Hemostasis and Thrombosis: Chemistry, Biology and Physiology

总体方案-摘要 血液凝固源自一系列特异性蛋白水解活化反应， 胰蛋白酶样丝氨酸蛋白酶的狭窄和确定的特异性。在一些情况下，这些蛋白酶 在膜组装酶复合物中的功能。独特的蛋白质底物特异性和 通过与膜和辅因子的相互作用来调节酶的功能是蛋白水解的标志。 凝血反应。目前对这些分子基础的理解存在重大差距， 这些独特的特征是止血反应功能的基础。该计划提出了一个 整合的方法集中在酶的功能和特异性的调制，独特地产生于 大分子相互作用是止血酶作用的基础。项目1（Krishnaswamy） 使用凝血酶原酶复合物作为范例来研究结构和功能机制， 从酶功能中辅因子、蛋白酶和底物之间的膜依赖性相互作用。 项目2（Camire）将研究因子V转化为辅因子的分子机制， 因子Va和这些机制揭示的令人惊讶的新生物学见解。项目3（硫化机） 使用RNA适体作为独特的探针，用于凝血所必需的大分子相互作用 酶的功能，并显示新的方法来干扰或增强这些 治疗增益的相互作用。项目4（Arruda）研究内在Xase的生物化学，重点是 具有上级功能的蛋白酶和辅因子变体的特性， 产生治疗血友病的治疗剂。四个项目的目标将得到支持 由管理核心（核心A），一个为分子生物学，蛋白质表达和 结构生物学（核心B）和一个核心，支持止血和 血栓形成（核心C）。总的来说，该项目将个别调查人员的专业知识应用于 解决止血和血栓形成方面的主要未回答问题，从生物化学的见解， 生物学功能和生理学。所提出的方法将提供新的见解的化学和 血液凝固反应的生物学与理解正常止血的含义， 血栓形成

Overall Program-Abstract Blood coagulation derives from a series of specific proteolytic activation reactions that are catalyzed with narrow and defined specificity by trypsin-like serine proteinases. In several instances, these proteinases function in membrane assembled enzyme complexes. Distinctive protein substrate specificities and the modulation of enzymic function by interactions with membranes and cofactors are hallmarks of the proteolytic reactions of blood coagulation. There are major gaps in the current understanding of the molecular bases for these unique features that underlie the function of the hemostatic reactions. This program proposes an integrated approach focused on the modulation of enzymic function and specificity that uniquely arises from macromolecular interactions that underlie the action of the hemostatic enzymes. Project 1 (Krishnaswamy) uses the prothrombinase complex as a paradigm to investigate structural and functional mechanisms deriving from the membrane-dependent interactions between cofactor, proteinase and substrate in enzyme function. Project 2 (Camire) will investigate molecular mechanisms at play in the conversion of factor V to the cofactor, factor Va and the surprising new biological insights that these mechanisms reveal. Project 3 (Sullenger) employs RNA aptamers as unique probes for the macromolecular interactions essential for coagulation enzyme function and to show the way forward for novel approaches to either interfere with or enhance these interactions for therapeutic gain. Project 4 (Arruda) investigates the biochemistry of intrinsic Xase with a focus on characterizing proteinase and cofactor variants with superior function and potential for use as the next generation of therapeutics in the treatment of hemophilia. The objectives of the four projects will be supported by an administrative core (Core A), a core that provides support for molecular biology, protein expression and structural biology (Core B) and a core that supports the program with mouse models of hemostasis and thrombosis (Core C). Overall, this project applies the expertise of the individual investigators towards addressing major unanswered questions in hemostasis and thrombosis extending from biochemical insights to biological function and physiology. The proposed approaches will provide new insights into the chemistry and biology of the blood coagulation reactions with implications for an understanding of normal hemostasis and thrombosis.