Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA.
J Thromb Haemost. 2013 Aug;11(8):1532-9. doi: 10.1111/jth.12307.
Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor (F) Va and FXa on the activated platelet surface. While the plasma-derived procofactor, FV, must be proteolytically activated by α-thrombin to FVa to function in prothrombinase, the platelet molecule is released from α-granules in a partially activated state, obviating the need for proteolytic activation.
The current study was performed to test the hypothesis that subsequent to its endocytosis by megakaryocytes, plasma-derived FV is proteolytically processed to form the platelet-derived pool.
METHODS & RESULTS: Subsequent to FV endocytosis, a time-dependent increase in FV proteolytic products was observed in megakaryocyte lysates by SDS-PAGE followed by phosphorimaging or western blotting. This cleavage was specific and resulted in the formation of products similar in size to FV/Va present in a platelet lysate as well as to the α-thrombin-activated FVa heavy chain and light chain, and their respective precursors. Other proteolytic products were unique to endocytosed FV. The product/precursor relationships of these fragments were defined using anti-FV heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte-derived FV fragments exhibited substantial FVa cofactor activity that was comparable to platelet-derived FV/Va.
Taken together, these observations suggest that prior to its packaging in α-granules endocytosed FV undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet-derived pool.
在血管损伤部位，适当的凝血酶形成的核心是血浆和/或血小板衍生的因子（F）Va 和 FXa 在激活的血小板表面的协同组装。虽然血浆来源的前体因子 FV 必须被α-凝血酶蛋白水解激活为 FVa 才能在凝血酶原酶中发挥作用，但血小板分子以部分激活状态从α-颗粒中释放出来，从而避免了蛋白水解激活的需要。
本研究旨在检验以下假说：FV 在被巨核细胞内吞后，会被蛋白水解加工形成血小板衍生池。
FV 内吞后，在巨核细胞裂解物中通过 SDS-PAGE 后磷成像或 Western blot 观察到 FV 蛋白水解产物随时间的增加。这种切割是特异性的，形成的产物大小与血小板裂解物中存在的 FV/Va 以及 α-凝血酶激活的 FVa 重链和轻链及其各自的前体相似。其他蛋白水解产物是内吞 FV 所特有的。使用具有定义表位的抗 FV 重链和轻链抗体定义了这些片段的产物/前体关系。活性测量表明，巨核细胞衍生的 FV 片段表现出相当大的 FVa 辅助因子活性，可与血小板衍生的 FV/Va 相媲美。
综上所述，这些观察结果表明，在被包装到α-颗粒之前，内吞 FV 会被一种或多种特定的巨核细胞蛋白酶进行蛋白水解，形成部分激活的血小板衍生池。
