THROMBIN-RECEPTOR AGONIST PEPTIDES, IN CONTRAST TO THROMBIN ITSELF, ARE NOT FULL AGONISTS FOR ACTIVATION AND SIGNAL-TRANSDUCTION IN HUMAN PLATELETS IN THE ABSENCE OF PLATELET-DERIVED SECONDARY MEDIATORS

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin rec eptor that is generated by thrombin's proteolytic activity, were repor ted to activate platelets equally with thrombin itself and are conside red to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using a spirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5' -[alpha-thio]triphosphate to prevent the secondary effects of the pote nt agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A(2)), we assessed the direct actions of thrombin and TRPs (i.e. TRP(42-47) and TRP(42-55)). Compared with thrombin, un der these conditions, TRPs: (I) failed to aggregate platelets complete ly; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60(c-src) with the cytoskeleton and (4) caused less alpha-granule secretion, phosphorylation of cytopl asmic phospholipase A(2), arachidonic acid release and phosphatidyl in ositol (PtdOH) production. Furthermore, TRPs induced transient increas es in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin we re greater and more sustained. Hirudin added after thrombin accelerate d protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally d esensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsiv e to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guano sine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. C hem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitizat ion in keeping with their lower intrinsic activity; (2) thrombin's eff ects in platelets, even in TRP-desensitized platelets, are entirely me diated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, m ay require the continued progressive proteolytic activation of naive t hrombin receptors.