Involvement of the "tethered-ligand" receptor in thrombin inhibition of platelet adenylate cyclase.
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.
Department of Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543.
S M
SM
Seiler
I M
IM
Michel
J W
JW
Fenton
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000097:blood*; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D000242:Cyclic AMP / Q000097:blood*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D016285:Iloprost / Q000037:antagonists & inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000502:physiology*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
D000067956:Adenylyl Cyclase Inhibitors; D008024:Ligands; D010446:Peptide Fragments; D010455:Peptides; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C072032:thrombin receptor peptide (42-55); C072028:BMS 180742; D000242:Cyclic AMP; D013917:Thrombin; D000262:Adenylyl Cyclases; D016285:Iloprost