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Human platelets containing dense granules labelled with 5-hydroxy[14C]tryptamine ([14C]5-HT) were permeabilized by exposure to streptolysin O (SLO) in the presence of 4 mM [gamma-32P]ATP. Addition of either 100 nM phorbol 12-myristate 13-acetate (PMA) or of Ca2+ (pCa 5) at the same time as SLO induced secretion of dense-granule [14C]5-HT and the phosphorylation of pleckstrin by protein kinase C (PKC). Ca2+ also induced phosphorylation of myosin P-light chains. Guanosine 5'-[gamma-thio]triphosphate (GTP[S], 100 microM) did not stimulate secretion from SLO-permeabilized platelets in the absence of Ca2+ (pCa>9), but greatly potentiated secretion in the presence of low PMA (10 nM) or low Ca2+ (pCa 6). However, GTP[S] did stimulate myosin P-light-chain phosphorylation in the absence of Ca2+, an effect that was associated with morphological changes, including granule centralization. Inhibition of PKC and of pleckstrin phosphorylation by Ro 31-8220 blocked secretion induced by PMA or by GTP[S] and PMA in the absence of Ca2+, but did not prevent the GTP[S]-induced phosphorylation of myosin P-light chains or secretion induced by Ca2+ at pCa 5. When the time period between exposure of platelets to SLO and challenge at pCa>9 with PMA or with GTP[S] and PMA was increased, there were rapid and parallel decreases in the secretion and pleckstrin phosphorylation responses, which were lost after 3-5 min. In contrast, the responsiveness of secretion to Ca2+ (pCa 5) or to GTP[S] and Ca2+ (pCa 6) persisted for at least 10 min after exposure of platelets to SLO, although the ability of pleckstrin to undergo phosphorylation was still lost after 3-5 min. Both PKC and pleckstrin were undetectable within platelets after 5 min exposure to SLO. The results suggest that the loss of responsiveness to PMA or to GTP[S] and PMA is attributable to the leakage of PKC (and possibly pleckstrin) from the platelets, whereas secretion stimulated by Ca2+ or by GTP[S] and Ca2+ utilizes membrane-associated Ca2+- and GTP-binding proteins and occurs independently of PKC activation.

cGMP enhances cAMP accumulation in platelets via cGMP-inhibited phosphodiesterase (PDE3) [Maurice and Haslam (1990) Mol. Pharmacol. 37, 671-681]. However, cGMP might also limit cAMP accumulation by activating cGMP-stimulated phosphodiesterase (PDE2). We therefore evaluated the role of PDE2 in human platelets by using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to inhibit this enzyme selectively. IC50 values for the inhibition of platelet PDE2 by EHNA, with 10 microM cAMP as substrate in the absence and in the presence of 1 microM cGMP, were 15 and 3 microM respectively. Changes in platelet cyclic [3H]nucleotides were measured after prelabelling with [3H]adenine and [3H]guanine. Nitroprusside (NP) caused concentration-dependent increases in [3H]cGMP and a biphasic increase in [3H]cAMP, which was maximal at 10 microM (49+/-6%) and smaller at 100 microM (32+/-6%) (means+/-S.E.). In the presence of EHNA (20 microM), which had no effects alone, NP caused much larger increases in platelet [3H]cAMP (125+/-14% at 100 microM). EHNA also enhanced [3H]cGMP accumulation at high NP concentrations. In accord with these results, EHNA markedly potentiated the inhibition of thrombin-induced platelet aggregation by NP. The roles of cAMP and cGMP in this effect were investigated by using 2', 5'-dideoxyadenosine to inhibit adenylate cyclase. This compound decreased the accumulation of [3H]cAMP but not that of [3H]cGMP, and diminished the inhibition of platelet aggregation by NP with EHNA. We conclude that much of the effect of NP with EHNA is mediated by cAMP. Lixazinone (1 microM), a selective inhibitor of PDE3, increased platelet [3H]cAMP by 177+/-15%. This increase in [3H]cAMP was markedly inhibited by NP; EHNA blocked this effect of NP. Parallel studies showed that NP suppressed the inhibition of platelet aggregation by lixazinone. EHNA enhanced the large increases in [3H]cAMP seen with 20 nM prostacyclin (PGI2), but had no effect with 1 nM PGI2. NP and 1 nM PGI2 acted synergistically to increase [3H]cAMP, an effect attributable to the inhibition of PDE3 by cGMP; EHNA greatly potentiated this synergism. In contrast, NP decreased the [3H]cAMP accumulation seen with 20 nM PGI2, an effect that was blocked by EHNA. The results show that, provided that cGMP is present, PDE2 plays a major role in the hydrolysis of low cAMP concentrations and restricts any increases in cAMP concentration and decreases in platelet aggregation caused by the inhibition of PDE3. At high cAMP, PDE2 plays the major role in cAMP breakdown, whether cGMP is present or not.

