Platelet-Leukocyte Physiology in Cardiopulmonary Bypass

描述(申请人提供)：与体外循环(CPB)相关的心脏手术的不良后果包括神经认知障碍、心肌缺血、围手术期出血和术后血栓形成。我们的总体目标是阐明CPB的潜在病理生理学，涉及细胞和可溶性止血和炎症系统的激活和相互作用，目的是设计治疗干预措施。我们的研究基于体外模型和临床资料。在过去这笔赠款的支持下，我们帮助定义了血小板-白细胞相互作用的正常生理学；显示CPB导致体外和体内白细胞上特定的p2整合素、血小板上的P-选择素上调，以及循环中白细胞-血小板结合物的形成；明确了特定补体和凝血激活产物在诱导血小板、中性粒细胞和单核细胞激活中的不同作用；表明在临床CPB中进行的C5补体阻断与体外试验中看到的相似的效果有关，并且在先导性研究中，CPB不良结果的减少；并发现在临床CPB中，血小板GP 111a的PL-A1/A2基因多态性与不同的神经学和心肌结局相关。我们的新的具体目标是：(1)利用体外模型，确定C5a和C3a受体在模拟体外循环(SECC)诱导的中性粒细胞、单核细胞和血小板激活中的作用；由于初步数据显示C5aR阻断取消血小板激活的意外发现，确定其相关机制；(1)通过(I)体内补体激活与患者体内甘露糖结合凝集素(MBL)水平和获得性CRP水平的遗传变异的相关性，(Ii)检测MBL在SECC中的作用，确定凝集素补体途径在CPB中的作用；(2a)确定CD64/CD16/CD14表达所定义的人类单核细胞亚群与组织因子调节的差异；(2b)检测体内和体外CPB期间单核细胞亚群的分布；利用表达谱阵列技术，确定SECC诱导的单核细胞炎症基因的表达模式；(3)通过体外模拟检测SECC对人内皮细胞的影响，特别是组织因子调节的变化和不同补体成分的影响

DESCRIPTION (provided by applicant): Adverse outcomes in cardiac surgery associated with cardiopulmonary bypass (CPB) include neurocognitive deficits, myocardial ischemia, peri-operative bleeding, and post-operative thrombosis. Our overall goal is to elucidate the underlying pathophysiology of CPB which involves activation of, and cross-talk between, cellular and soluble hemostatic and inflammatory systems, with the aim of devising therapeutic interventions. Our studies are based on both in vitro models and clinical material. Under the past aegis of this grant, we have helped define normal physiology of platelet-leukocyte interactions; shown that CPB results in upregulation of specific p 2 integrins on leukocytes, P-selectin on platelets, and formation of circulating leukocyte-platelet conjugates in vitro and in vivo; defined differential roles for specific complement and coagulation activation products in the induction of platelet versus neutrophil versus monocyte activation; shown that C5 complement blockade carried out in clinical CPB is associated with similar effects to those seen in vitro, and, in a pilot study, a reduction in CPB adverse outcomes; and discovered that the PL-A1/A2 genetic polymorphism of platelet gp llla is associated with different neurologic and myocardial outcomes in clinical CPB. Our new specific aims are: (la) using in vitro models, determine the role of the C5a and C3a receptors in neutrophil, monocyte and platelet activation induced by simulated extracorporeal circulation (SECC); since preliminary data suggests the unexpected finding that C5aR blockade abrogates platelet activation, determine the responsible mechanism; (1 b) determine the role of the lectin complement pathway in CPB by (i) correlating in vivo complement activation with interpatient genetic variability in mannose binding lectin (MBL) levels and acquired CRP levels, (ii) examining the role of MBL in SECC; (2a) determine differences in human monocyte subsets, defined by CD64/CD16/CD14 expression, with respect to p.2 in and tissue factor regulation; (2b) examine monocyte subset distribution during in vivo and in vitro CPB; using expression array technology, define the pattern of monocyte inflammatory gene expression induced by SECC; (3) examine the effects of SECC on human endothelial cells by in vitro modeling, specifically including alterations in tissue factor regulation and the effect of differential complement components