Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1

Activated protein C (APC) exerts endothelial protein C receptor (EPCR)-dependent neuroprotective effects in a brain focal ischemia model and direct cellular effects on human umbilical vein endothelial cells (HUVECs) via protease-activated receptor-1 (PAR-1). Because PAR receptors are expressed in brain endothelium and mediate intracellular calcium concentration ([Ca2+](i)) signaling, we hypothesized that APC may regulate intracellular [Ca2+] flux in human brain endothelial cells (BECs) via EPCR and PAR-1. Primary cortical BECs derived from human autopsies (early passage) and HUVECs were used for [Ca2+](i) imaging fluorometry. Cells were exposed for 1 minute to APC, protein C zymogen, or mutant Ser360Ala-APC, and [Ca2+](i) was monitored in the presence or absence of antibodies against PARA, PAR-2, PAR-3, or EPCR. APC, but not protein C zymogen or the active site mutant Ser360Ala-APC, induced close-dependent [Ca2+](I) release in human BECs (Delta[Ca2+](i) max = 278.3 +/- 19.5 nM; EC50 for APC = 0.23 +/- 0.02 nM, n = 70 measurements). APC-incluced [Ca2+](i) signaling was abolished by a cleavage site blocking anti-PAR-1 antibody, whereas anti-PAR-2 and -PAR-3 antibodies were without effect. Antibody RCR252 that ablates APC binding to EPCR blocked APC-mediated [Ca2+](i) signaling, whereas anti-EPCR antibody RCR92 that does not block APC binding did not abolish the APC-induced [Ca2+](i) response. Experiments using HUVECs confirmed the findings for BECs. Thapsigargin inhibited the APC-induced [Ca2+](i) signal, implicating the endoplasmic reticulum as a major source for the APC-induced [Ca2+](i) release. These data suggest that APC regulates [Ca2+](i) in human brain endothelium and in HUVECs by binding to EPCR and signaling via PAR-1. (Blood. 2003; 101:4797-4801).