Pérez-Casal Margarita, Downey Colin, Cutillas-Moreno Beatriz, Zuzel Mirko, Fukudome Kenji, Toh Cheng Hock
Department of Haematology, Royal Liverpool University Hospital, Prescot Street, Liverpool L7 8XP, United Kingdom.
Haematologica. 2009 Mar;94(3):387-94. doi: 10.3324/haematol.13547. Epub 2009 Feb 11.
The endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. Recently, we described that endothelial protein C receptor can be released in vitro in microparticulate form from primary endothelial cells by exogenous activated protein C. Activated protein C bound to this endothelial protein C receptor retains anticoagulant activity and we hypothesize that this microparticulate endothelial protein C receptor-activated protein C complex can also cleave endothelial protease-activated receptor 1 to modulate inflammation and increase cell survival. Our main objective was, therefore, to study the effect that microparticle-associated endothelial protein C receptor-activated protein C has on endothelial function.
Mini-arrays were used and probed with cDNA obtained from endothelial cells after treatment with microparticle-associated endothelial protein C receptor-activated protein C and results were confirmed by real time polymerase chain reaction. The functional relevance of changes at gene level were further analyzed by endothelial apoptosis and permeability assays, in the presence and absence of specific blockade of endothelial protein C receptor, protein C and protease-activated receptor 1.
Gene profiling of endothelial cells stimulated by 40 nmol/L activated protein C on microparticles showed significant changes in anti-apoptotic and inflammatory pathways. This was accompanied by protease-activated receptor 1-dependent anti-apoptotic and barrier protective effects, the latter of which also involved sphingosine 1-phosphate receptor and vascular endothelial growth factor receptor-2/ kinase insert domain receptor. Protein C blockade reversed these effects showing specificity for activated protein C on microparticles. Furthermore, confocal microscopy and enzyme-linked immunosorbent assay of plasma obtained from septic patients during recombinant activated protein C treatment showed evidence of their presence in vivo.
Activated protein C on microparticle-associated endothelial protein C receptor release can induce protease-activated receptor 1-dependent endothelial effects. The mechanisms underlying barrier protection involve sphingosine 1-phosphate receptor and kinase insert domain receptor.
内皮细胞蛋白C受体在蛋白C途径中对调节凝血和炎症起着重要作用。最近，我们发现外源性活化蛋白C可使原代内皮细胞以微粒形式在体外释放内皮细胞蛋白C受体。与该内皮细胞蛋白C受体结合的活化蛋白C保留抗凝活性，我们推测这种微粒状内皮细胞蛋白C受体 - 活化蛋白C复合物也能裂解内皮蛋白酶活化受体1，从而调节炎症并提高细胞存活率。因此，我们的主要目的是研究微粒相关的内皮细胞蛋白C受体 - 活化蛋白C对内皮功能的影响。
使用微阵列并用微粒相关的内皮细胞蛋白C受体 - 活化蛋白C处理后从内皮细胞获得的cDNA进行检测，结果通过实时聚合酶链反应得到证实。在存在和不存在内皮细胞蛋白C受体、蛋白C和蛋白酶活化受体1特异性阻断的情况下，通过内皮细胞凋亡和通透性测定进一步分析基因水平变化的功能相关性。
用40 nmol/L微粒上的活化蛋白C刺激内皮细胞的基因谱分析显示抗凋亡和炎症途径有显著变化。这伴随着蛋白酶活化受体1依赖性的抗凋亡和屏障保护作用，后者还涉及鞘氨醇1 - 磷酸受体和血管内皮生长因子受体 - 2/激酶插入结构域受体。蛋白C阻断逆转了这些作用，显示出对微粒上活化蛋白C的特异性。此外，共聚焦显微镜检查和对重组活化蛋白C治疗期间脓毒症患者血浆的酶联免疫吸附测定显示了它们在体内存在的证据。
微粒相关的内皮细胞蛋白C受体释放的活化蛋白C可诱导蛋白酶活化受体1依赖性的内皮效应。屏障保护的潜在机制涉及鞘氨醇1 - 磷酸受体和激酶插入结构域受体。
