Li J, Xia Y, Bertino A M, Coburn J P, Kuter D J
Hematology Oncology Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Transfusion. 2000 Nov;40(11):1320-9. doi: 10.1046/j.1537-2995.2000.40111320.x.
Although it is usually involved only in nucleated cells (NCs), artificially enucleated cells also lose viability by a programmed process of cell death called apoptosis. Because platelets undergo loss of viability during storage, an attempt was made to determine whether platelets contained the apoptotic mechanisms and whether it was activated during platelet storage.
Platelet viability was measured by reduction of a tetrazolium dye (MTS) and annexin V binding. Members of the death receptor, caspase, and Bcl-2 families were detected by RNase protection assay and Western blotting. Caspase 3 activation was measured by enzyme and Western blot assays and by cleavage of gelsolin.
After 5 days of storage under standard blood banking conditions, platelets display biochemical signs of apoptosis by losing MTS activity and increasing the amount of phosphatidylserine on their surface. The mRNA and the proenzyme for several members of the caspase, death receptor, and Bcl-2 families are expressed at high levels in platelets. An increase in caspase 3 activity and the amount of the biologically active p17 subunit of active caspase 3 were observed to coincide with the appearance of apoptotic markers during storage. These effects were not due to platelet activation. The caspase 3 substrate, gelsolin, began to undergo proteolysis after 3 to 4 days of storage, and the addition of the caspase inhibitor z-VAD-fmt substantially inhibited this process.
Platelets contain many of the components of the apoptotic mechanism and show activation of caspase 3 and consequent cleavage of gelsolin during storage, independent of platelet activation. Evaluation of the mechanism of apoptosis in platelets may provide a basis for developing novel strategies to enhance platelet viability during storage.
尽管凋亡通常仅发生于有核细胞（NCs），但人工去核细胞也会通过一种称为凋亡的程序性细胞死亡过程丧失活力。由于血小板在储存过程中会丧失活力，因此尝试确定血小板是否含有凋亡机制以及在血小板储存过程中该机制是否被激活。
通过四唑盐染料（MTS）还原法和膜联蛋白V结合法测定血小板活力。通过核糖核酸酶保护分析和蛋白质印迹法检测死亡受体、半胱天冬酶和Bcl-2家族成员。通过酶法、蛋白质印迹法以及凝溶胶蛋白的裂解来测定半胱天冬酶3的激活情况。
在标准血库条件下储存5天后，血小板通过丧失MTS活性和增加其表面磷脂酰丝氨酸的量而表现出凋亡的生化迹象。半胱天冬酶、死亡受体和Bcl-2家族的几个成员的mRNA和酶原在血小板中高水平表达。观察到储存期间半胱天冬酶3活性的增加以及活性半胱天冬酶3的生物活性p17亚基的量与凋亡标志物的出现相一致。这些效应并非由于血小板激活所致。凝溶胶蛋白作为半胱天冬酶3的底物，在储存3至4天后开始发生蛋白水解，并且添加半胱天冬酶抑制剂z-VAD-fmt可显著抑制这一过程。
血小板含有许多凋亡机制的成分，并且在储存期间显示出半胱天冬酶3的激活以及随之而来的凝溶胶蛋白的裂解，这与血小板激活无关。评估血小板凋亡机制可能为开发提高血小板储存期间活力的新策略提供依据。
