Abstract
BackgroundThe endothelial cell protein C receptor (EPCR) presents protein C to the thrombin:thrombomodulin complex on the endothelium of large vessels, and enhances the generation of activated protein C (APC) and activation of protease-activated receptor-1. A previous report has demonstrated binding of soluble (s) EPCR to activated neutrophils via surface proteinase 3 (PR3).MethodsWe now report further characterization of this interaction. Activated neutrophils and purified PR3 both decrease endothelial cell (EC) surface EPCR, suggestive of its proteolysis.ResultsWhen added to purified recombinant sEPCR, PR3 produced multiple cleavages, with early products including 20 kDa N-terminal and C-terminal (after Lys176) fragments. The binding of active site blocked PR3 to sEPCR was studied by surface plasmon resonance. Estimates of the KD of 18.5–102 nm were obtained with heterogeneous binding, suggestive of more than a single interaction site.ConclusionsThis work demonstrates PR3 binding to and proteolysis of EPCR and suggests a mechanism by which anticoagulant and cell protective pathways can be down-regulated during inflammation.
Highlights
The protein C anticoagulant pathway is one of the major coagulation regulatory mechanisms and modulates thrombin generation by degrading factors (F) Va and VIIIa [1]
By binding endothelial cell protein C receptor (EPCR) with high affinity [8,9], protein C is concentrated on the endothelial cell (EC) surface and the activation peptide orientated towards the active site of the thrombomodulinthrombin complex, which increases severalfold the generation of activated protein C (APC) [3]
In addition to its anticoagulant role, EPCR has been shown to facilitate APC-mediated activation of protease-activated receptor-1 [10], which stimulates neuroprotective/anti-apoptotic signalling pathways [11]. This activity of EPCR may be critical in the beneficial clinical effects of APC in the treatment of severe sepsis, as a sublethal dose of Escherichia coli administered to baboons became lethal when animals were simultaneously administered an anti-EPCR monoclonal antibody that blocks the binding of protein C to EPCR [12]
Summary
The protein C anticoagulant pathway is one of the major coagulation regulatory mechanisms and modulates thrombin generation by degrading factors (F) Va and VIIIa [1]. In addition to its anticoagulant role, EPCR has been shown to facilitate APC-mediated activation of protease-activated receptor-1 [10], which stimulates neuroprotective/anti-apoptotic signalling pathways [11]. This activity of EPCR may be critical in the beneficial clinical effects of APC in the treatment of severe sepsis, as a sublethal dose of Escherichia coli administered to baboons became lethal when animals were simultaneously administered an anti-EPCR monoclonal antibody (mAb) that blocks the binding of protein C to EPCR [12]
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