Cys27Ser突变影响凝血因子X激活和凝血功能的机制

Coagulation factor X (FX) is a key enzyme in common pathway of coagulation. Zymogen-like activated FX (FXa), with its features of long half-life period and in situ stypticity, has become a hotspot in the development of bypassing agents (BPA). The characteristics are attributed to the hindrance of the heavy chain N-terminal mutation on FX activation, but the mutation also reduces coagulation activity of FXa in prothrombinase. We have previously found that Cys27Ser (C27S) mutant FXa can resist the inactivation of antithrombin and exhibits moderate coagulation activity; Molecular dynamics simulation showed that the breaking of C22-C27 disulfide bond increases the swing flexibility of polypeptide on heavy chain N-terminal. Therefore, we proposed that the C27S mutation would impede the insertion of polypeptide chain into active site by breaking C22-C27 disulfide bond, thus hindering FX activation; The mutation did not affect the conformation of the active site, leading to a higher coagulation activity. This project intends to clarify the mechanism of C27S mutation affecting FX activation through molecular dynamics simulation and enzymatic experiments, and study the characteristics of mutant FXa such as catalytic activity, affinity for substrate and inorganic ion, half-life period, etc.; The clotting tests are used to further analyze the bypass clotting activity of mutant FXa in plasma of hemophilia A patients with high titer inhibitor. The solution of questions above will increase the understanding of FX activation and provide new targets and theoretical basis for BPA development with zymogen-like FXa.

凝血因子X（FX）是凝血共同途径的关键酶。酶原样活化FX（FXa）具备长半衰期和原位止血特性，是近年研制旁路制剂的热点。其特性来自重链N端突变对FX激活过程的阻碍，但突变也导致凝血酶原酶状态下的FXa凝血活性减低。申请人前期发现Cys27Ser（C27S）突变型FXa能抵抗抗凝血酶灭活且具有较高凝血活性；分子模拟显示C22-C27二硫键断裂会增加重链N端多肽链摆动灵活性。据此推测，C27S突变通过破坏C22-C27二硫键，降低多肽链插入活性中心的效率，阻碍FX激活并使其维持酶原样状态；突变不影响活性中心，因此对凝血活性影响较小。本项目拟通过分子模拟和酶学实验，阐明C27S突变影响FX激活及影响FXa催化活性、亲和力、灭活途径等凝血功能的机制；采用凝血实验进一步分析其对含抑制物血友病A患者血浆的旁路凝血活性。解答以上问题能增加对FX激活过程的认识，为酶原样FXa研制旁路制剂提供新靶点和依据。

在血友病A患者的替代治疗中，FⅧ相关抑制物（抗体）产生是临床面临的一大问题。对于免疫诱导耐受无效的患者，只能采用旁路制剂绕过内源途径，直接激活下游共同途径的凝血酶生成，起到止血效果。凝血因子X（FX）是共同途径的关键酶，酶原样活化FX（FXa）具备长半衰期和原位止血特性，是目前研制旁路制剂的热点。其机制在于阻碍FX激活过程并使其维持酶原样状态。本项目在1例无明显出血表现的FX缺陷症家系中发现新突变Cys27Ser（C27S），Cys27位于FX活化肽N端，通过Cys22-Cys27二硫键稳定多肽链局部结构，对FX激活具有重要调控作用。通过构建野生型和Cys27Ser突变型人FX的表达质粒以及稳转细胞株，对Cys27位氨基酸进行相关功能及结构研究。结果显示，突变导致部分FX滞留在胞内，使胞外FX抗原水平减低；突变型FX受TF-FVIIa复合物和FIXa-FVIIIa复合物激活的能力受损，但被RVV激活能力及其完全激活后的发色底物活性没有影响。在没有辅因子FVa的条件下，游离突变型FXa对凝血酶原的活化受损；但当其与FVa、磷脂和Ca2+装配为凝血酶原酶复合物时，凝血活性得到大幅纠正。突变对辅因子FVa和Na+的结合能力没有影响。生理性抑制物AT和TFPI对突变型FXa的抑制作用分别下降了7倍和15倍。分子动力学模拟显示，Cys22是FX活化过程中多肽链的摆动支点，C27S突变破坏了Cys27-Cys22二硫键，使其摆动灵活性增加，从而阻碍FX激活并使其维持酶原样状态。凝血酶生成实验结果显示，C27S对含抑制物血友病A患者血浆具有很强的凝血功能纠正能力，且半衰期可达约30min。综上所述，本项目从功能检测及分子动力学模拟两方面共同阐明了C27S影响FX激活和凝血功能的机制，也为为酶原样FXa研制旁路制剂提供新靶点和依据。