Michael C. Scrutton

Platelet β-adrenoceptors

1 Inhibition by isoprenaline of the aggregatory response of human and rat platelets induced by various excitatory agonists is blocked by β2‐adrenoceptor antagonists, β1‐Adrenoceptor antagonists are ineffective. 2 β2‐Adrenoceptor agonists cause inhibition of the response of human platelets to various excitatory agonists. The maximal extent of inhibition is less than that observed for isoprenaline. β1‐Adrenoceptor agonists fail to cause detectable inhibition of this response. Neither β1 nor β2‐adrenoceptor agonists cause inhibition of the response of rat platelets to excitatory agonists. Only β2‐adrenoceptor agonists block the inhibitory response to isoprenaline. 3 The extent of inhibition by isoprenaline is a function of the excitatory agonist used and in human platelets is correlated with the ability of that agonist to suppress elevated platelet cyclic adenosine 3′,5′‐monophosphate (cyclic AMP) levels. 4 Inhibition by isoprenaline is prevented in the presence of an inhibitor of adenylate cyclase. 5 Isoprenaline increases platelet cyclic AMP levels with an EC50 similar to that required to observe inhibition of the aggregatory response. 6 These data indicate that human platelets carry β2‐adrenoceptors whose occupancy causes inhibition of the response to excitatory agonists as a consequence of elevation of platelet cyclic AMP. The β‐adrenoceptor present on rat platelets also appears to be of the β2‐subtype.

Direct evidence for a role for Ca2+ in amine storage granule secretion by human platelets

Abstract Exposure of washed human platelets to a high voltage electric field renders these cells permeable to small molecules such as Ca EGTA. Such accessed platelets secrete 5-hydroxytryptamine (5HT) when challenged with 10- 5 M Ca 2+ but do not release lactate dehydrogenase under these conditions. The secretion of 5HT induced by Ca 2+ requires MgATP but is independent of synthesis of prostaglandin endoperoxides. Half maximal secretion of 5HT is observed in the presence of 1.9μM Ca 2+ . These data suggest a direct involvement of Ca 2+ in platelet amine granule secretion.

Effects of α2‐adrenoceptor agonists and of related compounds on aggregation of, and on adenylate cyclase activity in, human platelets

1 A range of 2–(,5‐dihydroimidazolyl)‐benzene, ‐quinoline, and ‐quinoxaline derivatives and 2‐morpholino‐4‐catechol have been characterized as agonists or partial agonists for human platelet aggregation; and for inhibition of adenylate cyclase by measurement of their effect on platelet [cyclic‐3′,5′‐AMP]. Antagonist activity for these compounds versus adrenaline as agonist has also been assessed for these two responses. 2 The compounds can be divided into 4 groups. Group I contains compounds that are agonists for both responses; group II, compounds that are agonists for inhibition of adenylate cyclase but antagonists for the aggregatory response; group III, compounds that are agonists for the aggregatory response but are antagonists for inhibition of adenylate cyclase by adrenaline; and group IV, compounds that are antagonists for both responses. 3 In group I the EC50 values for induction of aggregation are not significantly different from the EC50 values for inhibition of adenylate cyclase except for 2‐morpholino‐4‐catechol which is significantly more potent as an inhibitor of adenylate cyclase. 4 In group IV a linear correlation is observed between the K1 values for the two responses for 8 compounds but 2 other compounds do not conform to this correlation. 5 The data are not consistent with a model in which a single x2‐adrenoceptor mediates both the aggregatory response and inhibition of adenylate cyclase and hence support a model in which unique x2‐adrenoceptors mediate these two responses.

