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

凝血酶原上因子 Va 结合位点的鉴定和表征。

• 批准号: RGPIN-2017-05347
• 负责人: Kim, Paul
• 金额: $ 1.89万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=655572
• 关键词: Identification; characterization; factor; Va; binding

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.**

当血管损伤时，凝血系统被激活，以停止失血，开始伤口愈合和损伤修复的过程。凝血系统由酶复合体组成，这些酶复合体依次激活下游酶复合体。一个共同的特征是，这些复合体都含有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，其辅因子的存在也会使反应增强103-105倍。因此，了解这些辅因子发挥作用的平行机制对于我们理解凝血系统和潜在的治疗方案是至关重要的。*在这些复合体中，中心凝血酶是当其前体凝血酶原(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PT可以被FXA单独激活，但当FXA在Pase中时，该反应被增强了30万倍。尽管这些相互作用的重要性，以及这一主题背后数十年的研究工作，FVA如何发挥其辅助因子功能的确切机制仍然难以捉摸。这部分是由于Pase激活PT的潜在机制的复杂性。此外，FVA如何与PT结合并提交给FXA仍未得到回答。这一点特别重要，因为相对于FXA，Fv(A)的丰度决定了PT-FVA相互作用在将PT呈现给FXA时可能是至关重要的。因此，我们希望研究PT与FVA相互作用的区域，从而使FVA在Pase激活PT的过程中发挥其辅因子活性。*我们有初步数据表明，PT Kringle2结构域的外表面参与了FVA重链的结合。更具体地说，我们已经用核磁共振鉴定了PT上六个与FVA直接结合的残基。然后，我们用惰性残基取代了这六个残基，产生了PT变体。这种PT变异体的激活导致主要中间体的形成显著减少，凝血酶的生成也显著减少，这表明PT的激活机制发生了重大变化。在这项提案中，我们的目标是通过直接表征FVA-PT相互作用来进一步调查我们的初步发现。此外，代替通常用来模拟Pase组装到的激活细胞表面的带负电荷的合成脂泡，我们还将使用新鲜获得的激活的血小板。*因此，我们的研究将产生的潜在影响是更好地理解这些非酶蛋白辅助因子如何促进血液凝块的形成。通过回答这些长期存在的问题，我们的目标是在了解调节凝血的生化过程方面开辟令人兴奋的新天地。