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Prepublished online as a Blood First Edition Paper on February 13, 2003; DOI 10.1182/blood-2002-12-3680. This Article Full Text Full Text (PDF) All Versions of this Article: 2002-12-3680v1 101/12/4797    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Dömötör, E. Articles by Zlokovic, B. V. Search for Related Content PubMed PubMed Citation Articles by Dömötör, E. Articles by Zlokovic, B. V. Related Collections Hemostasis, Thrombosis, and Vascular Biology Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 June 2003, Vol. 101, No. 12, pp. 4797-4801 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY 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 Eszter Dömötör, Omar Benzakour, John H. Griffin, David Yule, Kenji Fukudome, and Berislav V. Zlokovic From the Frank P. Smith Neurosurgical Research Laboratory, Department of Neurosurgery and Division of Neurovascular Biology, Center of Aging and Development Biology, University of Rochester Medical Center, Rochester, NY; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA; Department of Physiology and Pharmacology, University of Rochester Medical Center, Rochester, NY; Department of Immunology, Saga Medical School, Saga, Japan; and Socratech Laboratories, Rochester, NY. 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 ([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. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: Y. Wang, M. Thiyagarajan, N. Chow, I. Singh, H. Guo, T. P. Davis, and B. V. Zlokovic Differential Neuroprotection and Risk for Bleeding From Activated Protein C With Varying Degrees of Anticoagulant Activity Stroke, May 1, 2009; 40(5): 1864 - 1869. [Abstract] [Full Text] [PDF] R. J. S. Preston, S. Tran, J. A. Johnson, F. N. 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