Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as
sepsis and
acute lung injury (ALI). Activated
protein C (APC), a
serine protease critically involved in the regulation of coagulation and inflammatory processes, improves
sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by
thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical
myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac
GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to
thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist,
sphingosine 1-phosphate (S1P), we explored potential cross-talk between
endothelial protein C receptor (
EPCR) and S1P1, the receptors for APC and S1P, respectively.
EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid,
EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on
threonine residues consistent with
S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between
EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using
siRNA significantly reduced APC-mediated barrier protection against
thrombin. These data suggest that novel
EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with
severe sepsis.