title: The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.
abstract: Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.
authors.affiliation: Department of Medicine, Cornell University Medical College, New York, New York 10021.
authors.forename: J R
authors.initials: JR
authors.lastname: Ngaiza
authors.forename: G
authors.initials: G
authors.lastname: Manley
authors.forename: J
authors.initials: J
authors.lastname: Grulich-Henn
authors.forename: J L
authors.initials: JL
authors.lastname: Krstenansky
authors.forename: E A
authors.initials: EA
authors.lastname: Jaffe
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D011464:Epoprostenol / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006629:Hirudins / Q000032:analysis / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000494:pharmacology; D013917:Thrombin / Q000032:analysis / Q000378:metabolism* / Q000502:physiology*
chemical_list: D006629:Hirudins; D010446:Peptide Fragments; D005340:Fibrinogen; D011464:Epoprostenol; D013917:Thrombin; C027962:diisopropylphosphoryl-thrombin; D002118:Calcium