凝血酶通过蛋白酶激活受体1介导对斑块稳定性的影响及其作用机制的研究

Thrombin, a multifunctional serine protease, plays important roles in many physiological and pathological processes, such as the coagulation cascade at the site of vascular injury. It activates blood platelets and elicits multiple effects on variant cell types associated with atherosclerosis, including endothelial cells, vascular smooth muscle cells (VSMCs) and monocytes. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), especially PAR1. Thrombin signaling in platelets contributes to haemostasis and thrombosis. PAR1 has become a therapeutic target to inhibit platelet aggregation and thrombosis. In normal arteries, PAR1 is mainly expressed in endothelial cells, while limited in VSMCs. Accumulating evidences have demonstrated that thrombin/PAR1 contribute to the pro-inflammatory phenotype in endothelial cells, VSMCs and macrophages. Based on these observations, thrombin/PAR1 might be a key element in the pathogenesis of atherosclerosis. However, the exact effects of thrombin on atherosclerosis pathogenesis are still unknown. In the following study, we will observe the effects of thrombin on plaque destabilization via PAR1 in vivo, and investigate the potential mechanisms involved in vitro, by using PAR1 antagonist and PAR1 agonist to inhibit and activate the function of PAR1, respectively. To avoid the mixed effects of the thrombin/PAR1/platelet activation signaling pathway, we will use ApoE-/- mice as an atherosclerosis model, for thrombin activates platelets via PAR3 and PAR4 in mice while via PAR1 in human. This study will provide more information about the thrombin mediated effects on atherosclerotic pathogenesis and might be helpful for the development of a new therapeutic target for atherosclerosis-related diseases.

急性冠脉综合征（ACS）患者血液凝血酶水平高于正常人。凝血酶是一种多功能丝氨酸蛋白水解酶，除了参与凝血反应外，还可通过特异性受体PAR1介导对多种细胞产生作用。为研究凝血酶通过PAR1介导对斑块稳定性的影响及其可能作用机制，本课题拟从体内采用本实验室构建的ApoE-/-小鼠易损斑块动脉模型，通过给予PAR1拮抗剂或激动剂分别抑制或激活PAR1，观察凝血酶-PAR1对斑块稳定性的影响；体外培养细胞，通过添加激动剂和抑制剂激活或者抑制凝血酶-PAR1及其下游通路，探讨凝血酶-PAR1对平滑肌细胞以及巨噬细胞功能的影响，以明确凝血酶-PAR1对斑块稳定性影响的分子作用机制。本研究结果将为探讨凝血酶-PAR1是否可以作为ACS治疗新靶点提供直接实验依据。

为研究凝血酶通过PAR1介导对斑块稳定性的影响及其可能作用机制，本课题分别从体内采用ApoE-/-小鼠易损斑块动脉模型，通过给予PAR1拮抗剂或激动剂分别抑制或激活PAR1，观察凝血酶-PAR1对斑块稳定性的影响；体外培养平滑肌细胞、巨噬细胞、血管内皮细胞，通过添加激动剂和抑制剂激活或者抑制凝血酶—PAR1及其下游通路，探讨凝血酶—PAR1对细胞功能的影响，以明确凝血酶—PAR1对斑块稳定性影响的可能作用机制。体内研究结果显示，与对照组相比，模型组小鼠斑块内胶原含量显著减少、而炎症因子浸润及新生血管形成显著增加，斑块不稳定性及斑块内出血发生率增加。给予PAR1拮抗剂SCH79797组小鼠颈动脉斑块胶原含量增加、炎症因子浸润减少、新生血管形成减少、斑块内出血减少，斑块稳定性增加。体外研究进一步明确了凝血酶通过PAR1介导增加斑块不稳定性的可能机制包括以下三方面：①凝血酶能够增加MMP-2、MMP-9表达及活化，促进斑块内胶原降解，同时抑制斑块内胶原蛋白合成，从而减少斑块内胶原含量，而PAR1拮抗剂SCH79797可抑制凝血酶这些作用；②凝血酶能够增加细胞中炎症因子IL-6、IL-1β表达，MAPK通路参与凝血酶-PAR1的促炎作用；SCH79797可抑制凝血酶的促炎作用；③凝血酶能够增加血管内皮细胞迁移、表达血管生成因子，促进内皮细胞体外成管，促进新生血管形成。PAR1拮抗剂SCH79797可抑制凝血酶的促新生血管形成作用。该研究为探讨凝血酶/PAR1途径是否能够成为急性冠脉综合征的新治疗靶点提供一定的实验基础。