脓毒症时血小板cGMP/GSK-3β信号途径调节血小板活化的分子机制及外源性CO的调控作用

Activation of platelets played a major role in sepsis, but its mechnisms remain to be investigated. Our previous studies have shown that the activity of GSK-3β, a key ser-thr kinase, was increased strongly and was associated with overactivation of coagβulation system. We thus hypothesis that exogenous CO can regulate activation of platelets through sGC/cGMP/GSK-3βpathway. This project employs CLP mice and LPS-stimulated platelets as sepsis models to examine the dynamic state of sGC/cGMP/GSK-3βpathway , especially the phosphorylation level of GSK-3β, and its relative regulatory kinases under the intervention of the exogenous CO liberated by CO-releasing molecules. These important factors will be investigated in the molecular and cellular levels through applying updated molecular biological techniques including gene analysis, measurement of protein expressions. The dynamic changes of important kinases (Akt, PKG) phosphorylation will be further explored. Thus, this project focuses on the relationship between the modification of GSK-3 β, and its relative regulatory kinases phosphorylation and activity of platelets during sepsis. Furthermore, the effects of exogenous CO on regulating GSK-3β phosphorylation in sepsis and potential mechnisms will be evaluted in this project. Therefor, we are in attempt to find which kinds of phosphorylation regulatory factors will be the novel targets of exogenous CO, in order to elucidate molecular mechnisms (even partly) responsible for the regulatory effects of CO on abnormal activity of platelets and signal transduction during sepsis and to provide theoretical basis and new idea for the research and treatment of sepsis.

血小板活化是脓毒症时凝血系统激活过程中的重要事件，但其调控机制还未被清楚阐明。我们的前期研究首次证实脓毒症早期血小板关键激酶GSK-3β活性明显提高，与血小板活化关系密切，外源性CO干预GSK-3β活性被下调。我们进一步推测CO通过sGC/cGMP/GSK-3β途径调节血小板活化。本研究采用CLP脓毒症动物模型和相关血小板模型，以新型外源性CO干预，运用基因分析、蛋白质水平测定等手段，在分子、细胞和整体等不同层面上，研究sGC/cGMP/GSK-3β信号途径对血小板活化的调节作用，尤其是重要信号分子（Akt,PKG）活性变化对GSK-3β的动态调控，旨在探明脓毒症血小板sGC/cGMP/GSK-3β信号途径顺序活化与调节激酶之间的相关关系，发现或探明GSK-3β及重要激酶是否是CO的作用对象及机制，明确CO调节脓毒症时血小板异常活化及信号传导的（部分）分子学基础，为脓毒症的治疗提供新思路。

血小板活化是脓毒症时凝血系统激活过程中的重要事件，但其调控机制还未被清楚阐明。我们的前期研究首次证实脓毒症早期血小板关键激酶GSK-3β活性明显提高，与血小板活化关系密切，外源性CO干预GSK-3β活性被下调。本研究在脓毒症动物模型和人血小板细胞模型上，外源性一氧化碳释放分子(CORM-2)进行干预,揭示了CORM-2基于对GSK-3β信号通路活化的抑制而抑制了脓毒症时血小板以及凝血系统过度活化。结果表明:（1）LPS刺激后，血小板的失去正常形态、伸出大量丝状伪足、增大其表面的粘附力；CORM-2的干预明显改善了血小板的形态学特征。（2）LPS刺激后，血小板的粘附率增大、侵袭到纤维蛋白原上的血小板数目增多、聚集率增高、ATP和P选择素的释放增强；CORM-2的干预明显降低了血小板的功能学指标。(3) LPS刺激后，血小板表面的integrinβ3、GPⅠbα和GPⅥ阳性率异常增加；CORM-2的干预显著地降低了血小板膜糖蛋白的阳性率。同时发现血小板中的cGMP含量在LPS刺激后明显下降，给予CORM-2的干预后，cGMP含量显著升高。(4) 脓毒症时血小板异常活化，其活化规律与PI3K/Akt/GSK-3β信号通路密切相关，外源性CO的干预可以通过升高血小板中的cGMP量来调控该信号通路，有效抑制血小板异常活化，改善血小板功能，从而提高脓毒症救治的成功率。本课题初步阐明了CORM-2对脓毒症时血小板和凝血系统过度活化的抑制作用，为临床脓毒症治疗提供了新思路。