The individual contributions of glycoprotein Ib (GPIb) and the seven transmembrane domain receptor (STDR) to increases in platelet [Ca2+]i induced by alpha-thrombin or the tethered ligand peptide (TLP; SFLLRNPNDKYEPF) have been determined in control platelets, in platelets where the thrombin binding site on GPIb was blocked with the monoclonal antibodies TM60 and LJ-Ib10, in platelets where access of thrombin to the STDR was blocked by polyclonal antipeptide antibodies, and in Bernard-Soulier platelets which constitutively lack GPIb. Curve-fitting analyses (LIGAND) showed that binding of PPACK-thrombin and alpha-thrombin to the moderate-affinity site was not detected in the best-fit model in the presence of anti-STDR antibodies although with alpha-thrombin there was also decreased binding at the high-affinity site. Conversely, TM60 blocked binding of alpha-thrombin to the high-affinity site but also decreased binding at the moderate affinity site. Separately, either TM60 or anti-TNA (150 micrograms/mL) reduced thrombin (0.5 nM)-induced elevations in [Ca2+]i to 50% of control values, but Ca2+ elevations were essentially abrogated (4.2 +/- 5%) when the two were added in combination. [Ca2+]i dose-response curves for alpha-thrombin were curvilinear and were only 50% of controls in the presence of anti-GPIb or anti-STDR antibodies at up to 10 nM alpha-thrombin, with their greatest sensitivity being below 2 nM. With Bernard-Soulier platelets, changes in [Ca2+]i were not detectable at < or = 0.5 nM alpha-thrombin but were also 50% of controls at 5-10 nM alpha-thrombin. [Ca2+]i responses to TLP (1-100 microM) of antibody-blocked platelets were identical to those of controls whereas responses were approximately 50% of controls in Bernard-Soulier platelets. The rate of increase in [Ca2+]i in controls was twice that seen in antibody-blocked platelets and about 5-fold greater than in Bernard-Soulier platelets. These results demonstrate that both GPIb and the STDR are required to ensure the optimal rate and extent of platelet activation over a range of alpha-thrombin concentrations (0.3-10 nM) and that the STDR corresponds to the previously described moderate-affinity thrombin receptor.

Previous results have shown that both GPIb and the seven transmembrane domain receptor (STDR) are required for optimal thrombin-induced platelet activation (Greco et al., 1996). Limited degradation (approximately 10%) of GPIb and the STDR by elastase reduced the Ca2+ response to 0.5 nM alpha-thrombin by only 10% whereas Serratia marcescens metalloprotease reduced the Ca2+ response by 80% and fully abrogated high-affinity thrombin binding and aggregation. vWF/ristocetin-induced agglutination was only slightly reduced (20%) while Ca2+ and aggregation response to higher thrombin concentrations were retained. At increasing elastase and Serratia protease concentrations, degradation of the STDR proceeded from the amino-terminal domain, but Ca2+ responses to the tethered ligand peptide SFLLRNPNDKYEPF were not affected by either protease. These results show that both putative thrombin receptors are susceptible to protease degradation and suggest that Serratia protease is able to differentiate the GPIb-mediated events associated with thrombin activation from those associated with ristocetin-induced agglutination.

Signal transduction components, incuding the Ras superfamily of low molecular weight GTP-binding proteins and the y subunits of heterotrimeric G proteins, are reversibly carboxyl methylated at C-terminal prenylcysteine residues. We have previously shown that the prenylcysteine analog N-acetyl-S-tans,trans-farnesyl-L-cysteine (AFC) inhibits carboxyl methylation of these proteins in human platelets. Here we show that concentrations of AFC that inhibit Ras carboxyl methylation also block responses to agonists such as ADP, collagen, arachidonic acid, U46619 (a stable analog of prostaglandin H2), thrombin, and guanoine 5'-[y-thioltriphosphate. AFC does not inhibit aggregation induced by effectors such as ionomycin, phorbol 12,13-dibutyrate, and bacterial phospholipase C that bypass G proteins to activate platelets at the level of cytosolic Ca2+ concentration and protein kinase C. These findings indicate that AFC inhibits agonist-receptor-mediated signal transduction in human platelets.The finding that Ras proteins and the heterotrimeric G proteins are methylated at the carboxyl group of the C terminus has raised the possibility that this type of reversible covalent modification may play a role in signal transduction (1, 2). These GTP-binding proteins function to relay information from membrane receptors to control the cytoplasmic levels of second messengers, such as cAMP, cytosolic Ca2W, and diacylglycerol (DAG) (3,4), that act in turn to regulate protein kinases. Ras proteins and the y subunits of G proteins are initially synthesized with a characteristic C-terminal sequence, CXXX (a cysteine followed by three, generally aliphatic, residues) (2, 5). CXXX sequences provide a recognition site for cytosolic prenyltransferase enzymes, which catalyze the attachment of farnesyl or geranylgeranyl polyisoprenoid groups in thioether linkage to the cysteine (2, 6). The three residues distal to the cysteine in prenylated CXXX sequences are removed by membrane-associated protease activities (7), and the exposed prenylcysteine a-carboxylate undergoes methylesterificationbyamembrane-bound S-adenosylmethionine:prenylcysteine methyltransferase (8,9).Results with inhibitors ofpolyisoprenoid synthesis, such as compactin and mevinolin (lovastatin), indicate that prenylation is essential for the function of Ras proteins and 'y subunits ofthe heterotrimeric G proteins (10-13). Prenylation is required for all subsequent steps in CXXX processing (13,14), and without prenylation, these proteins cannot localize to their sites of action at the plasma membrane (15-17). Classical heterotrimeric G proteins as well as low molecular weight G proteins such as rapla, rapib, rap2, and rap2b are present in human platelets (18)(19)(20)(21)(22)(23)(24)(25)(26)(27). To specifically investigate the role of carboxyl methylation in G-protein-dependent signal transduction, we have examined the effects of a prenylcysteine methyltransferase inhibitor, N-acetyl-Strans,trans-farnesyl-L-cysteine (AFC), on agonist-receptormediated signal transduction in ...

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