title: 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 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 Ca(2+)-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 micrograms/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 micrograms/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 micrograms/ml) inhibited these responses by 70 and 80%, respectively. However, AbL1 (300 micrograms/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.
authors.affiliation: Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.
authors.forename: L T
authors.initials: LT
authors.lastname: Nguyen
authors.forename: H
authors.initials: H
authors.lastname: Lum
authors.forename: C
authors.initials: C
authors.lastname: Tiruppathì
authors.forename: A B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D000906:Antibodies / Q000494:pharmacology*; D000918:Antibody Specificity; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007457:Iodine Radioisotopes; D008657:Metabolic Clearance Rate; D008833:Microcirculation; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D018179:Receptors, Thrombin / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D012709:Serum Albumin / Q000493:pharmacokinetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D012867:Skin / Q000098:blood supply; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D000906:Antibodies; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D018179:Receptors, Thrombin; D012709:Serum Albumin; D013917:Thrombin; D002118:Calcium