Inhibition of thrombin and SFLLR-peptide stimulation of platelet aggregation, phospholipase A2 and Na+/H+ exchange by a thrombin receptor antagonist.
A thrombin receptor has been described that is activated by thrombin cleavage generating a new N-terminus. The newly exposed SFLLR-containing "tethered-ligand" then activates the receptor. In these studies, we used 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn- Asp-Lys-amide (Mpapeptide) as a thrombin receptor antagonist. This compound was capable of preventing both thrombin- and SFLLR-peptide-induced platelet aggregation with little effect on collagen-induced platelet aggregation. It also prevented thrombin- and SFLLRNP-induced calcium mobilization with little effect on thromboxane receptor-activated platelet Ca2+ mobilization. Platelet membrane GTPase could be activated by peptides that activated the thrombin receptor, and the thrombin receptor antagonist also prevented receptor-stimulated GTPase activity. Platelet phospholipase A2 (PLA2) activity (measured as the release of radiolabeled arachidonic acid) and Na+/H+ exchange activation were stimulated by alpha-thrombin and by SFLLR-containing peptides. Activation of both processes with low concentrations of thrombin required thrombin's anion-binding exosite, as they were not activated by similar concentrations of gamma-thrombin, and the alpha- and zeta-thrombin activation was blocked by peptides mimicking the C-terminal region of hirudin. Stimulation of PLA2 and Na+/H+ exchange by both thrombin and SFLLR-containing peptides was inhibited by the thrombin receptor antagonist Mpa-peptide. These results support the hypothesis that thrombin stimulation of PLA2 activity and Na+/H+ exchange occurs via activation of the thrombin tethered-ligand receptor. Moreover, these data are consistent with the tethered-ligand receptor mediating most actions elicited by low concentrations of alpha-thrombin involved in human platelet activation.
Seiler
S M
SM
Department of Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543.
Peluso
M
M
Michel
I M
IM
Goldenberg
H
H
Fenton
J W
JW
Riexinger
D
D
Natarajan
S
S
D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*; D013917:Thrombin / Q000037:antagonists & inhibitors*
D000067956:Adenylyl Cyclase Inhibitors; C090291:C186 65; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D018179:Receptors, Thrombin; D017923:Sodium-Hydrogen Exchangers; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin