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

Abstract

Thrombin receptor is activated by thrombin-mediated cleavage of the receptor’s NH2 terminus between Arg-41 and Ser-42, generating a new NH2terminus 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 transendothelial125I-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+]iand 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+]iand 60% of the increased125I-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+]iand transendothelial permeability to albumin.