The coagulant and inflammatory exacerbation in sepsis is counterbalanced by the protective protein C (PC) pathway. Activated PC (APC) was shown to use the endothelial cell PC receptor (EPCR) as a coreceptor for cleavage of protease activated receptor 1 (PAR1) on endothelial cells. Gene profiling demonstrated that PAR1 signaling could account for all APC-induced protective genes, including the immunomodulatory monocyte chemoattractant protein-1 (MCP-1), which was selectively induced by activation of PAR1, but not PAR2. Thus, the prototypical thrombin receptor is the target for EPCR-dependent APC signaling, suggesting a role for this receptor cascade in protection from sepsis.

Endothelial cells normally form a dynamically regulated barrier at the blood-tissue interface, and breakdown of this barrier is a key pathogenic factor in inflammatory disorders such as sepsis. Pro-inflammatory signaling by the blood coagulation protease thrombin through protease activated receptor-1 (PAR1) can disrupt endothelial barrier integrity, whereas the bioactive lipid sphingosine 1-phosphate (

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)-factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF-VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF-VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF-VIIa-X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TFVIIa-Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF-VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.

Summary.  Coagulation serine proteases signal through protease‐activated receptors (PARs). Thrombin‐dependent PAR signaling on platelets is essential for the hemostatic response and vascular thrombosis, but regulation of inflammation by PAR signaling is now recognized as an important aspect of the pro‐ and anti‐coagulant pathways. In tissue factor (TF)‐dependent initiation of coagulation, factor (F) Xa is the PAR‐1 or PAR‐2‐activating protease when associated with the transient TF–FVIIa–FXa complex. In the anticoagulant protein C (PC) pathway, the thrombin–thrombomodulin complex activates PC bound to the endothelial cell PC receptor (EPCR), which functions as a required coreceptor for activated PC‐mediated signaling through endothelial cell PAR‐1. Thus, the pro‐ and anti‐inflammatory receptor cascades are mechanistically coupled to immediate cell signaling, which precedes systemic coagulant or anticoagulant effects. In contrast to the substrate‐like recognition of PARs by thrombin, TF‐ or EPCR‐targeted activation of PARs generates cell‐type specificity, PAR selectivity and protease receptor cosignaling with the G‐protein‐coupled PAR response. Protease receptors are thus major determinants of the biological outcome of coagulation factor signaling on vascular cells.

Activated protein C (APC) has anti-inflammatory and vascular protective effects independent of anticoagulation. We previously identified the prototypical thrombin receptor, protease-activated receptor-1 (PAR1), as part of a novel APC-endothelial cell protein C receptor (EPCR) signaling pathway in endothelial cells. Experiments in wild-type and PAR1؊/؊ mice demonstrated that intravenous injection of APC leads to PAR1-dependent gene induction in the lung. The vascular endothelium undergoes profound changes in severe sepsis, the approved therapeutic indication for APC. Similar to PAR1, APC activated PAR2 through canonical cleavage. Although PAR2 was up-regulated in cytokine-stimulated endothelial cells, APC signaling remained PAR1-dependent. Large scale gene expression profiling documented marked differences in both up-and down-regulated genes between APC and thrombin signaling in cytokine-stimulated cells. APC down-regulated transcripts for proapoptotic proteins including p53 and thrombospondin-1, but p53 was unchanged, and thrombospondin was even upregulated by thrombin. Concordant PAR1-dependent effects on protein levels were found. Thus, by signaling through the same receptor PAR1, APC, and thrombin can exert distinct biological effects in perturbed endothelium. These data may explain how APC can be therapeutically protective through the EPCR-PAR1 signaling despite ongoing thrombin generation due to disseminated intravascular coagulopathy.