Involvement of the "tethered-ligand" receptor in thrombin inhibition of platelet adenylate cyclase

• Published in: Biochemical and biophysical research communications Vol. 182; no. 3; p. 1296
• Main Authors: Seiler, S M, Michel, I M, Fenton, 2nd, J W
• Format: Journal Article
• Language: English
• Published: United States 14-02-1992
• Subjects: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases - blood; Amino Acid Sequence; Blood Platelets - drug effects; Blood Platelets - enzymology; Blood Platelets - physiology; Cyclic AMP - blood; Dose-Response Relationship, Drug; Humans; Iloprost - antagonists & inhibitors; Iloprost - pharmacology; In Vitro Techniques; Kinetics; Ligands; Molecular Sequence Data; Peptide Fragments - pharmacology; Peptides - pharmacology; Receptors, Cell Surface - drug effects; Receptors, Cell Surface - physiology; Receptors, Thrombin; Thrombin - isolation & purification; Thrombin - metabolism; Thrombin - pharmacology
• ISSN: 0006-291X
• DOI: 10.1016/0006-291X(92)91873-O

Thrombin is thought to activate platelets through multiple signaling pathways. Recently a new thrombin receptor was identified (Vu et al., Cell 64:1057-1068, 1991) that recognizes alpha-thrombin's anion-binding exosite. Thrombin cleaves this receptor generating a new N-terminal ("tethered-ligand") that activates the receptor. We report here that this receptor is involved in alpha-thrombin inhibition of platelet adenylate cyclase, a process thought mediated by thrombin's high-affinity pathway. In gel-filtered human platelets, iloprost-stimulated cAMP levels were lowered by alpha- and zeta-thrombin addition and, to a much lesser extent, by gamma-thrombin. The alpha- and zeta-thrombin mediated decreases in cAMP were prevented by the thrombin anion-binding exosite inhibitor, BMS 180742, implying that binding to thrombin's anion-binding exosite was required. The iloprost-stimulated increase in cAMP was also reversed (in a concentration-dependent fashion) by a peptide mimicking the new N-terminal of the "tethered-ligand" thrombin receptor (SFLLRNPNDKYEPF). In broken cell preparations, platelet adenylate cyclase activity was also inhibited by SFLLRNPNDKYEPF (but not by a similar peptide used as a control, FSLLRNPNDKYEPF). These results support the hypothesis that thrombin inhibition of platelet adenylate cyclase activity is mediated, at least in part, via the "tethered-ligand" receptor. Moreover, this data is consistent with the "tethered-ligand" receptor mediating the high affinity actions of alpha-thrombin.