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We have tested the hypothesis that phospholipase D (PLD) is the effector of the unidentified G protein (GE) mediating Ca(2+)-independent exocytosis in platelets. Although GTP gamma S, and to a lesser extent phorbol 12-myristate 13-acetate (PMA), caused some secretion of 5-HT from electropermeabilized human platelets in the effective absence of Ca2+ (pCa > 9), these stimuli had much more potent synergistic effects when added together. In all cases, secretion of 5-HT was closely correlated to the stimulus-induced formation of [3H]phosphatidic acid ([3H]PA) from [3H]arachidonate-labelled phospholipids. Addition of ethanol inhibited both secretion and [3H]PA formation and led to the accumulation of [3H]phosphatidylethanol ([3H]PEt), indicating that [3H]PA was formed largely by activation of PLD. BAPTA and analogues caused dose-dependent inhibitions of both GTP gamma S-induced secretion and PLD activity in the permeabilized platelets. This action of BAPTA did not appear to be mediated by chelation of Ca2+ or by direct inhibition of protein kinase C (PKC). The results suggest that PLD is the target of GE in platelets and that BAPTA can block PLD activation.

Polyclonal antibodies raised against specific recombinant low molecular mass GTP-binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn-proteins (i.e. proteins that bind [alpha-32P]GTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn-protein (Gn27). In further analysis by two-dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn-protein forms, seven of 27 kDa (Gn27a-g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a-d). The ralA antibody reacted strongly with the five most basic Gn27 species (a-e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a-d. We conclude that ral gene products account for some but probably not for all of the platelet Gn-proteins.

Arg8-vasopressin inhibited the adenylate cyclase activity of human platelet particulate fraction up to a maximum of 27% (IC50 = 1.2 nM). This inhibition required the presence of 10 microM GTP and was optimal with 100 mM NaCl. Orn8-vasopressin had similar effects. 1-Deamino-Val4, D-Arg8-vasopressin did not by itself affect adenylate cyclase activity but competitively inhibited the action of Arg8-vasopressin (pA2 = 7.74). Arg8-vasopressin did not inhibit adenylate cyclase in intact platelets but instead caused platelet aggregation, an effect that was also competitively inhibited by 1-deamino-Val4, D-Arg8-vasopressin (pA2 = 7.82). Thus, platelets possess vasopressin receptors of the V1 type that, under appropriate conditions, can mediate either inhibition of platelet adenylate cyclase or platelet aggregation.

The 27 kDa platelet membrane protein (Gn27) that binds [alpha-32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617-620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22-23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431-439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of [32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.

Human platelets containing granule-bound [14C]serotonin were permeabilized, equilibrated at 0 degrees C with ATP and with various Ca2+ buffers and guanine nucleotides, and then incubated at 25 degrees C with or without a stimulatory agonist. Ca2+ alone induced the ATP-dependent secretion of [14C]serotonin (50% at a pCa of 5.1) but the sensitivity of secretion to Ca2+ was greatly enhanced by guanine nucleotides [6-fold by 100 microM GTP, 100-fold by 100 microM guanyl-5'-yl imidodiphosphate and greater than 500-fold by 100 microM guanosine 5'-O-(3-thiotriphosphate)] or by stimulatory agonists (10-fold by 2 units thrombin/ml and 4-fold by 1 microM 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine). When both GTP and a stimulatory agonist were added, they had synergistic effects on secretion. Cyclic GMP and GMP acted similarly to GTP. The effects of all these guanine nucleotides were inhibited by guanosine 5'-O-(2-thiodiphosphate), whereas those of stimulatory agonists were not. Our results demonstrate the presence in platelets of guanine nucleotide-dependent and independent mechanisms regulating the sensitivity of secretion to Ca2+.

NaCl stimulated the adenylate cyclase activities of human and rabbit platelet particulate fractions prepared in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetate, but inhibited the activities of particulate fractions proteolysed by endogenous Ca2+-activated protease or treatment with alpha-chymotrypsin. Studies with other monovalent cations showed that LiCl had weak effects similar to those of NaCl, whereas KCl inhibited the enzyme in both proteolysed and non-proteolysed preparations. The results suggest that NaCl exerts stimulatory and inhibitory effects through different sites. NaCl potentiated and proteolysis greatly reduced the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor).