Factor VIIa Bound to Endothelial Cell Protein C Receptor Activates Protease Activated Receptor 1-Mediated Cell Signaling and Barrier-Protective Response In Endothelial Cells
Authors: Sen Prosenjit , Gopalakrishnan Ramakrishnan , Kothari Hema , Clark Curtis , Pendurthi Usha , Rao L. Vijaya Mohan 
Published: October 2019

Abstract 346

Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C and activated protein C (APC). In addition to controlling coagulation by modulating the protein C-mediated anticoagulant pathway, EPCR has been shown to play a critical role in supporting APC-induced cell signaling, which could be responsible for some of the non-hemostatic functions of EPCR and APC. Recent studies from our laboratory and others have shown that factor VIIa (FVIIa), a coagulation factor whose primary function is to initiate tissue factor (TF)-dependent coagulation, also binds to EPCR on endothelium. At present, the physiological significance of this interaction is unclear. APC binding to EPCR has been shown to provide cytoprotective effects via protease activated receptor (PAR) 1-mediated cell signaling. In earlier studies using exogenously expressed PAR1 and PAR2 reporter constructs in a heterologus cell model system, we were unable to find measurable n-terminal cleavage (activation) of PARs by FVIIa bound to EPCR. It is possible that transfected PAR constructs may segregate differently on the cell surface membrane than that of endogenous PARs, and thus may have decreased susceptibility for cleavage by FVIIa-EPCR. In the present study, we have investigated whether FVIIa, upon binding to EPCR on endothelial cells, activates endogenous PAR1 and induces PAR1-mediated cell signaling. To determine whether FVIIa cleaves endogenously expressed PAR1 on endothelial cells, unperturbed cultures of human umbilical vein endothelial cells (HUVEC) were exposed to varying concentrations of FVIIa (0-40 nM) and the cleavage of PAR1 at the cell surface was measured quantitatively in a cell-surface ELISA using a cleavage-specific PAR1 monoclonal antibody. The data show that FVIIa, in a dose- and time-dependent manner, cleaves PAR1 on endothelial cells. FVIIa cleavage of PAR1 on endothelial cells is dependent on FVIIa binding to EPCR, as prevention of FVIIa binding to EPCR by pretreating HUVEC with EPCR polyclonal antibody completely abolished FVIIa cleavage of PAR1. Similarly, silencing EPCR with EPCR-specific siRNA fully attenuated FVIIa cleavage of PAR1. FVIIa cleavage of PAR1 on endothelial cells is independent of TF as pretreatment of HUVEC with anti-TF antibodies or transduction of HUVEC with adenovirus encoding TF had no significant effect on FVIIa cleavage of PAR1. The efficiency of PAR1 cleavage by FVIIa appears to be comparable to that of APC, as both at 10 nM cleave PAR1 to a similar extent. FVIIa (10 nM) cleaves only a fraction of PAR1 (∼25 to 30%) on endothelial cell surface; increasing either FVIIa concentration or duration of treatment has not resulted in additional cleavage of remaining PAR1. Low expression of PAR2 in endothelial cells and lack of cleavage specific antibodies to PAR2 prevented us from determining whether FVII bound to EPCR also cleaves PAR2. FVIIa (10 nM) induced p44/42 MAPK activation in HUVEC and this activation was dependent on EPCR and PAR1 but not PAR2, as silencing EPCR or PAR1 but not PAR2 attenuated FVIIa-induced p44/42 MAPK phosphorylation. In additional studies, FVIIa (10 nM) was found to elicit protection against thrombin-induced barrier disruption in endothelial cells as analyzed in a dual-chamber system using Evans blue-labeled BSA or measurements of transendothelial electrical resistance. FVIIa-induced barrier-protective effect is EPCR-dependent. F-actin staining of HUVEC exposed to thrombin showed formation of transcellular actin stress fibers, cellular contractions and paracellular gap formation. Pretreatment of HUVEC with FVIIa maintained actin at the cell periphery, and reduced formation of central stress fibers and paracellular gaps. FVIIa-induced p44/42 MAPK activation and barrier protective effect are mediated via Rac1, as specific inhibitors against Rac1 or transduction of Rac1 dominant negative mutant abolished these FVIIa-induced effects. Consistent with in vitro findings, in vivo studies in mice showed that administration of FVIIa prior to LPS attenuated the LPS-induced vascular leakage in lung and kidney. Overall, our present data provide strong and convincing evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier protective effect. These findings are novel and assume a great clinical significance as FVIIa is used prophylactically for prevention of bleeding in hemophiliacs.

Disclosures:

No relevant conflicts of interest to declare.