We have used reverse transcriptase PCR, platelet mRNA and degenerate primers based on platelet peptide sequences, to amplify a fragment of platelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). Sequence analysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR product representing the C-terminal half of platelet PDE3 cDNA and corresponding to amino acid residues 560-1141 of the cardiac enzyme, was cloned and expressed in Escherichia coli cGI-PDEDelta1. Further deletion mutants were constructed by removing either an additional 100 amino acids from the N-terminus (cGI-PDEDelta2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDEDelta1Deltai). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by [32P]cGMP and for the effects of PDE3 inhibitors. The Km values for hydrolysis of cAMP and cGMP by immunoprecipitates of cGI-PDEDelta1 (182+/-12 nM and 153+/-12 nM respectively) and cGI-PDEDelta2 (131+/-17 nM and 99+/-1 nM respectively) were significantly lower than those for immunoprecipitates of intact platelet PDE3 (398+/-50 nM and 252+/-16 nM respectively). Moreover, N-terminal truncations of platelet enzyme increased the ratio of Vmax for cGMP/Vmax for cAMP from 0.16+/-0.01 in intact platelet enzyme, to 0.37+/-0.05 in cGI-PDEDelta1 and to 0.49+/-0.04 in cGI-PDEDelta2. Thus deletion of the N-terminus enhanced hydrolysis of cGMP relative to cAMP, suggesting that N-terminal sequences may exert selective effects on enzyme activity. Removal of the 44-amino-acid insert generated a mutant with a catalytic domain closely resembling those of other PDE gene families but despite a limited ability to be photolabelled by [32P]cGMP, no cyclic nucleotide hydrolytic activities of the mutant were detectable. Mutation of amino acid residues in putative beta-turns at the beginning and end of the 44-amino-acid insert to alanine residues markedly reduced the ability of the enzyme to hydrolyse cyclic nucleotides. The PDE3 inhibitor, lixazinone, retained the ability to inhibit cAMP hydrolysis and [32P]cGMP binding by the N-terminal deletion mutants and the site-directed mutants, suggesting that PDE3 inhibitors may interact exclusively with the catalytic domain of the enzyme.

The major substrate of protein kinase C(PKC) in platelets is the 40 kDa protein, pleckstrin. Addition of the homobifunctional reagent, bis(sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fraction or to electropermeabilized platelets resulted in cross-linking of pleckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100-120 kDa respectively, which were visualized by immunoblotting with an anti-pleckstrin antibody. Higher levels of cross-linking were observed in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of platelet lysate or lysis of electropermeabilized platelets and, in the case of the 90 kDa and 100-120 kDa species, after activation of PKC by phorbol 12-myristate 13-acetate. Similar experiments with purified pleckstrin indicated that the 90 kDa and 100-120 kDa species consist, at least in part, of pleckstrin dimers and higher oligomers. After incubation of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25% of the protein was present in cross-linked species. The results indicate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts with an unidentified platelet protein of about 28 kDa.

In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding protein or Ca2+.

Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.

The photoaffinity labelling of platelet cyclic GMP (cGMP)-binding proteins by [32P]cGMP was studied; at least five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet cytosol and four (80, 65, 49 and 38 kDa) in platelet membranes. The 110 kDa species was identified as cGMP-inhibited cyclic AMP (cAMP) phosphodiesterase (PDE III) by immunoprecipitation and by the inhibition of photolabelling by specific inhibitors of this enzyme. Similarly, the 80 kDa species was identified as cGMP-dependent protein kinase by immunoprecipitation and by the effects of cGMP analogues on photolabelling. Addition of cAMP greatly enhanced the labelling of this 80 kDa protein, implying the existence of a potentially important interaction between the effects of cGMP and cAMP. The 65 kDa photolabelled protein appears to be a novel platelet cyclic-nucleotide-binding protein. In contrast, the 49 and 55 kDa photolabelled species are probably the RI and RII regulatory subunits of cAMP-dependent protein kinase, and the 38 kDa protein(s) may be proteolytic fragment(s) of RI and/or RII.

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.

