From the Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322.
From the Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322,; the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30322, and.
J Biol Chem. 2013 Oct 18;288(42):30672-30681. doi: 10.1074/jbc.M113.472688. Epub 2013 Sep 6.
When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin αIIbβ3 or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin αIIbβ3 and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin αIIbβ3 epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin αIIbβ3 function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β3 cytoplasmic domain, coincided closely with integrin αIIbβ3 inactivation. Calpain inhibition blocked integrin β3 cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β3 and associated regulatory proteins, resulting in integrin αIIbβ3 inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.
当血小板受到强烈刺激时，会形成一个促凝的血小板亚群，其特征是暴露磷脂酰丝氨酸 (PS) 和整合素 αIIbβ3 的表位调节或丧失与激活依赖性抗体的结合。对于促凝血小板的形成，线粒体通透性转换孔 (mPTP) 的形成是必不可少的，而在缺乏亲环素 D (CypD) 的情况下，mPTP 的形成会受到损害。在这里，我们研究了导致整合素 αIIbβ3 发生这些促凝血小板相关变化的机制，以及促凝血小板形成在调节血小板聚集中的生理作用。在强烈刺激的黏附血小板中，整合素 αIIbβ3 表位变化、mPTP 形成、PS 暴露和血小板变圆紧密相关。此外，血小板 mPTP 的形成导致其募集额外血小板的能力降低。在 CypD 缺乏的情况下，整合素 αIIbβ3 的功能在静态和流动条件下都得到了增强，并且在 CypD 血小板特异性缺乏的小鼠体内，发生了一种促血栓形成的表型。CypD 依赖性蛋白水解事件，包括整合素 β3 胞质结构域的裂解，与整合素 αIIbβ3 的失活密切相关。钙蛋白酶抑制阻断了整合素 β3 的裂解和失活，但不阻断 mPTP 的形成或 PS 的暴露，这表明整合素失活和 PS 暴露是 mPTP 形成后通过不同途径介导的。促凝血小板中发生 mPTP 依赖性碱化，这表明在促凝血小板中增强钙蛋白酶活性的可能存在另一种机制。综上所述，这些结果表明，在强烈刺激的血小板中，mPTP 的形成引发钙蛋白酶依赖性的整合素 β3 及其相关调节蛋白的裂解，导致整合素 αIIbβ3 失活，并证明了一种新的 CypD 依赖性负反馈机制，该机制限制了血小板聚集和血栓闭塞。
