Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo

Abstract

SummaryThe endothelial protein C receptor (EPCR) plays an anticoagulant role by improving protein C activation. Although low levels of activated protein C (APC) constitute a thrombosis risk factor, the relationship between modulating EPCR function and thrombosis has not been addressed so far. Monoclonal antibodies (mAb) against murine EPCR were raised, and their ability to block protein C/APC binding was tested. The ferric chloride carotid artery injury model in mice was chosen to test the effect of anti-EPCR mAb on thrombus formation. The time to total occlusion of the vessel was analysed in three groups, given an isotype control mAb (IC), a blocking (RCR-16) or a non-blocking (RCR-20) anti-EPCR mAb. RCR-16 prevented the interaction between protein C/APC and EPCR as demonstrated by surface plasmon resonance and flow cytometry, and inhibited the activation of protein C on the endothelium. IC and RCR-20 were unable to induce such effects. In vivo, RCR-16 shortened the time to total vessel occlusion with respect to IC [13.4 ± 1.0 (mean ± SD) and 17.8 ± 3.2 minutes, respectively, p<0.001]. Occlusive thrombi lasting for more than one hour were observed in all RCR-16-treated animals, but only in 43% of IC-treated ones. Results with RCR-20 were indistinguishable from those observed with IC. For the first time, a direct relationship between blocking EPCR and thrombosis is demonstrated. Blocking anti-EPCR autoantibodies can predispose to thrombosis episodes and may constitute a new therapeutic target.

The Endothelial Protein C Receptor Supports Tissue Factor Ternary Coagulation Initiation Complex Signaling through Protease-activated Receptors

“…We confirm these results and previous conclusions that complex formation of FVIIa with TF does not diminish the interaction of the FVIIa Gla domain with EPCR (16). Although a previous study raised doubts about the cross-species relevance of FVIIa binding to EPCR (18), our experiments demonstrate that EPCR is a receptor for FVIIa and the TF-FVIIa complex in the mouse.…”

“…The major difference between FX and the sTF-FVIIa complex was a slower association rate for FX, whereas the dissociation rates were similar for the human proteins. Although previous studies did not detect binding of murine FVIIa to EPCR (18), murine sTF-FVIIa (Fig. 2, E and F) bound to sEPCR albeit with somewhat lower affinity (K D ϳ 160 nM) than human counterparts.…”

“…Human FVIIa interacts with murine EPCR (17), and the clearance of human FVIIa in mice is regulated by EPCR (14). However, murine FVIIa apparently lacks high affinity for murine EPCR (18), raising questions about the physiological significance of interactions of endogenous FVIIa with EPCR. Although FX inefficiently competes with FVIIa binding to cells (12), FXa activation of PAR1 has recently been shown to depend on EPCR expressed by endothelial or Chinese hamster ovary (CHO) cells (19), adding yet another potential mechanism by which EPCR may regulate procoagulant proteases.…”

sPLA2-V inhibits EPCR anticoagulant and antiapoptotic properties by accommodating lysophosphatidylcholine or PAF in the hydrophobic groove

“…Sections from carotid arteries from mice subjected to a laser injury-induced thrombosis 16 and from human left atrial appendages were analyzed for the presence of sPLA 2 -V using an anti-sPLA 2 -V Ab followed by the antirabbit Dako Envisionϩ System-HRP, and for the presence of fibrin deposits using Masson trichrome staining.…”

A Review of Radiation-Induced Coagulopathy and New Findings to Support Potential Prevention Strategies and Treatments

“…One example is the endothelial cell product known as endothelial protein C receptor; this substance activates protein C, which is produced in the liver. Blocking the protein C receptor promotes thrombosis (77). It has been shown that the activity of the protein-C system is particularly important in radiation-induced adverse effects, including death (78).…”