Thrombin activation of protein C requires prior processing by a liver proprotein convertase
Thrombin activation of protein C requires prior processing by a liver proprotein convertase
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DOI：
10.1074/jbc.m116.770040
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发表时间：
2017-06-23
影响因子：
4.8
通讯作者：
Seidah, Nabil G.
中科院分区：
文献类型：
--
作者：
Essalmani, Rachid;Susan-Resiga, Delia;Seidah, Nabil G.
蛋白C是一种分泌型维生素K依赖性抗凝丝氨酸蛋白酶，可使因子Va/VIIIa失活。它仅在肝细胞中作为无活性酶原（前蛋白C）合成。在人类中，凝血酶在PR 221处切割前肽导致活化的蛋白C（APC;残基222-461）。然而，前肽也被弗林蛋白酶样前蛋白转化酶（PC）在KKRSHLKR 199（加下划线的对PC识别至关重要的碱性残基）处切割，但切割顺序未知。在此，我们提出的证据表明，在COS-1细胞的表面，小鼠前蛋白C是第一次切割的转化酶弗林蛋白酶，PC 5/6A，和PACE 4。在小鼠中，这种切割发生在等效位点KKRKILKR 198，并且需要P1处存在Arg（198），以及P2（Lys（197））、P6（Arg（193））或P8（Lys（191））位置处存在两个其他碱性残基的组合。值得注意的是，凝血酶抗性R221 A突变体仍然被这些PC裂解，这表明转化酶裂解可以先于凝血酶活化。这一结论得到了以下事实的支持：COS-1细胞培养基中的APC特异性活性完全依赖于转化酶的预先切割，因为R198 A和R221 A都缺乏蛋白C活性。来自野生型或肝细胞特异性弗林蛋白酶，PC 5/6，或完全PACE 4基因敲除小鼠的肝细胞的原代培养物表明，过度表达的蛋白C前体的裂解主要是由弗林蛋白酶细胞内和所有三个前蛋白转化酶在细胞表面进行。事实上，单前蛋白转化酶敲除小鼠的血浆分析显示，肝细胞中转化酶弗林蛋白酶或PC 5/6的缺失导致APC水平降低约30%，而PACE 4没有显著贡献。我们的结论是，在肝细胞中蛋白C的转化酶裂解是至关重要的凝血酶激活。
Protein C, a secretory vitamin K-dependent anticoagulant serine protease, inactivates factors Va/VIIIa. It is exclusively synthesized in liver hepatocytes as an inactive zymogen (proprotein C). In humans, thrombin cleavage of the propeptide at PR221 results in activated protein C (APC; residues 222-461). However, the propeptide is also cleaved by a furin-like proprotein convertase(s) (PCs) at KKRSHLKR199 (underlined basic residues critical for the recognition by PCs), but the order of cleavage is unknown. Herein, we present evidence that at the surface of COS-1 cells, mouse proprotein C is first cleaved by the convertases furin, PC5/6A, and PACE4. In mice, this cleavage occurs at the equivalent site, KKRKILKR198, and requires the presence of Arg(198) at P1 and a combination of two other basic residues at either P2 (Lys(197)), P6 (Arg(193)), or P8 (Lys(191)) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these PCs, revealing that convertase cleavage can precede thrombin activation. This conclusion was supported by the fact that the APC-specific activity in the medium of COS-1 cells is exclusively dependent on prior cleavage by the convertases, because both R198A and R221A lack protein C activity. Primary cultures of hepatocytes derived from wild-type or hepatocyte-specific furin, PC5/6, or complete PACE4 knock-out mice suggested that the cleavage of overexpressed proprotein C is predominantly performed by furin intracellularly and by all three proprotein convertases at the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase furin or PC5/6 in hepatocytes results in a approximate to 30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase cleavage of protein C in hepatocytes is critical for its thrombin activation.