Is APC activation of endothelial cell PAR1 important in severe sepsis?: Yes

A primary response to intravascular thrombin generation is thrombomodulin-dependent activation of protein C (PC). Generation of activated PC (APC) in vivo is facilitated by the endothelial cell protein C receptor (EPCR) and APC dampens coagulation by inactivating cofactors Va and VIIIa [1]. During pregnancy implantation, the thrombomodulin/EPCR receptor system similarly counterbalances coagulation activation by the tissue factor (TF) pathway [2]. EPCR binds PC as well as APC and serves as the co-receptor for APC to elicit direct, proteasedependent cell signaling responses that are endothelial protective and anti-inflammatory. Based on analogy to the well-characterized anticoagulant activities of the PC pathway, it is reasonable to assume that an important function of APC signaling is to re-balance adverse cellular effects of coagulation activation. Activated protein C administration attenuates the severity of ischemic stroke [3,4] and improves survival in severe sepsis with coagulopathy andmultiple organ failure [5]. The importance of coagulation driven inflammatory and thrombotic pathways to sepsis lethality is well documented in primate models, but efficient anticoagulation per se does not guarantee reduction of lethality [6]. Clinical trials with antithrombin or TFPI in severe sepsis have not achieved survival benefit comparable with APC therapy, suggesting that APC’s efficacy in sepsis involves actions independent of its anticoagulant activity. Activated protein C exerts direct cellular effects on cell types that are highly relevant for the pathology of severe systemic inflammation, including monocytes, neutrophils and endothelial cells. APC is anti-inflammatory by suppressing NF-jB induction in endothelial cells and reduces TF in monocytes [7–10]. Together, these effects achieve dampening of the systemic inflammatory and coagulant response. APC inhibits neutrophil activation and migration to a variety of stimuli [11–14]. This is relevant for reducing the severity of acute lung injury and of organ dysfunction. Improvements in cardiovascular function upon APC therapy can result from APC’s endothelial and vascular protective properties. APC increases endothelial cell barrier function through sphingosine-1 phosphate signaling pathways [15,16] and protects endothelial cells from apoptosis [3,7,17,18]. The essential role for EPCR in APC’s direct cellular effects is documented extensively [3,9,12,15–21] and EPCR is not only expressed by endothelial cells [22], but also by monocytes [9,23] and neutrophils [12,13]. Active site modification of APC has been employed to demonstrate proteolytic cell signaling mechanisms of EPCRbound APC [3,14,20,21]. Thus, APC acts as an anticoagulant enzyme as well as a signaling protease in complex with EPCR. Our original study demonstrated that EPCR serves as a coreceptor for APC-mediated activation of protease activated receptor (PAR) 1 in endothelial cells [21] and this pathway is now generally accepted [3,17,18,20,24,25]. In vivo, PAR1 is required for APC’s neuroprotective activities [3,4] and APCmediated upregulation of target genes [18]. Thus, PAR1 is a relevant signaling receptor for APC in vivo, but alternative pathways may contribute to APC’s cell signaling effects. Neuroprotection by APC is also reduced in PAR3-deficient animals [4] and EPCR-bound APC efficiently activates PAR2 in heterologous over-expression systems [21] through canonical scissile bond cleavage [18]. In addition, antibody blockade of PAR1 did not reduce endothelial cell proliferation [20] or neutrophil chemotaxis [12]. These studies did not evaluate whether the antibodies had sufficient potency to block an established APC signaling response over prolonged time, nor was alternative PAR cleavage addressed experimentally. However, effects of APC on leukocytes may not be restricted to direct cell signaling pathways and involve, e.g. interference with complex formation of EPCR/proteinase 3/b2 integrins [26]. PAR1 was also variably implicated in endothelial cell barrier protection by APC, based on experiments using different methodology and endothelial cell sources [15,16]. However, the conclusion that APC induces cross-activation of sphingosine 1-phosphate receptors solely by binding to EPCR appears premature without additional control experiments using active site blocked APC [16]. Thus, PAR1 is a predominant receptor for endothelial cell APC-EPCR signaling. PAR1 is emerging as the target for various proteases. However, in the context of sepsis, special attention should be given to thrombin and possibly TF-VIIa-Xa signaling [27]. Thrombin activates PAR1 to elicit a multitude of Correspondence: Wolfram Ruf, Department of Immunology, SP258, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel. : +1 858 784 2748; fax: +1 858 784 8480; e-mail: ruf@scripps.edu Journal of Thrombosis and Haemostasis, 3: 1912–1914