Mammalian platelet adrenoceptors

1 Mammalian platelets vary widely in their responses to catecholamines in part because these agonists can act via excitatory α‐ and inhibitory β‐adrenoceptors. In the absence of antagonists, adrenaline enhances the response of rabbit platelets to an excitatory agonist, e.g. adenosine‐5′‐O‐(1‐thiodiphosphate) (ADP‐α‐s) acting at another receptor, but has no effect on the response of rat or guinea pig platelets to such an agonist. In the presence of a β‐adrenoceptor antagonist, adrenaline enhances the response of rat, but not guinea‐pig platelets to ADP‐α‐S and the extent of the enhanced effect on rabbit platelets is increased. In the presence of an α‐adrenoceptor antagonist, adrenaline inhibits the response of rabbit and rat platelets to ADP‐α‐S but has no such effect on the response of guinea‐pig platelets. 2 Studies using selective agonists and antagonists demonstrate that the excitatory response of rat platelets to adrenaline is mediated by an α2‐adrenoceptor, and the inhibitory response of rabbit platelets to adrenaline by a β2‐adrenoceptor as is the case in other species which have been examined. 3 The mean α2‐adrenoceptor density on human, rabbit, rat and guinea‐pig platelets as assessed using [3H]‐yohimbine as radioligand is obtained as 258, 270, 42 and < 10 receptors per platelet. 4 The mean β2‐adrenoceptor density on human, rabbit, rat and guinea‐pig platelets as assessed using (‐)‐[125I]‐iodocyanopindolol is obtained as 66, 14, 41 and < 5 receptors per platelet. 5 The nature of the effect of adrenaline on the response of mammalian platelets to other excitatory agonists, e.g. ADP‐α‐S, appears therefore to be largely determined by the absolute number of α2‐adrenoceptors and by the relative content of α2‐ and β2‐adrenoceptors on these cells for the species which have been examined in this analysis.

Particle volume changes associated with light transmittance changes in the platelet aggregometer: dependence upon aggregating agent and effectiveness of stimulus.

The increase in light transmittance of aspirin-treated platelet-rich plasma caused by addition of non-saturating doses of ADP and, at earlier times, of adrenaline is correlated with formation of aggregates having a volume in the range 490-8580 fl. and containing 100-2000 platelets. The disappearance of single platelets and the formation of aggregates having volumes less than 490 fl. makes no significant contribution to the increase in light transmittance. Similar relationships are observed on addition of saturating doses of ADP and adrenaline except that the formation of aggregates larger than 8580 fl. contributes significantly to the initial phase of the increase in light transmittance and is more closely correlated with the overall change in this parameter.

Effect of various excitatory agonists on the secretion of 5-hydroxytryptamine from permeabilised human platelets induced by Ca2+ in the presence or absence of GTP

Addition of GTP markedly enhances the ability of thrombin to cause a leftward shift in the Ca2+ dose/ response curve for 5‐hydroxytryptamine secretion from permeabilised human platelets. Little effect is observed on addition of GTP in the absence of thrombin. Neither ADP nor adrenaline, in the presence or absence of GTP, causes such a shift, whereas 5‐hydroxytryptamine does so to a small extent but only in the presence of GTP. The leftward shift in the Ca2+ dose/response curve induced by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate or 1‐oleyl‐2‐acetylglycerol is not enhanced by addition of GTP. The thrombin concentration required for half‐maximal enhancement of the responce to Ca2+ is markedly reduced by addition of GTP. The results support the postulate that the effects of excitatory agonists in this system correlate with their ability to activate phospholipase C and provide further evidence for a role for GTP in signal transduction between the receptor and phospholipase C.

Identification of small peptide analogues having agonist and antagonist activity at the platelet thrombin receptor.

