Biochemistry of Intrinsic Xase

项目4 -摘要 增强FVIII和FIX生物活性的新方法是关键的努力，具有潜在的 改善血友病的蛋白质和基因治疗。我们已经确定了新的变体和新的 对FVIII和FIX的生物活性具有积极影响的策略。有前途的临床前研究 在小型和大型动物模型中的有效性和安全性为使用这些药物的转化研究提供了基础。 蛋白质;特别是FIX-帕多瓦（R338 L）变体已经处于早期基因治疗临床试验中， 血友病B。本申请的主要目标是了解变异体的生物化学， 新变异的情况下，评估其在血友病模型中的潜力。我们试图用这种方法来揭示 机制方面的内在Xase功能，从而提供了基于证据的见解的潜力， 这些变异在人类疾病的治疗中。我们实验策略的核心原则是 事实上，即使是轻微的促凝血功能增强，从生化指标判断， 从这个角度来看，可以对治疗方法的成功产生非常重要的影响。在目标1中， 假设具有修饰PACE/弗林蛋白酶切割位点的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。第二类变体基于 缺乏B结构域和酸性区域3的分子，具有增加的比活性。这些发现表明， vWF参与虽然是FVIII循环半衰期的重要决定因素，但是否限制了 通过FXa激活，如果通过解除这种限制，我们可以提高可用的FVIIIa水平，以促进凝血 阵在目标2中，我们将描述FIX-帕多瓦变体的特征，其增强的分子基础 功能不确定。我们将使用生物化学和生物物理学方法来确定 超功能分子来自FXa适体的阻止其结合的新结构信息 蛇FVa-FX的已知结构暗示了Xa的一个区域，该区域以前不被认为是FVa的中心。 Va绑定。我们假设FIXa和FVIIIa之间的类似相互作用可能通过以下方式增强： 肝素结合外部位的修饰。如果成功，我们的目标是将联合收割机与修改后的FIX-帕多瓦结合起来 以产生具有增强的生物学特性的FIX变体。在目标3中，我们将追求 使用FIX变体进行的研究，这些变体在活化后产生具有增加的酶原样特征的产物， 由于它们对蛋白酶抑制的抗性而长寿命，但可能在它们的 组装成内在的Xase。生物化学研究将测试这些想法之后的方法， 评估这些变异体，以及那些在FIX-帕多瓦背景下结合的变异体，是否对以下疾病有治疗价值： 血友病B。这些方法将为内在Xase调节提供新的机制见解 和活性，并为开发出血性疾病的新治疗剂提供平台。

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.