Arg67His突变凝血酶活化PAR1诱导G蛋白信号通路导致血管发育异常的机制

研究表明凝血酶活化血管内皮细胞表面蛋白酶激活肽1（PAR1）诱导G蛋白信号是正常血管发育所必需。凝血酶活化PAR1的特定构象决定其诱导4种不同G蛋白信号通路，但如何调控血管新生功能与机制尚不明确。本项目组首次发现一种凝血酶原突变（Arg67His）导致血管发育异常。凝血酶阴离子结合外位点1（ABE-I）与PAR1水蛭素样结构结合后活化PAR1，Arg67位于ABE-1上。据前期工作我们推测突变可能对活化PAR1构象造成特定影响，导致PAR1诱导Gαq蛋白信号减弱。拟通过3种（His 67、Cys67和Ala67）突变凝血酶活化PAR1，评估其对血管内皮细胞血管形成功能影响；分子动力学模拟分析突变对活化PAR1构象影响；Gαq蛋白信号及4种G蛋白信号定量分析，阐明该突变导致血管发育异常机制，利用Arg67His突变小鼠模型活体内印证临床发现及机制。本项目将为血管发育异常机制研究开辟新途径。

Studies showed that the signaling pathway of activation of proteinase-activated receptor 1 (PAR1) by thrombin is essential to the maintenance of normal vascular development. Thrombin cleaves PAR1 at Arg41-Ser42 peptide bond leading to the activation of distinct four G protein subtype signal pathways by initiating conformational change of activated PAR1, but how these pathways to keep the normal formation of vasculature by crosstalk are still unkown. Here our team for the first time reported that prothrombin mutation (Arg67His) was associated with vascular dysplasia. The thrombin anion binding exosite I (ABE-I ) binds to the hirudin-like domain of PAR1 to effectively activate PAR1. Located at the crucial site of ABE-I, residue Arg67 plays an overriding role maintaining the spatial stability of the binding affinity. Based on our previous works, we predicted the mutant prothrombin will reduce the Gαq protein signal pathway by regulating the conformation of the activated PAR1. By the means of activation of PAR1 using 3 mutant recombinant proteins (Arg67His, Arg67Cys and Arg67Ala) and wild-type prothrombin, we aim to study the effect of mutative signaling on vascular formation by endothelial cell; analysis of Gαq protein signal pathway and quantitative analysis of four G protein signal pathways by phosphoproteomics, computer-assisted structural modeling analysis of effect of mutants on the conformation of activated PAR1. Based on all these results, we try to elucidate the possible mechanism of vascular dysplasia led by prothrombin mutation, which would be further discussed in vivo by Arg67His mutant mouse model in this study. This project will open a new way to investigate the mechanism of abnormal vascular development.