Human platelet activation by an alkylacetyl analogue of phosphatidylcholine

The present study has evaluated the influence of semi-synthetic platelet-aggregating factor, (PAF) i.e., alkylacetylglycerophosphocholine, on human platelet morphology, biochemistry and function in order to determine if PAF serves as the corrective factor restoring sensitivity to refractory platelets after treatment with epinephrine. Threshold concentrations of PAF caused irreversible platelet aggregation which could be blocked by agents elevating endogenous levels or cyclic AMP or inhibited by antagonists of platelet prostaglandin synthesis and secretion. PAF did not stimulate platelets through α-adrenergic receptors or receptors for arachidonate, endoperoxides or thromboxanes. 24 h after aspirin ingestion, platelets could be aggregated irreversibly by high concentrations, but not by threshold amounts of PAF, even though they were still insensitive to arachidonate. Another less potent PAF derivative, alkenylacetylglycerophosphocholine, blocked aggregation of 24-h aspirin platelets by PAF, but did not inhibit restoration of arachidonate sensitivity and irreversible aggregation when the samples were treated first with epinephrine. Our findings indicate that threshold amounts of PAF activate human platelets in a physiologic manner and cause irreversible aggregation which is dependent on prostaglandin synthesis and the release reaction. The results do not support the concept that PAF is the mediator of the mechanism of membrane modulation through which epinephrine induces correction of the refractory state in prostaglandin I₂-treated or dissociated platelets, or cells obtained from individuals following aspirin ingestion. Thus, the mechanism of platelet membrane modulation is capable of securing irreversible aggregation of secretion, prostaglandin synthesis or PAF formation.     

Effects of N-acetylglucosamine and platelet inhibitors on the synergistic interaction of platelets and aggregating agents in the presence of wheat germ agglutinin

Low concentrations of wheat germ agglutinin (4 micrograms/ml) have been shown to act synergistically to induce platelet aggregation with epinephrine, collagen, arachidonate and ionophore A23187. Aggregation ceased on the addition of the haptenic sugar N-acetylglucosamine at any time following the onset of aggregation with these agonists and a small degree of disaggregation was observed during the reversible first wave with the biphasic aggregating agents epinephrine and ADP. Cyclooxygenase inhibitors such as indomethacin and aspirin blocked the second wave of aggregation with the biphasic aggregating agents epinephrine and ADP but a synergistic response continued to be shown with the first wave in the presence of these inhibitors. Release of [14C]serotonin and the mobilization of [3H]arachidonate by epinephrine and collagen were markedly stimulated in the presence of wheat germ agglutinin but there was no increase of either radiolabel in the case of ADP. Platelet shape change, but not aggregation, occurred with low levels of wheat germ agglutinin and the synergistic response with ADP, collagen or ionophore A23187 occurred without further shape change. Wheat germ agglutinin did not affect the basal or stimulated levels of cyclic AMP. The membrane fluidity of platelets was not affected by the lectin or by thrombin as shown by the lack of change in fluorescence polarization with diphenylhexatriene. It is suggested that the binding of wheat germ agglutinin to the platelet surface induces platelet activation by mechanisms similar to those of other agonists and that it may affect the distribution of membrane-bound Ca2+ by a reversible perturbation of the platelet membrane.     

Inhibition of platelet aggregation and arachidonate metabolism in platelets by procyanidins

The effects of procyanidins on platelet aggregation and arachidonate metabolism in platelets were studied. Nine procyanidins were used in this investigation. Procyanidins B-2-S, EEC and C-1 significantly induced the inhibition of platelet aggregation, and the potency of inhibition was comparable with aspirin. Procyanidin B-2-S was used as a representative of procyanidins for further studies on the effect on arachidonate metabolism. In arachidonate metabolism by fatty acid cyclooxygenase pathway, B-2-S inhibited TXB2 and HHT formation by intact platelets treated with exogenous arachidonic acid. It also inhibited TXB2 formation measured by a specific radioimmunoassay when the cells were challenged with calcium ionophore A23187. In cell-free system, B-2-S inhibited both TXB2 and 12-HETE bioxynthesis in platelet microsome and cytosol, respectively. The inhibitory effect on thromboxane biosynthesis might explain the inhibitory effect of procyanidins on platelet aggregation.     

