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

Abstract

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 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. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors.

TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

“…Several authors have suggested that TRAP is only a partial agonist because it induces less expression of activated GPIIb-IIIa, fails to induce platelet prothrombinase activity, and causes weaker responses in terms of Ca ϩϩ mobilization, arachidonate production, and serotonin release. 8,9,28,29 Our studies do not fully support these concepts. In our hands, concentrations of thrombin and TRAP that induced similar aggregation responses in washed platelets resulted in nearly equivalent levels of phosphorylation at tyrosine residues of proteins present in whole platelet lysates.…”

“…TRAP is able to induce aggregation and secretion by similar mechanisms than thrombin, 4 it is also involved in inositol phosphatase metabolism, 6 in inhibition of adenylcyclase, 7 and in protein phosphorylation. 8 Although it is generally accepted that TRAP acting on PAR-1 has the ability to induce many of the cellular responses observed after thrombin activation, comparative studies show that TRAP seems to be less efficient to induce the whole sequence of responses caused by thrombin. 4,9 -13 Considering that thrombin has an additional receptor on GPIb␣, differential responses to both agonists should be expected.…”

Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

“…The recent finding that neither thrombin receptor directly couples to G i indicates that thrombin operates a G i -independent mechanism for platelet activation (22). However, it is also well known that the G i independence of thrombin-induced platelet response is correlated to the concentration of the agonist, as ADP scavengers are able to affect platelet activation by low, but not high, doses of thrombin (22,29). In this work we report that activation of Rap1B by 0.1 unit/ml thrombin is only minimally affected by preincubation with ADP scavengers apyrase or creatine phosphate-creatine phosphokinase.…”

“…Specific peptides reproducing the sequence of the new N terminus of activated thrombin receptors are potent and selective activators of PAR-1 and PAR-4 and have been shown to trigger all of the platelet responses elicited by thrombin (24,28). However, several findings indicate that, in contrast to thrombin, thrombin receptor-activating peptides are not full agonists for platelet activation and that the support of secreted ADP is necessary to achieve complete platelet response (29,30). For instance, maximal doses of PAR-1-activating peptide (TRAP-1) require secreted ADP to trigger irreversible platelet aggregation, whereas ADP scavengers inhibit platelet aggregation only at low, but not high, doses of thrombin (29,30).…”

“…However, several findings indicate that, in contrast to thrombin, thrombin receptor-activating peptides are not full agonists for platelet activation and that the support of secreted ADP is necessary to achieve complete platelet response (29,30). For instance, maximal doses of PAR-1-activating peptide (TRAP-1) require secreted ADP to trigger irreversible platelet aggregation, whereas ADP scavengers inhibit platelet aggregation only at low, but not high, doses of thrombin (29,30). Similarly, it has also been shown that the late activation of phosphatidylinositol 3-kinase induced by TRAP-1 is dependent on secreted ADP (30).…”