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  • Poster presentation
  • Open Access

Regulation of endothelial function by coagulation proteases in sepsis

  • 1,
  • 2,
  • 1,
  • 1,
  • 1 and
  • 2
Critical Care201115 (Suppl 1) :P251

  • Published:


  • Thrombin
  • Transendothelial Electrical Resistance
  • Intracellular Calcium Mobilization
  • Bioluminescent Resonance Energy Transfer
  • Tethered Ligand


Thrombin and activated protein C (aPC) are two pleiotropic proteases whose opposing functions in hemostasis and endothelial function are dysregulated during sepsis. Exogenous supplementation of aPC, the ligand for endothelial protein C receptor (EPCR), is the only known therapeutic shown to reduce mortality in severe septic patients. Paradoxically, both thrombin and aPC signal the endothelium via the same receptor, protease-activated receptor-1 (PAR-1), by cleaving its N-terminus to produce an identical tethered ligand, yet result in opposing signaling networks. Once activated, PAR-1 triggers at least three separate signaling pathways (Gi, Gq, G13) and it is the relative contribution of each pathway that determines the endothelial response. Thrombin is a potent proinflammatory, endothelial barrier disruptive agonist, while aPC induces an anti-inflammatory and barrier protective phenotype, thought to be important to its therapeutic mechanism. We hypothesized that when bound to its ligand, aPC, EPCR functionally dimerizes with activated PAR-1, thereby altering its specificity for Gq, an important mediator of proinflammatory pathways in endothelial cells.


We used bioluminescent resonance energy transfer to dynamically monitor the interaction of recombinant PAR-1 and EPCR in HEK cells. The effect of EPCR on PAR-1 G-protein selectivity was determined by EPCR siRNA knock down in cultured endothelial cells. Relative activation of Gq was determined by assaying agonist-induced intracellular calcium mobilization. G13 activation was determined by monitoring agonist-induced changes transendothelial electrical resistance across monolayers.


We found that in the absence of protease ligands, unactivated PAR-1 dimerizes with EPCR. However, proteolytically activated PAR-1/EPCR interaction was maintained with aPC but not thrombin. Both aPC and thrombin induced G13 signaling; however, aPC failed to activate Gq compared with thrombin. aPC-induced PAR-1/Gq signaling appears to be impaired by aPC-bound EPCR and is relieved when EPCR is depleted using siRNA.


aPC-bound EPCR neutralizes the proinflammatory function of PAR-1 signaling by maintaining interaction with activated PAR-1, thereby abrogating Gq signaling. Thus it is not the difference in protease activation between thrombin and aPC, but rather the ability of aPC to direct PAR-1/EPCR dimerization that controls PAR-1 signaling, and thereby provides the therapeutic barrier protective/anti-inflammatory effects associated with aPC treatment.

Authors’ Affiliations

University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
University of Illinois at Chicago, IL, USA


© McLaughlin et al. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.