Activated protein C (APC) cleaves protease-activated receptor 1 (PAR1) in vitro at R46 to initiate beneficial cell signaling; however, thrombin and APC can cleave at R41. To elucidate PAR1-dependent aspects of the pharmacologic in vivo mechanisms of APC, we generated C57BL/6 mouse strains carrying QQ41 or QQ46 point mutations in PAR1 (F2r gene). Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against ischemic stroke when mice carried PAR1-homozygous mutations that prevent cleavage at either R41 or R46. Intercrossing PAR1+/R46Q mice generated expected numbers of PAR1+/+, PAR1+/R46Q, and R46Q/R46Q offspring whereas intercrossing PAR1+/R41Q mice gave decreased R41Q/R41Q homozygotes (resembling intercrossing PAR1+/PAR1-knockout mice). QQ41-PAR1 and QQ46-PAR1 brain endothelial cells showed the predicted retention or loss of cellular responses to thrombin receptor-activating peptide, thrombin, or APC for each PAR1 mutation. In sepsis studies, exogenous APC reduced mortality from 50% to 10% in Escherichia coli-induced pneumonia for wild-type (Wt) PAR1 and QQ41-PAR1 mice (P < .01) but had no benefit for QQ46-PAR1 mice. In transient distal middle cerebral artery occlusion stroke studies, exogenous APC significantly reduced infarct size, edema, and neuronal apoptosis for Wt mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb-asymmetry and foot-fault tests at 24 hours after stroke induction, signaling-selective APC was beneficial for Wt and QQ41-PAR1 mice but not QQ46-PAR1 mice. These results support the concept that APC-induced, PAR1-dependent biased signaling following R46 cleavage is central to the in vivo benefits of APC.

中文翻译：

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PAR1 偏向信号传导是活化蛋白 C 对脓毒症和中风的体内益处所必需的


激活蛋白 C (APC) 在体外裂解蛋白酶激活受体 1 (PAR1) 的 R46，启动有益的细胞信号传导；然而，凝血酶和 APC 可以在 R41 处裂解。为了阐明 APC 体内药理学机制的 PAR1 依赖性方面，我们生成了 PAR1（F2r 基因）中携带 QQ41 或 QQ46 点突变的 C57BL/6 小鼠品系。使用这些菌株，我们确定当小鼠携带防止 R41 或 R46 裂解的 PAR1 纯合突变时，重组鼠信号选择性 APC 突变体是否会减少败血症死亡或提供针对缺血性中风的神经保护。 PAR1+/R46Q 小鼠交叉产生预期数量的 PAR1+/+、PAR1+/R46Q 和 R46Q/R46Q 后代，而 PAR1+/R41Q 小鼠交叉产生 R41Q/R41Q 纯合子减少（类似于 PAR1+/PAR1 敲除小鼠交叉）。对于每个 PAR1 突变，QQ41-PAR1 和 QQ46-PAR1 脑内皮细胞显示出对凝血酶受体激活肽、凝血酶或 APC 的细胞反应的预测保留或丧失。在脓毒症研究中，外源 APC 将野生型 (Wt) PAR1 和 QQ41-PAR1 小鼠大肠杆菌诱导的肺炎死亡率从 50% 降低至 10% (P < .01)，但对 QQ46-PAR1 小鼠没有任何益处。在短暂性远端大脑中动脉闭塞中风研究中，外源性 APC 显着减少了 Wt 小鼠和 QQ41-PAR1 小鼠的梗塞面积、水肿和神经元凋亡，但对携带 QQ46-PAR1 的小鼠没有明显的益处。在中风诱导后 24 小时的前肢不对称和足部错误测试的功能研究中，信号选择性 APC 对 Wt 和 QQ41-PAR1 小鼠有益，但对 QQ46-PAR1 小鼠无效。 这些结果支持这样的概念：R46 裂解后 APC 诱导的 PAR1 依赖性偏向信号传导对于 APC 的体内益处至关重要。