Thrombin receptor activation by thrombin and receptor-derived peptides in platelet and CHRF-288 cell membranes: receptor-stimulated GTPase and evaluation of agonists and partial agonists.
Thrombin receptor activation, by thrombin or SFLLR-containing peptides, stimulates GTPase activity in platelet and CHRF-288 membranes. Polyclonal antibodies to peptides derived from the thrombin receptor (anti-TR52-69 and anti-TR36-49), which block many of thrombin's actions on platelets and endothelial cells, also block thrombin activation of membrane GTPase (as does thrombin active site and anion-binding exosite inhibitors). Most of the receptor-activated GTPase, stimulated by both thrombin and SFLLRNP in platelet membranes, was inhibited by prior treatment with pertussis toxin or N-ethylmaleimide, suggesting that under these conditions much of the thrombin receptor-stimulated GTPase in platelet membranes is a member of the pertussis toxin-sensitive G alpha i family. In platelet membrane preparations, the peptide agonists stimulated approximately twice as much GTPase activity as stimulated by alpha-thrombin. In contrast, the membranes prepared from CHRF-288 cells showed similar maximal SFLLRNP- and alpha-thrombin-stimulated GTPase activity. Stimulation of the platelet membrane GTPase by a variety of different peptide agonists correlated with their ability to stimulate platelet aggregation. Several peptide-based agonists were more potent than the wild-type sequence. The most potent was Ser-(p-fluoro-Phe)-(2-Napthyl-Ala)-Leu-Arg-NH2, which stimulated platelet aggregation (EC50 = 80 nM) and GTPase activity (EC50 = 110 nM). The peptide YFLLRN stimulated GTPase activity but only to approximately 40% of the activity observed with optimal concentrations of other receptor agonists. YFLLRN also limited the stimulation observed with SFLLRNP in a competitive fashion, indicating that YFLLRN is a competitive partial agonist at the thrombin receptor. These studies show that the tethered-ligand receptor mediates the GTPase activation by thrombin in platelet and CHRF-288 cell membranes, and this provides a specific, reliable, and convenient cell-free assay system with which one can evaluate agonists and partial agonists.
Seiler
S M
SM
Department of Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA.
Peluso
M
M
Tuttle
J G
JG
Pryor
K
K
Klimas
C
C
Matsueda
G R
GR
Bernatowicz
M S
MS
D000595:Amino Acid Sequence; D000906:Antibodies / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology / Q000494:pharmacology*; D037342:Pertussis Toxin; D018179:Receptors, Thrombin / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000906:Antibodies; D010455:Peptides; D018179:Receptors, Thrombin; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D013917:Thrombin; D020558:GTP Phosphohydrolases; D005033:Ethylmaleimide