Effects of N-acetylglucosamine and platelet inhibitors on the synergistic interaction of platelets and aggregating agents in the presence of wheat germ agglutinin

Low concentrations of wheat germ agglutinin (4 μg/ml) have been shown to act synergistically to induce platelet aggregation with epinephrine, collagen, arachidonate and ionophore A23187. Aggregation ceased on the addition of the haptenic sugar N-acetylglucosamine at any time following the onset of aggregation with these agonists and a small degree of disaggregation was observed during the reversible first wave with the biphasic aggregating agents epinephrine and ADP. Cyclooxygenase inhibitors such as indomethacin and aspirin blocked the second wave of aggregation with the biphasic aggregating agents epinephrine and ADP but a synergistic response continued to be shown with the first wave in the presence of these inhibitors. Release of [¹⁴C]serotonin and the mobilization of [³H]arachidonate by epinephrine and collagen were markedly stimulated in the presence of wheat germ agglutinin but there was no increase of either radiolabel in the case of ADP. Platelet shape change, but not aggregation, occurred with low levels of wheat germ agglutinin and the synergistic response with ADP, collagen or ionophore A23187 occurred without further shape change. Wheat germ agglutinin did not affect the basal or stimulated levels of cyclic AMP. The membrane fluidity of platelets was not affected by the lectin or by thrombin as shown by the lack of change in fluorescence polarization with diphenylhexatriene. It is suggested that the binding of wheat germ agglutinin to the platelet surface induces platelet activation by mechanisms similar to those of other agonists and that it may affect the distribution of membrane-bound Ca²⁺ by a reversible perturbation of the platelet membrane.     

Stimulation of platelet protein phosphorylation by arachidonic acid and endoperoxide analogs

The present study has investigated the influence of arachidonate, endoperoxide analogs, and the calcium ionophore A23187 on platelet aggregation and on the phosphorylation of platelet proteins. Following stimulation of platelets by these agents a rapid increase in phosphorylation of three proteins was observed which began at the same time as the initial formation of platelet aggregates. These three proteins were the 260,000 dalton actin-binding protein, a 40,000 dalton protein of unknown function, and the 20,000 dalton myosin light chain. When extensive aggregation was reached, the extent of phosphorylation returned toward baseline. Pretreatment of platelets with aspirin completely inhibited both aggregation and protein phosphorylations induced by arachidonate, but had only partial inhibitory effects on endoperoxide analogs or A23187. Since endoperoxide analogs and A23187 may trigger endogenous production of prostaglandin endoperoxides and thromboxane A₂, in addition to having a direct effect of their own, it is probable that the partial inhibition seen was due to inhibition of that component of their effect due to this endogenous production, through other effects of aspirin can not be entirely ruled out. Since recent evidence shows that phosphorylation of myosin light chain results from calcium stimulation of a protein kinase in the presence of calmodulin, the results are consistent with mobilization of calcium as the primary role of the arachidonate-endoperoxide-thromboxane pathway.     

Platelet aggregation independent of adp release or prostaglandin synthesis in patients with Hermansky-Pudlak syndrome

Platelets from patients with the Hermansky-Pudlak (HPS) syndrome are deficient in the storage pool of adenine nucleotides and serotonin. As a result, the storage pool deficient (SPD) platelets develop only single waves of clumping when stimulated by threshold concentrations of aggregating agents which cause irreversible, biphasic aggregation of normal platelets. Yet, patients with HPS either have no bleeding problems or only mild symptoms.In the present study we have evaluated the importance of prostaglandin synthesis and secretion to the irreversible aggregation of HPS platelets. Results of the study demonstrate that aspirin-treated SPD platelets, which cannot form thromboxane or undergo the release reaction on stimulation by arachidonate, can still undergo irreversible aggregation in response to thrombin and ADP if treated first with epinephrine. A mechanism of membrane modulation mediated by a-adrenergic receptors cooperatively linked to the endoperoxide and thromboxane receptor can secure irreversible aggregation of normal or abnormal platelets despite absence of secretion and prostaglandin synthesis.     

