PKC/PDE3A信号通路调控糖蛋白GPIbα介导的血小板凋亡的分子机制研究

Platelets membrane glycoprotein GPIbα (GPIbα)-dependent platelets apoptosis can be triggered by pathological and physiological stimuli leading to platelets dropping as well as the occurrence of hemorrhagic diseases. Our previous findings reported that protein kinase A (PKA) inhibition induced platelets apoptosis. Preliminary experiments showed that GPIbα-mediated apoptotic platelets had reduced PKA activity as well as activaiton of PKC/PDE3A signaling pathway. Moreover, inhibtion of PKC/PDE3A significantly elevated PKA activity, inhibited GPIbα-mediated platelets apoptosis. Thus, we hypothesized that PKC/PDE3A signaling was involved in GPIbα-mediated platelets apoptosis by mediating PKA activity. Based on the above data, the strategies of Western blot, washed platelets and gene konckout mice were used to explore the role and related molecular meachnisms of PKC/PDE3A in GPIbα-mediated platelets apoptosis. The expected findings will enrich the theoretic basis of prevention and treatment for platelet dropping-related diseases.

生理或病理性因素可导致血小板膜糖蛋白Glycoprotein Ibα（GPIbα）依赖的血小板凋亡，引起血小板数量减少，进而造成出血性疾病的发生。申请人已发现并报道，蛋白激酶A（Protein kinase A, PKA）活性降低可导致血小板凋亡。预实验结果显示，GPIbα介导的血小板凋亡伴随PKA活性降低及蛋白激酶C/磷酸二酯酶3A（PKC/PDE3A）信号通路的活化；而抑制PKC/PDE3A可显著提高PKA活性，阻止GPIbα介导的血小板凋亡。因此，我们认为：PKC/PDE3A信号通路通过调控PKA活性参与GPIbα介导的血小板凋亡过程。本研究拟在预实验基础上，利用Western blot、洗涤血小板、基因敲除鼠等技术手段从分子、细胞、动物等三个层面探明PKC/PDE3A信号通路在GPIbα介导的血小板凋亡中的作用及其分子机制，为血小板减少性相关疾病的预防及治疗等方面提供理论依据。

血小板凋亡作为体内血小板清除的一种方式，常见于肿瘤、败血症和糖尿病等机体多种病理性过程；然而调控血小板凋亡的机制仍不明确。本项目研究了血小板膜糖蛋白Glycoprotein Ibα（GPIbα）调控的血小板凋亡及分子机制，发现GPIbα/PKC/PDE3A/PKA信号轴参与调控血小板凋亡，在调控血小板寿命和肿瘤转移等过程中起关键作用。研究结果显示，PKCα与14-3-3ζ蛋白具有相同的GPIbα胞内段结合域；当GPIbα胞外段被胞外配体激活，诱导GPIbα胞内段与14-3-3ζ结合，导致PKCα被竞争性释放入胞并激活其活性；活化的PKCα进而激活下游PDE3A信号，引起细胞内cAMP水平降低，抑制PKA酶活性，最终诱导血小板凋亡。在GPIbα，Prkca和Bad敲除小鼠中进行验证实验发现，抑制GPIbα，PKCα或Bad可显著抑制血小板凋亡，显著延长血小板寿命，提示调控GPIbα/PKC/PDE3A/PKA信号通路在调控血小板凋亡中的潜在作用。因此，本项目研究成果对有效抑制血小板凋亡，显著降低血小板储存过程中造成的血小板质量及功能损伤，提高血小板储存时间具有重要的现实意义。对认识血小板减少相关疾病的病理、生理意义，寻找有效防治血小板数量减少相关性疾病的新靶点、新方法具有重要的理论及现实意义。