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Am J Physiol Cell Physiol 273: C1756-C1763, 1997;
0363-6143/97 $5.00
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Vol. 273, Issue 5, C1756-C1763, November 1997

Site-specific thrombin receptor antibodies inhibit Ca2+ signaling and increased endothelial permeability

Lan T. Nguyen1, Hazel Lum2, Chinnaswamy Tiruppathì2, and Asrar B. Malik2

2 Department of Pharmacology, College of Medicine, The University of Illinois, and 1 Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612

Thrombin receptor is activated by thrombin-mediated cleavage of the receptor's NH2 terminus between Arg-41 and Ser-42, generating a new NH2 terminus that functions as a "tethered ligand" by binding to sites on the receptor. We prepared antibodies (Abs) directed against specific receptor domains to study the tethered ligand-receptor interactions required for signaling the increase in endothelial permeability to albumin. We used polyclonal Abs directed against the peptide sequences corresponding to the extracellular NH2 terminus [residues 70-99 (AbDD) and 1-160 (AbEE)] and extracellular loops 1 and 2 [residues 161-178 (AbL1) and 244-265 (AbL2)] of the seven-transmembrane thrombin receptor. Receptor activation was determined by measuring changes in cytosolic Ca2+ concentration ([Ca2+]i) in human dermal microvascular endothelial cells (HMEC) loaded with Ca2+-sensitive fura 2-acetoxymethyl ester dye. The transendothelial 125I-labeled albumin clearance rate (a measure of endothelial permeability) was determined across the confluent HMEC monolayers. AbEE (300 µg/ml), directed against the entire extracellular NH2-terminal extension, inhibited the thrombin-induced increases in [Ca2+]i and the endothelial 125I-albumin clearance rate (>90% reduction in both responses). AbDD (300 µg/ml), directed against a sequence within the NH2-terminal extension, inhibited 70% of the thrombin-induced increase in [Ca2+]i and 60% of the increased 125I-albumin clearance rate. AbL2 (300 µg/ml) inhibited these responses by 70 and 80%, respectively. However, AbL1 (300 µg/ml) had no effect on either response. We conclude that NH2-terminal extension and loop 2 are critical sites for thrombin receptor activation in endothelial cells and thus lead to increased [Ca2+]i and transendothelial permeability to albumin.

intracellular calcium concentration; tethered ligand


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