Michael E. DeBakey Veterans Affairs Medical Center, Department of Pathology, Baylor College of Medicine.
Transfusion. 2010 Oct;50(10):2167-75. doi: 10.1111/j.1537-2995.2010.02676.x. Epub 2010 Oct 4.
The exposure of phosphatidylserine occurs during platelet (PLT) activation and during in vitro storage. Phosphatidylserine exposure also occurs during apoptosis after the release of mitochondrial cytochrome c. We have examined the role of cytochrome c release, mitochondrial membrane potential (ΔΨm), and cyclophilin D (CypD) in phosphatidylserine exposure due to activation and storage.
The exposure of phosphatidylserine and the loss of ΔΨm were determined in a flow cytometer using fluorescein isothiocyanate-lactadherin and JC-1, a lipophilic cationic reporter dye. The role of CypD was determined with cyclosporin A and CypD-deficient murine PLTs. Cytochrome c-induced caspase-3 and Rho-associated kinase I (ROCK1) activation were determined by immunoblotting and using their inhibitors.
Collagen- and thrombin-induced exposure of phosphatidylserine was accompanied by a decrease in ΔΨm. Cyclosporin A inhibited the phosphatidylserine exposure and the loss of ΔΨm. CypD(-/-) mice had decreased loss of ΔΨm and impaired phosphatidylserine exposure. Collagen and thrombin did not induce the release of cytochrome c nor the activation of caspase-3 and ROCK1. In contrast, in PLTs stored for more than 5 days, the phosphatidylserine exposure was associated with cytochrome c-induced caspase-3 and ROCK1 activation. ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis, induced cytochrome c release and activation of caspase-3 and ROCK1 and phosphatidylserine exposure independent of CypD.
These results show that in stored PLTs cytochrome c release and the subsequent activation of caspase-3 and ROCK1 mediate phosphatidylserine exposure and it is distinct from activation-induced phosphatidylserine exposure.
血小板（PLT）激活和体外储存过程中会出现磷脂酰丝氨酸暴露。线粒体细胞色素 c 释放后发生细胞凋亡时也会出现磷脂酰丝氨酸暴露。我们已经研究了细胞色素 c 释放、线粒体膜电位（ΔΨm）和亲环蛋白 D（CypD）在激活和储存过程中导致磷脂酰丝氨酸暴露的作用。
使用荧光素异硫氰酸酯-乳白蛋白和亲脂性阳离子报告染料 JC-1，在流式细胞仪中测定磷脂酰丝氨酸的暴露和 ΔΨm 的丧失。用环孢素 A 和 CypD 缺陷型小鼠 PLT 确定 CypD 的作用。通过免疫印迹和使用其抑制剂测定细胞色素 c 诱导的半胱天冬酶-3 和 Rho 相关激酶 I（ROCK1）激活。
胶原和凝血酶诱导的磷脂酰丝氨酸暴露伴随着 ΔΨm 的降低。环孢素 A 抑制了磷脂酰丝氨酸暴露和 ΔΨm 的丧失。CypD(-/-) 小鼠 ΔΨm 丧失减少，磷脂酰丝氨酸暴露受损。胶原和凝血酶不会诱导细胞色素 c 的释放或半胱天冬酶-3 和 ROCK1 的激活。相比之下，在储存超过 5 天的 PLT 中，磷脂酰丝氨酸暴露与细胞色素 c 诱导的 caspase-3 和 ROCK1 激活有关。ABT737，一种诱导线粒体凋亡途径的 BH3 模拟物，可诱导细胞色素 c 释放以及 caspase-3 和 ROCK1 的激活和磷脂酰丝氨酸暴露，而与 CypD 无关。
这些结果表明，在储存的 PLT 中，细胞色素 c 的释放以及随后的 caspase-3 和 ROCK1 的激活介导了磷脂酰丝氨酸的暴露，与激活诱导的磷脂酰丝氨酸暴露不同。
