Identification and characterization of the factor Va binding site on prothrombin.

在血管损伤时，凝血系统被激活，以停止失血，并开始伤口愈合和损伤修复的过程。凝血系统由酶复合物组成，酶复合物依次激活下游酶复合物。一个共同的特点是这些复合物都含有1)酶，2)辅因子，3)钙和4)带负电荷的细胞表面。在所有情况下，即使酶能够单独作用于底物，其辅因子的存在将反应增强103- 105倍。因此，了解这些辅助因子发挥作用的平行机制对于我们了解凝血系统和潜在的治疗方案至关重要。***在这些复合体中，中心凝血酶凝血酶是在其前体凝血酶原（PT）被凝血酶原复合体（Pase）激活时产生的。Pase由酶因子(F) Xa、辅因子FVa、钙和细胞表面组成。虽然FXa可以单独激活PT，但当FXa在Pase中时，该反应增强了30万倍。尽管这些相互作用很重要，并且在这个主题背后进行了数十年的研究工作，但FVa如何发挥其辅因子功能的确切机制仍然难以捉摸。这部分是由于PT被Pase激活的潜在机制的复杂性。此外，FVa如何结合PT并呈递给FXa仍未得到解答。这一点尤其重要，因为FV(a)相对于FXa的丰度表明，PT- fva相互作用可能是将PT呈递给FXa的关键。因此，我们希望研究PT中负责与FVa相互作用的区域，从而使FVa在PT被Pase激活时发挥其辅因子活性。***我们有初步的数据表明，PT的kringle 2结构域的外表面参与了FVa重链的结合。更具体地说，我们已经确定了六个残基PT直接结合FVa使用核磁共振。然后，我们用惰性残基取代了这六个残基，生成了一个PT变体。这种PT变体的激活导致一种主要中间体的形成大幅减少，凝血酶的生成也显著减少，这表明PT激活的机制发生了重大变化。在这项提议中，我们的目标是通过直接表征FVa-PT相互作用来进一步研究我们的初步发现。此外，代替通常用于模拟Pase组装的活化细胞表面的带负电荷的合成脂质囊泡，我们还将使用获得的新鲜活化血小板。因此，我们研究的潜在影响是更好地理解这些非酶蛋白辅助因子是如何促进血凝块形成的。通过回答这些长期存在的问题，我们的目标是在理解调节凝血的生化过程方面开辟令人兴奋的新天地

Upon vascular injury, the coagulation system is activated in order to stop the blood loss and begin the process of wound healing and damage repair. The clotting system consists of enzyme complexes that sequentially activate downstream enzyme complexes. One characteristic in common is that these complexes all contain 1) enzyme, 2) cofactor, 3) calcium, and 4) negatively charged cell surface. In all instances, even though the enzyme is able to act on the substrate alone, the presence of its cofactor enhances the reaction by 103- to 105-fold. Therefore, understanding the parallel mechanism by which these cofactors exert their effect is critical in our understanding of the coagulation system and potentially developing treatment options.***Of these complexes, the central clotting enzyme thrombin is generated when its precursor prothrombin (PT) is activated by the prothrombinase complex (Pase). Pase consists of the enzyme factor (F) Xa, cofactor FVa, calcium and cell surface. While PT can be activated by FXa alone, this reaction is enhanced by 300,000-fold when FXa is in Pase. Despite the importance of these interactions, and decades of research work behind this topic, the exact mechanism of how FVa exerts its cofactor function remains elusive. This is partly due to the complexity in the underlying mechanism of PT activation by Pase. Furthermore, how FVa binds PT and is presented to FXa remains unanswered. This is particularly of importance since the abundance of FV(a) relative to FXa dictates that PT-FVa interaction may be crucial in presenting PT to FXa. Therefore, we wish to investigate the region of PT that is responsible for interacting with FVa, and thus allowing FVa to exert its cofactor activity during PT activation by Pase.***We have preliminary data that demonstrate that the outer face of kringle 2 domain of PT is involved in binding the heavy chain of FVa. More specifically, we have identified six residues on PT that directly bind FVa using NMR. We then generated a PT variant with these six residues substituted with inert residues. Activation of this PT variant led to a substantial decrease in the formation of a major intermediate as well as a significant decrease in thrombin generation, suggesting that there has been a major change in the mechanism of PT activation. In this proposal, we aim to further investigate our initial findings by directly characterizing the FVa-PT interaction. Furthermore, in place of the negatively charged synthetic lipid vesicles that are typically used to mimic the activated cell surface to which Pase is assembled, we will also use activated platelets that are obtained fresh.***Therefore, the potential impact our research will have is a better understanding of how these non-enzymatic protein cofactors work to enhance blood clot formation. By answering these long standing questions, we aim to break exciting new ground in understanding the biochemical processes that modulate coagulation.**