制剂的复杂行为，包括对FVIII的部分抑制和复杂的非二级动力学。此外，还将通过定点突变对抗C2表位进行高分辨率映射。这项建议具有潜在的临床意义，包括开发新的重组FVIII分子，以改善对血友病A患者的止血效果，并改进对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.