Tyr319Cys突变影响凝血因子X功能的机制及其应用

Direct inhibitors of activated coagulation factor X (FXa) are promising oral anticoagulants in treatment and prevention of thromboembolism. However, there raises an increasing safety concern as clinical need for a agent to rapidly reverse the effects of anticoagulants is unmet, especially when serious bleeding occurs or urgent medical procedures are underwent. The crystal structure and site direction mutagenesis studies of FX indicated that Tyr319 residue was critical for specificity of chromogentic substrate. A novel FX Tyr319Cys mutation located this residue was identified in a patient with severe bleeding diathesis in this project. The mutant FX function of activation of prothrombin was impaired but cleavage of chromogenetic substrate was normal. We predicted that the mutant FX could connect with its inhibitor. By means of in vitro expression and activation of the mutant protein, we propose an overall study of activation of the mutant, its coagulation activity and enzymatic inhibition to further elucidate the possible mechanism resulting in hemorrhage, thus exhibiting an overview of the possible relationships between protein structural alteration and functional abnormality. Moreover, pharmacodynamics of FX Tyr319Cys mutant binding anti-FXa anticoagulants are carried out in wildtype mice, as well as tail-bleeding mice and thrombosis-induced mice model to comprehensively explore the efficacy of FXa Tyr319Cys in mitigating the anticoagulant FXa.

抗活化凝血因子X（FXa）药物为人工合成的FX抑制剂，是最具潜力的血栓病防治药物，但患者使用中发生严重出血和/或需紧急手术时，目前尚无快速终止该药抗凝作用的拮抗剂，是临床亟待解决的问题。晶体结构和定点突变研究显示FX的Tyr319位氨基酸在决定酶底物特异性和催化活性中起重要作用，本项目在1例有出血表现的患者中发现了该位点的1种新突变（Tyr319Cys），该突变的FX不能将凝血酶原活化为凝血酶但能够裂解其特异性小分子底物，说明该突变不影响FX的活化中心结构，推测该突变FX保留与其抑制物结合特性。本项目通过对该突变的FX活化，凝血活性及灭活的酶学特性分析，阐明突变导致凝血功能障碍的机制及突变蛋白结构与功能的关系。在此基础上，通过小鼠体内试验分析突变FXa与抗FXa药物结合的药代动力学行为；利用出血和血栓模型阐明突变FXa拮抗抗FXa药物的能力，探讨该突变FXa作为抗FXa药物拮抗剂的可行性。

利伐沙班等抗活化凝血因子X(FXa)药物是目前最具潜力的血栓病防治药物，但患者使用中发生严重出血和/或需紧急手术时尚无快速终止该药抗凝作用的拮抗剂，是临床亟待解决的问题。晶体结构和定点突变研究显示，FX的Tyr319位氨基酸在决定酶底物特异性和催化活性中起重要作用。本项目在1例有出血表现的患者中发现了该位点新突变（Tyr319Cys），通过构建2种突变型（Tyr319Cys和Tyr319Ala）及野生型人FX的表达质粒以及稳转细胞株，对Tyr319位氨基酸突变型FX的相关功能以及结构进行研究。结果显示，两种突变型能够被FXFVIIa-TF复合物和Russell蝰蛇毒正常活化；突变导致FXa裂解小分子底物的能力受损，但当其与组装为凝血酶原酶复合物时（FXa- FVa-磷脂-Ca2+）催化能力得到部分纠正；突变型FXa对凝血酶原的活化作用严重受损。分子动力学模拟显示，Tyr319位氨基酸位于S1口袋背侧，且突变型FXa的S4口袋面积未改变，推测突变不影响FXa与利伐沙班结合，且不影响FXa被抗凝血酶正常灭活；通过与AT和利伐沙班的结合实验证实了分子动力学模拟结果。此外，通过凝血酶原时间（PT）、凝血酶生成实验（TGT）和小鼠断尾出血模型评估了Tyr319Cys突变型FXa拮抗过量利伐沙班并纠正凝血功能的潜力。综上所述，本项目从结构检测及分子模拟两方面共同阐明了Tyr319位氨基酸突变导致II型FX缺陷症的分子致病机制，并为新型利伐沙班拮抗剂的研制提供理论依据。