体外循环中凝血酶控制血小板双向调节活化白细胞的机制研究

Systemic inflammatory response (SIR), characterized by leukocyte activation, mainly contributes to the high morbidity and mortality after cardiopulmonary bypass (CPB). During SIR, platelet may bind to, and then regulate the activated leukocyte, which may impact prognosis of CPB patients. Because both anti and pro inflammatory molecules are stored simultaneously in platelet alpha granules, platelets may bi-directly regulate leukocyte activation. Our pilot study suggests that thrombin formation controls this bidirectional regulation of platelet during CPB. In the present proposal, therefore, thrombin levels and leukocyte activation would be evaluated, and prognosis would be investigated to determine the relationships among them. To determine whether thrombin controls platelet to bi-directionally regulate leukocyte activation, thrombin would be blocked by bivalirudin in vitro and in vivo, and both pro and anti inflammatory molecular in alpha granules would be investigated. Furthermore, determine the release of pro or anti inflammatory cytokines after stimulation by tumor necrosis factor alpha and extracorporeal circulation with and without thrombin block, platelet would be investigated after immunofluorescence staining. Finally, the adhesion between platelet and leukocyte would be observed, and their adhesive molecular would be blocked to elucidate the mechanism of platelet bidirectional regulation on leukocyte. Obviously, our research would be helpful to understand the balance between anti and pro-inflammatory response, which may provide an evidence to develop specific drugs or therapies to reduce CPB associated SIR, and thus improve prognosis.

体外循环（CPB）中，以白细胞活化为特征的全身炎性反应（SIR）是导致术后患者高死亡率和并发症发生率的重要原因。血小板通过与白细胞粘附而密切调节其功能，并影响着患者预后。然而血小板同时具有抑炎/促炎细胞因子，提示可能对白细胞发挥抑制/激活的双向作用。我们的前期研究发现，凝血酶可能控制着血小板的双向作用。为此，本研究拟通过观察围术期凝血酶浓度、白细胞激活和患者预后，研究凝血酶是否可能通过血小板调节白细胞激活，从而影响患者预后；通过阻断凝血酶，研究其是否控制着血小板的双向作用；通过观察凝血酶依赖的血小板抑炎、促炎因子释放、粘附分子表达，研究血小板双向作用的物质基础；最后通过阻断凝血酶诱导的血小板-白细胞粘附位点，研究血小板双向作用的分子机制。本课题为深刻理解机体的抑炎/促炎平衡机制，从而为合理控制CPB相关SIR、改善患者预后开发特异性药物或治疗手段提供理论和实验依据。

正如我们所预期，血小板存在双向调节炎性反应的作用，而凝血酶是这种抗炎-促炎转化的关键。利用旁路循环小鼠肺损伤模型，我们发现未激活的静息血小板具有较好的抗炎作用。膜糖蛋白GPIIbIIIa是其发挥作用的主要物质。利用此蛋白，血小板进入肺组织，通过释放抗炎细胞因子TGF-β而抑制肺炎性反应。同时，我们还发现，血小板能够黏附于激活中性粒细胞的头端而形成复合体，并将胞浆内物质释放进入中性粒细胞，随后中性粒细胞去极化，从而阻断白细胞的爬行、穿膜作用。但这一作用机制仍待进一步研究。.利用小鼠急性肺损伤模型，我们发现，凝血酶激活的血小板则发挥较强的促炎作用。其具体机制是：凝血酶通过PAR4激活血小板，从而上调细胞膜表面CD31表达。借助于CD31-CD31之间的相互作用，血小板黏附于内皮细胞的管腔面，随后内皮细胞位于细胞连接处的CD31向管腔面转移，从而导致细胞连接发生断裂，内皮漏形成，肺水肿产生。.总之，血小板，如同方向盘，在炎性反应中具有极其重要的双向调控作用。本研究中，我们详细研究了凝血酶激活的血小板上调血管内皮屏障通透性，从而引起肺水肿形成的机制。这为炎症相关血管内皮通透性增高的治疗提供了新的思路。同时我们发现，未激活血小板不仅通过释放TGF-β抑制炎性反应，更有趣的是，它能够直接导致激活中性粒细胞的去极化。鉴于目前临床无任何药物能够有效控制炎性反应，研究中性粒细胞去极化的机制具有极为重要的科学和临床意义。