Feistritzer Clemens, Schuepbach Reto A, Mosnier Laurent O, Bush Leslie A, Di Cera Enrico, Griffin John H, Riewald Matthias
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Biol Chem. 2006 Jul 21;281(29):20077-84. doi: 10.1074/jbc.M600506200. Epub 2006 May 18.
Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.
活化蛋白C（APC）具有内皮屏障保护作用，这种作用需要与内皮蛋白C受体（EPCR）结合并裂解蛋白酶活化受体-1（PAR1），且可能在APC的抗炎作用中发挥作用。在本研究中，我们调查了内皮细胞表面凝血酶对蛋白C（PC）的激活是否可能与有效的保护信号传导相关。为了尽量减少凝血酶对内皮通透性的直接影响，我们使用了抗凝双突变凝血酶W215A/E217A（WE）。WE在内皮细胞表面激活PC产生具有高屏障保护活性的APC。外源性APC产生可比的屏障保护作用所需的APC浓度要高4倍。为了确凿证明在存在WE的情况下保护作用是由APC生成介导的，而非WE的直接信号传导，我们使用了一种将活性位点丝氨酸替换为丙氨酸的PC变体（PC S360A）。低浓度外源性APC的屏障保护作用被野生型PC和PC S360A均阻断，这与其作为APC与EPCR结合的竞争性抑制剂的预期作用一致。WE仅在存在野生型PC时诱导保护信号，而在PC S360A存在时则不诱导，且这些保护作用需要PAR1裂解。这些数据表明，内皮细胞表面的内源性PC激活途径在机制上与生成的APC的PAR1依赖性自分泌屏障保护信号传导相关。WE可能通过内源性生成的APC的信号传导在全身炎症中发挥强大的保护作用。
