Yang Aizhen, Chen Fengwu, He Chao, Zhou Junsong, Lu Yi, Dai Jihong, Birge Raymond B, Wu Yi
Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, China.
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, United States.
Front Immunol. 2017 Sep 25;8:1188. doi: 10.3389/fimmu.2017.01188. eCollection 2017.
Apoptotic cells, by externalizing phosphatidylserine (PS) as a hallmark feature, are procoagulant. However, the mechanism by which apoptotic cells activate coagulation system remains unknown. Intrinsic coagulation pathway is initiated by coagulation factor XII (FXII) of contact activation system. The purpose of this study was to determine whether FXII is involved in procoagulant activity of apoptotic cells. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells, but not with viable cells, resulted in rapid cleavage and activation of FXII in the presence of prekallikrein and high molecular weight kininogen (HK), other two components of contact activation system. As detected by flow cytometry, FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. Direct association of FXII with PS was confirmed in a surface plasmon resonance assay. Clotting time of FXII-deficient plasma induced by apoptotic cells was significantly prolonged, which was fully reversed by replenishment with FXII. Corn trypsin inhibitor, a FXII inhibitor, completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which was not affected by an inhibitory anti-tissue factor antibody. However, blocking of PS by annexin V, inhibition of FXII, or the deficiency of FXII suppressed apoptotic cells-induced thrombin generation. Addition of purified FXII to FXII-deficient plasma recovered thrombin generation to the normal plasma level. In conclusion, FXII binds to apoptotic cells PS and becomes activated, thereby constituting a novel mechanism mediating the procoagulant activity of apoptotic cells.
凋亡细胞通过将磷脂酰丝氨酸（PS）外翻作为标志性特征，具有促凝作用。然而，凋亡细胞激活凝血系统的机制仍不清楚。内源性凝血途径由接触激活系统的凝血因子XII（FXII）启动。本研究的目的是确定FXII是否参与凋亡细胞的促凝活性。通过蛋白质印迹法和发色底物测定，我们发现与凋亡细胞孵育，而不是与活细胞孵育，在存在前激肽释放酶和高分子量激肽原（HK）（接触激活系统的其他两个成分）的情况下，会导致FXII迅速裂解和激活。通过流式细胞术检测，FXII以浓度依赖性方式与凋亡细胞结合，这被膜联蛋白V和PS脂质体抑制。在表面等离子体共振测定中证实了FXII与PS的直接结合。凋亡细胞诱导的FXII缺陷血浆的凝血时间显著延长，补充FXII可完全逆转。玉米胰蛋白酶抑制剂，一种FXII抑制剂，完全阻止了凋亡细胞诱导的内源性凝血酶原酶复合物形成。一致地，凋亡细胞显著增加正常血浆中的凝血酶产生，这不受抑制性抗组织因子抗体的影响。然而，膜联蛋白V阻断PS、抑制FXII或FXII缺乏会抑制凋亡细胞诱导的凝血酶生成。向FXII缺陷血浆中添加纯化的FXII可将凝血酶生成恢复到正常血浆水平。总之，FXII与凋亡细胞的PS结合并被激活，从而构成介导凋亡细胞促凝活性的新机制。
