title: 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.
abstract: 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 PAR-1, PAR-2, PAR-3, or EPCR. APC, but not protein C zymogen or the active site mutant Ser360Ala-APC, induced dose-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-induced [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.
authors.affiliation: Frank P. Smith Neurological Research Laboratory, Department of Neurosurgery and Division of Neurovascular Biology, University of Rochester Medical Center, NY 14642, USA.
authors.forename: Eszter
authors.initials: E
authors.lastname: Dömötör
authors.forename: Omar
authors.initials: O
authors.lastname: Benzakour
authors.forename: John H
authors.initials: JH
authors.lastname: Griffin
authors.forename: David
authors.initials: D
authors.lastname: Yule
authors.forename: Kenji
authors.initials: K
authors.lastname: Fukudome
authors.forename: Berislav V
authors.initials: BV
authors.lastname: Zlokovic
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D015703:Antigens, CD; D001921:Brain / Q000098:blood supply*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D000074283:Endothelial Protein C Receptor; D016232:Endothelins / Q000276:immunology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000737:chemistry / Q000648:ultrastructure*; D006023:Glycoproteins; D006801:Humans; D011486:Protein C / Q000378:metabolism* / Q000494:pharmacology*; D044463:Receptor, PAR-1 / Q000276:immunology / Q000378:metabolism*; D011956:Receptors, Cell Surface; D015398:Signal Transduction / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology; D014471:Umbilical Veins
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; D016232:Endothelins; D006023:Glycoproteins; C478370:PROCR protein, human; D011486:Protein C; D044463:Receptor, PAR-1; D011956:Receptors, Cell Surface; D019284:Thapsigargin; D002118:Calcium