Immunoblot analysis showed that the 47 kDa platelet substrate of protein kinase C (P47) was expressed at low levels in undifferentiated HL-60 leukaemia cells. Treatment of these cells with dimethyl sulphoxide, 1 alpha,25-dihydroxycholecalciferol or retinoic acid caused progressive increases in P47 content. Retinoic acid (1 microM) elicited the largest response, a 4-fold increase in P47 protein after 7 days that was accompanied by an increase in translatable P47 mRNA. The induction of P47 by retinoic acid preceded cessation of cell proliferation and development of the capacity to reduce Nitro Blue Tetrazolium, indicating that its expression is an early event in the myeloid differentiation of HL-60 cells.

Cyclic AMP levels per mg of cell protein were higher in late-passage (senescent) fibroblasts than in early-passage (young) fibroblasts both during growth and stationary phase, but, because the protein concentration per unit volume in senescent cells was lower than in young cells, the molar concentrations of intracellular cyclic AMP were very similar in the two cell types. In both young and senescent fibroblasts cyclic AMP levels declined during growth and no increase in intracellular cyclic AMP occurred in association with density-dependent inhibition of growth. These results indicate that changes in cyclic AMP concentration do not play a role in controlling the growth or in the senescent decline of human fibroblasts. Prostaglandin E(1) (1mum) caused maximal increases in fibroblast cyclic AMP concentration of 60-500-fold after 10-30min, and adrenaline (epinephrine) (10mum) caused maximum increases of 5-25-fold after 2-10min, depending on both the number of passages and the period after subculture. The cyclic AMP level in confluent young cells increased more with prostaglandin E(1) and far less with adrenaline than the cyclic AMP level in confluent senescent cells. During growth to confluence the cyclic AMP response to adrenaline declined in young cells and increased in senescent cells. As these responses to prostaglandin E(1) and to adrenaline changed independently of each other and of the basal cyclic AMP concentration, it is suggested that the expression of hormone receptors is altered both during growth to confluence and during senescence of human fibroblasts.

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5'-[gamma-thio]triphosphate, but not by GMP or adenosine 5'-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).

A method is described for isolating cyclic [(3)H]GMP from platelets preincubated with [(3)H]guanine. Collagen increased and aspirin decreased the cyclic [(3)H]GMP concentration in platelets. The results are consistent with a role for cyclic GMP in the platelet release reaction.

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.

Human platelets that had been preincubated with 5-hydroxy[(3)H]tryptamine and [(32)P]P(i) were stirred with various agents; the secretion of 5-hydroxy[(3)H]tryptamine from platelet granules and the radioactivity of platelet [(32)P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[(3)H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E(1), which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[(3)H]tryptamine. Prostaglandin E(1) also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca(2+) concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714-722; (1978) Thromb. Res.12, 619-628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E(1) may initiate processes that decrease the Ca(2+) concentration in the cytosol, so inhibiting both the Ca(2+)-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E(1) did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca(2+) concentration in the platelet cytosol. As prostaglandin E(1) did inhibit the release of 5-hydroxy[(3)H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.

After human platelets have been rendered permeable to small molecules by high voltage electric discharges, addition of buffered micromolar concentrations of Ca2+ causes an ATP-dependent secretion of dense granule serotonin [Knight & Scrutton (1980) Thromb. Res. 20, 437-446]. In the present study, platelets permeabilized by this technique were found to show an up to 10-fold increase in their sensitivity to Ca2+ after exposure to thrombin. In permeabilized platelets, as in the intact cells, release of serotonin was associated with the Ca2+-dependent phosphorylation of 47 000 and 20 000 Da polypeptides (P47 and P20). Thrombin markedly increased the phosphorylation of P47 in the presence of 0.1-1.0 microM-Ca2+ free but had a much smaller effect on phosphorylation of P20. Thrombin also stimulated the formation of 1,2-diacylglycerol in the presence of 0.1 microM-Ca2+ free and was even more effective with 1.0 microM-Ca2+ free, suggesting that receptor-activated hydrolysis of phosphoinositides to 1,2-diacylglycerol was preserved in permeabilized platelets and was potentiated by low intracellular concentrations of Ca2+. The increase in phosphorylation of P47 on addition of thrombin may therefore be accounted for by the stimulatory action of 1,2-diacylglycerol on Ca2+-activated, phospholipid-dependent protein kinase. However, in both the presence and absence of thrombin, higher Ca2+ concentrations were required for optimal secretion than for maximal phosphorylation of both P47 and P20, indicating that additional actions of Ca2+ and thrombin, perhaps also mediated by 1,2-diacylglycerol formation, may be involved in the release of serotonin.

Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90 000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.