Two tripeptide analogues (N-[3-methyl-1-S[[2-S [(methyl-amino)carbonyl]-1-pyrrolidinyl] carbonyl]butyl-D-analine) (SC40476) and N-[3-methyl-S-(1-pyrrolidinylcarbonyl)butyl]-D-alanine, ethyl ester, hydrochloride (SC42619], inhibit aggregation of, and secretion from, human platelets induced by thrombin but cause no significant inhibition of esterolysis or fibrin formation catalysed by this enzyme. Inhibition by SC40476 of the aggregatory response induced by thrombin is incomplete. Neither peptide analogue inhibits aggregation induced by ADP, collagen, vasopressin or 11,9-epoxymethanoprostaglandin H2 (U-46619). Enhancement of the response is observed when nonsaturating concentrations of these agonists are employed. SC42619 causes a parallel shift to the right in the concentration-response curve describing aggregation induced by thrombin. The Schild plot of these data has a slope of 1.05 and the pA2 is 2.9 +/- 0.1. Both SC40476 and SC42619 induced a small but significant decrease in the single platelet content of platelet suspensions. Neither peptide analogue increases platelet cytosolic [Ca2+] measured using quin 2 or Fura 2. Both analogues cause inhibition of the increase in cytosolic [Ca2+] induced by thrombin. Inhibition by SC42619 is competitive with respect to thrombin when the extracellular [Ca2+] is reduced to less than 0.1 microM but is non-competitive in the presence of 1 mM Ca2+. SC42619 also inhibits the increase in cytosolic [Ca2+]induced by ADP in the presence of 1 mM Ca2+ but not the smaller increase caused by this agonist when the medium contains less than 0.1 microM Ca2+. SC42619 inhibits Mn2+ influx induced by thrombin and ADP. SC40476 and SC42619 inhibit the enhanced incorporation of [32P] into phosphatidic acid observed on stimulation by thrombin of platelets pre-labelled with [32P]-phosphate. Addition of the peptide analogues alone fails to increase significantly the 32P content of phosphatidate, phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. SC40476 causes no detectable hydrolysis of glycoprotein V as detected by release of the proteolytic product (glycoprotein VFR). The results indicate that SC40476 and SC42619 interact selectively with the platelet thrombin receptor. Both peptide analogues act as effective antagonists for this receptor but also possess weak agonist activity which may also result from interaction with the thrombin receptor. The molecular basis for this latter activity has not been defined. SC42619 non-selectively inhibits Ca2+ influx induced by several agonists but this effect does not appear to contribute to the observed inhibition of the aggregatory and secretory responses.

Responses of rabbit platelets to adrenaline induced by other agonists

Abstract ADP, thrombin, 5HT, arachidonate, collagen and stable prostaglandin endoperoxide/thromboxane A 2 analogues all induce rabbit platelets to aggregate in response to subsequent addition of adrenaline as a result of occupancy of an α 2 -adrenoceptor. The maximal rate and extent of the induced aggregatory response to adrenaline varies with the inducing agonist. The aggregatory response to adrenaline induced by collagen, arachidonate or U-46619 is associated with secretion of both 5HT and ADP. Indomethacin or N(1-carboxyheptyl-) imidazole inhibit the response to adrenaline induced by collagen or arachidonate. No secretion accompanies the aggregatory response to adrenaline induced by ADP or thrombin. These results demonstrate for the first time that occupancy of the rabbit platelet α 2 -adrenoceptor can give rise to a secretory response. They also explain the inconsistent reports of direct aggregatory responses to adrenaline observed in rabbit platelet-rich plasma or washed rabbit platelets.

Synergistic responses and receptor occupancy in rabbit blood platelets

Several observations indicate that simultaneous receptor occupancy is necessary for generation of a synergistic response of rabbit platelets to two excitatory agonists. First, an aggregatory response to adrenaline induced by prior addition of 5HT reverses rapidly unless stabilised by inhibition of 5HT uptake. The extent of response correlates with the extracellular 5HT concentration. Second, addition of an ADPase following adrenaline enhances the rate of disaggregation if the response to this agonist has been induced by prior addition of ADP. Third, disaggregation is induced, or enhanced, if an antagonist selective to the inducing agonist is added following completion of the induced aggregatory response. These data suggest marked lability in the mechanisms responsible for generation of synergistic responses.

Secretion of 5-hydroxytryptamine from electropermeabilised human platelets Effects of GTP and cyclic 3′, 5′-AMP

Enhancement by thrombin of Ca2+ ‐dependent 5HT secretion in the absence of added GTP decreases as the time between electropermeabilisation and addition of thrombin is increased. No decrease occurs if thrombin is added with GTP. Observation of apparent GTP‐independent receptor/phospholipase C coupling may result from the presence of bound GTP in the preparation. Enhancement by GTP of Ca2+ ‐dependent 5HT secretion occurs with a significant lag indicating an agonist‐independent effect. Cyclic 3′,5′‐AMP inhibits enhancement by GTP of Ca2+ ‐dependent 5HT secretion while having no effect on enhancement induced by GTPγS. Hence cyclic AMP may impair receptor/phospholipase C coupling by enhancing Np GTPase activity.