Measurement of ionized calcium in blood platelets with a new generation calcium indicator

Fura 2, a new generation calcium indicator, has a 30 fold brighter fluorescence than Quin 2, shows wavelength shifts upon calcium binding and has a relatively low buffering capacity for free calcium. Quin 2, the most widely used fluorophore, on the other hand, shows no wavelength shifts and has a very high affinity for free calcium. Therefore, we have compared the relative merits of these two fluorophores for monitoring agonist induced alterations in platelet cytosolic calcium. Platelets loaded with Fura 2 showed a significant rise in cytosolic calcium when stirred with agonists such as epinephrine, arachidonate and thrombin, whereas Quin 2 loaded platelets demonstrated a rise in cytosolic calcium only with thrombin stimulation. A rise in agonist induced calcium in Fura 2 loaded platelets was prevented when the cells were exposed first to antagonists such as aspirin or prostaglandin E1. Arachidonate refractory platelets, upon stirring with a single agonist, did not show a significant elevation in cytosolic calcium. However, when refractory platelets were first exposed to epinephrine and then challenged with arachidonate, they revealed a significant elevation in cytosolic calcium. Unlike Quin 2, Fura 2 at the highest concentration tested did not inhibit platelet function. Improved properties of Fura 2 suggest that it may be a useful agent to study agonist induced alterations in cytosolic calcium levels in blood platelets.     

The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.     

The influence of epinephrine on prostacyclin (PGI2) induced dissociation of adp aggregated platelets

Prostacyclin (PGI₂) is a powerful inhibitor of platelet function and recent studies have indicated that it can dissociate aggregated platelets in vivo. The present investigation has evaluated the mechanism involved in the dissociation of ADP aggregated platelets by PGI2, the influence of epinephrine on the process of PGI₂ mediated disaggregation, and effects of the catecholamine on the refractory state of PGI2 dispersed cells. Dissociation of ADP aggregates by PGI₂ was concentration and time dependent, caused complete disappearance of sensitive platelet clumps and restored platelet discoid form. The dispersed platelets were refractory to further stimulation by ADP, thrombin, arachidonate or the calcium ionophore, A23187. Measurement of cAMP revealed no change in control levels after aggregation by ADP, but a sharp increase during and after dissociation by PGI₂. Epinephrine added to ADP aggregates before or after PGI2 blocked disaggregation, or stopped dissociation if already in progress, and caused a rapid fall in the high levels of cAMP stimulated by PGI₂. Agents to which PGI2 dissociated platelets were refractory, including ADP, arachidonate and A23187 caused no lowering of elevated cAMP levels. Reduction of high levels of CAMP in PGI₂ dispersed platelets by epinephrine did not result in reaggregation. However, epinephrine addition to refractory platelets restored in large measure their sensitivity to aggregation by arachidonate, thrombin, ADP and A23187.     

Unsaturated fatty acids inhibit ADP- arachidonate-induced platelet aggregation without affecting thromboxane synthesis

The inhibitory effects of three cis-unsaturated C18 fatty acids (oleic, linoleic, and linolenic acids, sodium salts) on ADP- and sodium-arachidonate-induced aggregation of washed rabbit platelets were investigated. When the platelets were suspended in protein-free medium containing dextran, it was found that these fatty acids at very low concentrations (2-45 microM) were potent inhibitors of platelet responsiveness and the inhibitory effect occurred within seconds. The inhibition of ADP-induced aggregation was not affected by abolishing the activity of platelet cyclooxygenase using aspirin. Human serum albumin relieved the inhibition caused by fatty acids for both ADP- and arachidonate-induced aggregation. The inhibitory effect of fatty acids does not seem to be due to decreased thromboxane formation (except possibly in the case of linolenate), and the relief of fatty acid inhibition by albumin does not potentiate thromboxane B2 formation from exogenous arachidonate. It is suggested that the inhibitory effect of polyunsaturated fatty acids on platelet aggregation is specific and not related to a general surfactant effect, since inhibition occurs far below the critical micelle concentration of fatty acid soaps.     

Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclo-oxygenase activity

Aspirin and indomethacin do interact with the same site on cyclo-oxygenase. This suggestion is based on in vitro studies on ram seminal vesicles and in vivo drug interaction studies on rat platelets. The purpose of the present study was to ascertain whether the same interaction also occurred after administration of both drugs to human volunteers. Platelet aggregation induced by sodium arachidonate or by collagen, and formation of platelet MDA and TxB2 were measured before, two and 48 hours after ingestion of either indomethacin (50 mg) or aspirin (500 mg) or of both drugs (30 minutes apart). While the inhibitory effect of indomethacin on these parameters was short lasting, that of aspirin persisted for at least 48 hours. However, when both drugs were given concurrently, the long-lasting effect of aspirin was no longer detectable. Since competition at levels other than platelets was unlikely, this study indicates that indomethacin and aspirin inhibit human platelet cyclo-oxygenase by the same basic mechanism. Acetylation of the enzyme appears to be a secondary mechanism which makes the inhibitory effect of aspirin persistent.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclo-oxygenase activity

Aspirin and indomethacin do interact with the same site on cyclo-oxygenase. This suggestion is based on studies on ram seminal vesicles and drug interaction studies on rat platelets. The purpose of the present study was to ascertain whether the same interaction also occurred after administration of both drugs to human volunteers.Platelet aggregation induced by sodium arachidonate or by collagen, and formation of platelet MDA and TxB₂ were measured before, two and 48 hours after ingestion of either indomethacin (50 mg) or aspirin (500 mg) or of both drugs (30 minutes apart).While the inhibitory effect of indomethacin on these parameters was short lasting, that of aspirin persisted for at least 48 hours. However, when both drugs were given concurrently, the long-lasting effect of aspirin was no longer detectable. Since competition at levels other than platelets was unlikely, this study indicates that indomethacin and aspirin inhibit human platelet cyclo-oxygenase by the same basic mechanism. Acetylation of the enzyme appears to be a secondary mechanism which makes the inhibitory effect of aspirin persistent.     

Extract of a spice--omum (Trachyspermum ammi)-shows antiaggregatory effects and alters arachidonic acid metabolism in human platelets

An ethereal extract of omum (Trachyspermum ammi; Hindustani: ajwan)--a frequently consumed spice--was found to inhibit platelet aggregation induced by arachidonic acid (AA), epinephrine and collagen; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by omum could be explained by its effect on platelet thromboxane production as suggested by the following experimental observation. (i) Omum reduced TxB2 formation in intact platelet preparations from added arachidonate, and (ii) it reduced the formation of TxB2 from AA-labelled platelets after stimulation with Ca2+-ionophore A23187 by a direct action on cyclooxygenase as it did not affect the release of AA from labelled platelets. An increased formation of lipoxygenase-derived products from exogenous AA in omum-treated platelets was apparently due to redirection of AA from cyclooxygenase to the lipoxygenase pathway.     

The influence of lysophosphatidic acid on platelet protein phosphorylation

Lysophosphatidic acid (LPA) is a lysophospholipid that is produced during thrombin stimulation of platelets, which can promote platelet aggregation. The mechanism of the effect of LPA was explored in normal platelets and in platelets from a patient with a storage pool deficiency (SPD). A comparison with other lysophospholipids showed that only LPA exerted significant effects to cause or potentiate platelet aggregation. Aspirin, an inhibitor of prostaglandin endoperoxide synthetase, had little effect on LPA-induced aggregation, but completely blocked LPA-induced serotonin secretion. LPA also promoted phosphorylation of myosin light chain (MLC), a 47 kilodalton (kDa) protein, and actin-binding protein. Aspirin significantly inhibited the phosphorylation of the 47-kDa and actin-binding proteins at 3-8 min after the addition of LPA, but had no effect on protein phosphorylation within the 1st min and had no significant effect on MLC phosphorylation. In SPD platelets, aspirin partially inhibited both aggregation and phosphorylation of the 47-kDa protein (less than 30% inhibition) and MLC (less than 40% inhibition) at time points of 1 min or less. The addition of ADP to SPD platelets enhanced the LPA response in platelets either pretreated or not pretreated with aspirin. Studies with SPD platelets indicate that thromboxane and secreted ADP contribute to, but are not necessary for, LPA-induced aggregation and phosphorylation. A23187 (a calcium ionophore) and LPA showed some selectivity to promote MLC as opposed to the 47-kDa protein phosphorylation, particularly at low concentrations of agonists and at earlier time points. The protein phosphorylation changes seen are consistent with a role for MLC phosphorylation in the granule centralization promoted with LPA.     

