Structural determinants of PAR1 cleavage by activated protein C.

IF 5.5 2区医学 Q1 HEMATOLOGY

Journal of Thrombosis and Haemostasis Pub Date : 2025-06-09 DOI:10.1016/j.jtha.2025.05.034

Bosko M Stojanovski, Enrico Di Cera

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引用次数: 0

Abstract

Background: Activated protein C (APC) performs cytoprotective functions mediated by cleavage of the protease activated receptor 1 (PAR1) in the presence of the endothelial protein C receptor (EPCR) and signaling through b-arrestin-2. APC cleaves PAR1 at R41 and R46, but the specificity of the reaction is low. In contrast, thrombin cleaves PAR1 at R41 only in a reaction that is independent of EPCR, produces a pro-inflammatory response mediated by signaling through G-protein intermediates and features high specificity. The molecular basis of this difference between APC and thrombin remains unknown.

Objective: To identify the structural determinants of APC that influence PAR1 specificity.

Methods: Using available structural information, we engineered thrombin determinants of PAR1 recognition into APC. Specifically, we replaced T99 with Leu and swapped the entire 37- and 60- loops of APC with those of thrombin.

Results: The engineered APC variants feature up to 80-fold enhanced specificity toward PAR1 mediated by increased cleavage at R41 and decreased cleavage at R46. Notably, the variants APC_60/T99L and APC_37/60/T99L also show significantly reduced activity toward factor Va.

Conclusion: The 37-, 60-, and 99- segments of APC determine the cytoprotective and anticoagulant properties of the enzyme.

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活化蛋白C裂解PAR1的结构决定因素。

背景：在内皮蛋白C受体（EPCR）存在的情况下，活化蛋白C （APC）通过蛋白酶活化受体1 （PAR1）的裂解和b-arrestin-2的信号传导来发挥细胞保护功能。APC在R41和R46位点切割PAR1，但特异性较低。相比之下，凝血酶仅在R41位点切割PAR1的反应独立于EPCR，通过g蛋白中间体产生信号介导的促炎反应，具有高特异性。APC和凝血酶之间这种差异的分子基础尚不清楚。目的：探讨影响PAR1特异性的APC结构决定因素。方法利用现有的结构信息，将PAR1识别凝血酶决定因素改造成APC。具体来说，我们将T99替换为Leu，并将APC的整个37和60环替换为凝血酶的环。结果：经过工程修饰的APC变体对PAR1的特异性提高了80倍，这种特异性是通过R41位点的切割增加和R46位点的切割减少介导的。值得注意的是，变异APC60/T99L和APC37/60/T99L对va因子的活性也显著降低。结论：APC的37、60和99段决定了该酶的细胞保护和抗凝血特性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Thrombosis and Haemostasis 医学-外周血管病

CiteScore

24.30

自引率

3.80%

发文量

321

审稿时长

1 months

期刊介绍： The Journal of Thrombosis and Haemostasis (JTH) serves as the official journal of the International Society on Thrombosis and Haemostasis. It is dedicated to advancing science related to thrombosis, bleeding disorders, and vascular biology through the dissemination and exchange of information and ideas within the global research community. Types of Publications: The journal publishes a variety of content, including: Original research reports State-of-the-art reviews Brief reports Case reports Invited commentaries on publications in the Journal Forum articles Correspondence Announcements Scope of Contributions: Editors invite contributions from both fundamental and clinical domains. These include: Basic manuscripts on blood coagulation and fibrinolysis Studies on proteins and reactions related to thrombosis and haemostasis Research on blood platelets and their interactions with other biological systems, such as the vessel wall, blood cells, and invading organisms Clinical manuscripts covering various topics including venous thrombosis, arterial disease, hemophilia, bleeding disorders, and platelet diseases Clinical manuscripts may encompass etiology, diagnostics, prognosis, prevention, and treatment strategies.