Biochemistry of Intrinsic Xase

项目4-摘要 增强FVIII和FIX生物活性的新方法是具有潜在潜力的关键努力 改进基于蛋白质和基因的血友病治疗方法。我们已经确定了新的变种和新的 对FVIII和FIX生物活性有积极影响的策略。前景看好的临床前研究 在小动物和大动物模型中的有效性和安全性为使用这些技术进行转化性研究提供了基础 蛋白质；特别是Fix-Padua(R338L)变体已经处于早期基因治疗的临床试验阶段 血友病B。这个应用程序的主要目标是了解变异体的生物化学和在 在新变种的情况下，评估它们在血友病模型中的潜力。我们试图用这种方法来揭示 内在XASE函数的机械方面，从而提供了对 这些变种在治疗人类疾病中的作用。我们实验策略的中心原则是基于 事实上，即使是促凝血功能的轻微增强，从生化反应来判断 从不同的角度来看，这可能对治疗方法的成功产生非常重要的影响。在目标1中，我们 假设具有修饰的Pace/Furin裂解中心的FVIII衍生物导致其差异加工 通过凝血酶和/或FXA产生更稳定/活性的辅因子。第二类变量基于 缺少B结构域和酸性区域3的分子比活性增加。这些发现提出了 质疑vWF参与，尽管是FVIII循环半衰期的重要决定因素，是否限制了 由FXA激活，如果通过解除这一限制，我们可以提高可用的FVIIa水平以促进血栓形成 队形。在目标2中，我们将表征FIX-PADUA变体，其分子基础增强 功能不确定。我们将使用生化和生物物理方法来确定 功能超强的分子。来自阻止其结合的FXA适配子的新结构信息 与FVA和蛇FVA-FX的已知结构有关，涉及Xa的一个区域，该区域以前不被认为是 VA绑定。我们假设FixA和FVIIa之间的类似相互作用可能会通过以下方式增强 肝素结合外切酶的修饰。如果成功，我们的目标是将FIX-PADUA与修改相结合 以产生具有增强的生物特性的固定变体。在目标3中，我们将追求 对FIX的变体的研究，这些变体在激活时产生具有增加的酶原性质的产物，是 长寿是因为它们对蛋白酶抑制具有抵抗力，但在功能上可能会被拯救 组装成固有的XASE。测试这些想法的生物化学研究之后将有方法来 评估这些变异，以及那些结合在FIX-PADUA背景下的变异，是否具有治疗价值 血友病B。结合起来，这些方法将为内在Xase调节提供新的机械性见解。 并为开发新的出血性疾病治疗剂提供了一个平台。

Project 4 - Abstract Novel approaches to enhance the biologic activity of FVIII and FIX are critical endeavors with potential for improving protein- and gene-based therapy for hemophilia. We have identified novel variants and new strategies that have a positive effect on the biological activity of FVIII and FIX. Promising preclinical studies on efficacy and safety in small and large animal models provide the basis for translational studies using these proteins; in particular the FIX-Padua (R338L) variant is already in early phase gene therapy clinical trials for hemophilia B. The main goal of this application is to understand the biochemistry of the variants and in the case of new variants, evaluate their potential in hemophilic models. We seek to use this approach to reveal mechanistic aspects of intrinsic Xase function and thus provide evidence-based insights into the potential of these variants in the treatment of human disease. The central tenets of our experimental strategies are based on the fact that even modest enhancements of procoagulant function, as judged from a biochemical perspective, can have a very significant impact on the success of therapeutic approaches. In aim 1 we hypothesize that a FVIII derivative with a modified PACE/furin cleavage site results in its differential processing by thrombin and/or FXa yielding a more stable/active cofactor. A second class of variant is based on a molecule lacking the B-domain and acid region 3 with increased specific activity. These findings pose the question whether vWF engagement, while an important determinant of FVIII circulating half-life, limits activation by FXa, and if by relieving this constraint we can enhance available FVIIIa levels to promote clot formation. In aim 2 we will characterize the FIX-Padua variant for which the molecular basis for its enhanced function is uncertain. We will use biochemical and biophysical approaches to identify the mechanism of the hyperfunctional molecule. Emerging structural information derived from FXa aptamers that prevent its binding to FVa and the known structure of snake FVa-FX implicate a region of Xa not previously considered central in Va binding. We hypothesize that the analogous interaction between FIXa and FVIIIa may be enhanced by modifications in the heparin-binding exosite. If successful, our goal is to combine FIX-Padua with modifications from the FIX-loop 90 to generate FIX variants with enhanced biologic properties. In aim 3 we will pursue studies with variants of FIX that upon activation yield products with increasing zymogen-like character, are long-lived because of their resistance to protease inhibition but may be functionally rescued upon their assembly into the intrinsic Xase. Biochemical studies testing these ideas will be followed by approaches to assess if such variants, and those combined on the FIX-Padua background, have therapeutic value for hemophilia B. Together these approaches will provide new mechanistic insights into intrinsic Xase regulation and activity as well as provide a platform for the development of new therapeutic agents for bleeding disorders.