Vitamin E exerts antiaggregatory effects without inhibiting the enzymes of the arachidonic acid cascade in platelets

Vitamin E (α-tocopherol) and tocopherol acetate produced a slightly increased amount of thromboxane in treated compared to untreated platelets. In tocopherol acetate-treated platelets significantly more lipoxygenase products were produced. α-tocopherol induced an increased, but not significant, production of thromboxane B₂ during blood clotting. α-tocopherol was not found to affect platelet phospholipase activity as determined by its effect on the release of labelled arachidonic acid from platelet phospholipids by challenging the platelets with calcium ionophore A23,187. α-tocopherol potentiated the incorporation of labelled arachidonate in the platelet phospholipids. Inspite of having no effect on the arachidonic acid cascade in platelets, α-tocopherol inhibited aggregation induced by several aggregating agents including A23,187. Inhibition of aggregation may be explained by the ability of α-tocopherol to inhibit intracellular mobilization of sequestered calcium from the dense tubular system to the cytoplasm.     

Effect of single oral administrations of non steroidal antiinflammatory drugs to healthy volunteers on arachidonic acid metabolism in peripheral polymorphonuclear and mononuclear leukocytes

The effects of a single oral administration of acetylsalicylic acid (500 mg), indomethacin (50 mg) and piroxicam (40 mg) to healthy volunteers on functional and biochemical parameters of platelets, polymorphonuclear (PMN) and mononuclear (MNL) leukocytes were evaluated. Blood was collected before and two hours after the drug intake and blood cells separated according to conventional techniques. The considered drugs almost completely suppressed the aggregation of platelets, whereas they did not affect either PMN and MNL aggregation. Superoxide anion generation by leukocytes was (PMN), or no effect (MNL) was observed after piroxicam and indomethacin respectively. The formation of arachidonate metabolites via the 5-lipoxygenase pathway by PMN and MNL challenged with 10 microM A23187 was unchanged following aspirin and indomethacin. In this respect a selective increase of 5-HETE and LTC4 synthesis by MNL only was detected after piroxicam administration. The three drugs similarly reduced TXB2 synthesis by platelets and PMN (-80% for aspirin and indomethacin, and -40% for piroxicam). As far as MNL is concerned, aspirin inhibited this metabolite by 80%, while indomethacin reduced it by 40% only. In contrast piroxicam increased TXB2 synthesis by stimulated MNL. It can be concluded that the considered antiinflammatory drugs 1) differently affect the cyclooxygenase enzyme in platelets and leukocytes; 2) at variance with the situation in platelets, the inhibition of thromboxane formation by leukocytes is not related to modifications of cellular function; 3) the formation of arachidonate metabolites via the 5-lipoxygenase pathway is affected by piroxicam only.     

High concentrations of exogenous arachidonate inhibit calcium mobilization in platelets by stimulation of adenylate cyclase.

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.     

Response of human platelets exposed to arachidonate upon stimulation with other agonists: possible role of Ca2+i movement

Arachidonate (25 microM) induced only 20% maximal platelet aggregation but 80% maximal cytoplasmic ionized calcium movement (as measured by the photoprotein aequorin). Preincubation with 25 microM arachidonate prevented platelet aggregation and cytoplasmic ionized calcium movement induced by threshold concentrations of the thromboxane A2 analogue U-46619, but did not prevent platelet aggregation induced by threshold concentrations of thrombin, despite a marked reduction of cytoplasmic ionized calcium movement. These findings suggest that thrombin may employ pools of cytoplasmic ionized calcium and/or mechanisms to mobilize it different from those utilized by arachidonate and its metabolites.     

Platelet ADP receptor and alpha 2-adrenoreceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding

The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen-binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2-adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide.