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

Am J Physiol. 1997 Nov;273(5):C1756-63. doi: 10.1152/ajpcell.1997.273.5.C1756.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antibodies / pharmacology*
  • Antibody Specificity
  • Binding Sites
  • Calcium / metabolism*
  • Cell Membrane Permeability / drug effects
  • Cell Membrane Permeability / physiology*
  • Cells, Cultured
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / physiology*
  • Humans
  • Iodine Radioisotopes
  • Metabolic Clearance Rate
  • Microcirculation
  • Models, Molecular
  • Peptide Fragments / chemistry
  • Peptide Fragments / immunology
  • Receptors, Thrombin / chemistry
  • Receptors, Thrombin / immunology
  • Receptors, Thrombin / physiology*
  • Serum Albumin / pharmacokinetics
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • Skin / blood supply
  • Thrombin / pharmacology*


  • Antibodies
  • Iodine Radioisotopes
  • Peptide Fragments
  • Receptors, Thrombin
  • Serum Albumin
  • Thrombin
  • Calcium