Characterization of mechanisms through which coagulation proteases differentially regulate hyperglycemia and hyperlipidemia induced accelerated atherosclerosis.

凝血蛋白酶的作用，特别是蛋白酶依赖的信号，在动脉粥样硬化的建立是未知的。凝血酶和活化蛋白C （PC）是凝血系统的两个关键调节因子，在凝血激活方面具有相反的作用。尽管激活了相同的受体，蛋白酶激活受体1 (PAR-1)，它们仍会引起细胞内的差异信号传导。凝血酶和活化PC的活性是由凝血酶结合内皮蛋白凝血调节素（TM）决定的，TM抑制凝血酶并激活PC。因此，TM在决定凝血酶活性、活化的PC和通过这些蛋白酶介导的次生效应中起关键作用。凝血蛋白酶受体（PAR´s， TM， EPCR（内皮蛋白C受体））的表达已经在心血管疾病相关的细胞类型中建立。然而，TM、凝血酶和活化的PC引起细胞内差异信号传导的机制及其与慢性心血管疾病的关系尚不清楚。我们已经能够证明TM在体内调节动脉粥样硬化和心脏肥厚。体外实验结果表明，TM可能通过两种机制调节细胞功能（如NF-kB激活，粘附分子的表达）：(A)抑制凝血酶依赖的细胞激活和(B)促进激活的PC依赖的细胞保护。该提案的目的是确定通过TM调节差异细胞活化和心血管疾病的机制。通过体内和体外实验，我们打算：1)确定凝血酶和活化的PC通过不同的机制调节NF-kB的活化和粘附分子的表达；2)确定TM是否具有直接信令特性；3)明确中药依赖性凝血酶和活化PC在体内动脉粥样硬化中的作用；4)确定TM-PC系统是间接（通过调节动脉血压）调控心肌肥厚还是通过受体依赖机制直接调控。目前的提案将提供一个深入的机制，通过凝血酶，活化PC和TM调节心血管疾病。将使用加速动脉粥样硬化（高脂肪饮食或高血糖）和基因改变TM-PC系统活性的动物模型。这些体内实验将与补充的体外研究相结合。拟议研究的长期目标是深入了解凝血系统调节心血管疾病的机制。这项工作将为基于凝血蛋白酶调节机制的新型靶向治疗干预奠定基础，同时避免出血风险增加等相关问题。

The role of coagulation proteases, in particular protease dependent signalling, for the establishment of atherosclerosis is unknown. Thrombin and activated protein C (PC) are two key regulators of the coagulation system with opposing effects in regard to coagulation activation. They provoke differential intracellular signalling despite activation of the same receptor, protease activated receptor 1 (PAR-1). Activity of thrombin and activated PC is determined by the thrombin binding endothelial protein thrombomodulin (TM), which inhibits thrombin and activates PC. Thus, TM has a key role in determining activity of thrombin, activated PC and secondary effects mediated through these proteases. Expression of receptors for coagulation proteases (PAR´s, TM, EPCR (endothelial protein C receptor)) has been established in cell-types relevant for cardiovascular disease. However, the mechanisms through which TM, thrombin and activated PC evoke differential intracellular signalling and the relation to chronic cardiovascular disease are not understood. We have been able to show that TM modulates atherosclerosis and cardiac hypertrophy in vivo. In vitro results suggest that TM might modulate cellular function (e.g. NF-kB activation, expression of adhesion molecules) through two mechanisms: (A) inhibition of thrombin dependent cell activation and (B) promotion of activated PC dependent cytoprotection. The aim of the proposal is to identify the mechanism through which TM modulates differential cell activation and cardiovascular disease. Using in vivo and in vitro experiments we intend to: 1) Identify the mechanism through which thrombin and activated PC differentially regulate NF-kB activation and expression of adhesion molecules; 2) Determining whether TM has direct signalling properties; 3) Define the role of TM-dependent regulation of thrombin and activated PC for atherosclerosis in vivo; 4) Determine whether the TM-PC system regulates cardiac hypertrophy indirectly (via regulation of arterial blood pressure) or directly through receptor dependent mechanisms. The current proposal will provide an in depth analysis of the mechanisms through which thrombin, activated PC, and TM regulate cardiovascular disease. Animal models of accelerated atherosclerosis (high fat diet or hyperglycemia) and with genetically altered activity of the TM-PC system will be used. These in vivo experiments will be paired with complementary in vitro studies. The long term aim of the proposed studies is an in depth understanding of the mechanisms through which the coagulation system regulates cardiovascular disease. This work will lay ground for novel targeted therapeutic intervention based on the mechanisms regulated by coagulation proteases, but circumventing associated problems such as increased risk of hemorrhage.