Structure and Stability Studies of Factor VIII to Overcome the Immune Response

说明(申请人提供)：凝血因子VIII是一种非酶糖蛋白辅因子，对适当调节凝血级联反应是必不可少的。因子VIII的遗传缺陷会导致血友病A，全世界每5000名男性中就有一人受到影响。血友病A的治疗包括反复输注重组或血浆衍生因子，这可能导致抑制抗体的产生，在接受治疗的血友病A患者中约有三分之一会出现这种抗体。第VIII因子的C2结构域是与von Willebrand因子和活化的血小板膜表面正确相互作用所必需的，是抑制性抗体识别的主要表位。最近的研究表明，抗体反应的高发生率导致抗体阻断由丝氨酸蛋白酶、凝血酶或凝血因子Xa激活的因子VIII。这些“非经典”抗体具有高水平的致病性，并与“经典”抗体表现出正的协同性，导致第VIII因子活性水平急剧下降。致病反应的大部分效力可能对应于人类第VIII因子蛋白的边际稳定性。事实上，具有更高稳定性的第八因子结构，如猪第八因子，可以克服免疫反应，并导致第八因子在循环中适度的活性。这项建议旨在利用基于结构的计算蛋白质设计和X射线结晶学来更好地了解因子C2结构域的稳定性及其与“非经典”抗体抑制剂相互作用的性质。此外，我们建议了解针对第VIII因子C2结构域的“经典”和“非经典”抗体类型之间的协同作用的结构基础。该项目的具体目标是：(1)比较研究人和猪FVIII C2结构域的热力学和功能性质；(2)基于结构的计算蛋白质设计以设计热稳定的FVIII C2结构域；(3)人FVIII C2结构域/非经典抗体复合体的X射线晶体结构；(4)C2结构域/经典/非经典抗体三元复合体的X射线晶体结构。 与公共卫生相关：血友病A是一种出血性疾病，全世界每5000名男性中就有一人受到影响。在使用因子VIII治疗时，会产生抑制抗体，这往往会使他们的病情恶化，并使治疗变得更加困难和昂贵。这项建议旨在更好地了解这些抗体是如何致病的，并将导致开发更好的治疗血友病A患者的方法。血友病A是由编码凝血因子的基因破坏引起的，是最常见的血友病形式，全世界每5000名男性中就有一人受到影响。血友病A治疗中最重要的并发症是对第八因子的免疫抑制反应。研究第八因子抗体识别的结构基础，以及利用蛋白质工程来提高第八因子的稳定性，可能会导致全世界血友病治疗的改进。

DESCRIPTION (provided by applicant): Blood coagulation factor VIII is a non-enzymatic glycoprotein cofactor that is essential for proper regulation of the blood coagulation cascade. Genetic deficiencies in factor VIII cause hemophilia A, which affects 1 in 5,000 males worldwide. Treatment for hemophilia A consists of repeated infusions of recombinant or plasma-derived factor VIII, which can result in the development of inhibitor antibodies that arise in approximately one-third of the hemophilia A patient population that receives therapy. The C2 domain of factor VIII is essential for proper interactions with von Willebrand factor and the membrane surface of activated platelets, and is a major epitope that is recognized by inhibitory antibodies. Recent studies indicate that a high incidence of the antibody response result in antibodies that block the activation of factor VIII by the serine proteases, thrombin or factor Xa. These "non-classical" antibodies possess a high level of pathogenicity and display positive cooperativity with "classical" antibodies, resulting in a dramatic decrease in the level of factor VIII activity. Much of the potency of the pathogenic response may correspond to the marginal stability of the human factor VIII protein. Indeed, factor VIII constructs that have increased stability, such as porcine factor VIII, can overcome the immune response and result in a modest level of factor VIII activity in circulation. This proposal aims to use structure-based computational protein design and x-ray crystallography to better understand the stability of the factor VIII C2 domain and the nature of its interactions with "nonclassical" antibody inhibitors. Additionally, we propose to understand the structural basis for cooperativity between the "classical" and "nonclassical" antibody types that target the factor VIII C2 domain. The specific aims of this project are: (1) Comparative study of the thermodynamic and functional properties of human and porcine FVIII C2 domains; (2) Structure-based computational protein design to engineer a thermo-stabilized FVIII C2 domain; (3) X-ray crystal structure of a human FVIII C2 domain/nonclassical antibody complex; and (4) X-ray crystal structure of a C2 domain/classical/nonclassical antibody ternary complex. PUBLIC HEALTH RELEVANCE: Hemophilia A is a bleeding disorder that affects 1 in 5,000 males worldwide. Upon treatment with factor VIII, inhibitor antibodies develop which often worsens their condition and makes therapy more difficult and expensive. This proposal aims to provide a better understanding of how these antibodies become pathogenic and will lead to the development of better treatments for hemophilia A patients. Hemophilia A is caused by the disruption of the gene encoding blood coagulation factor VIII and is the most common form of hemophilia, affecting 1 in 5,000 males worldwide. The most significant complication in the management of hemophilia A is the development of an inhibitory immune response to therapeutically infused factor VIII. Studying the structural basis of antibody recognition of factor VIII as well as using protein engineering to improve the stability of factor VIII may lead to improved therapies for hemophiliacs worldwide.