Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

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.     

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

The equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 microM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets. The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8. A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 microM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation and ADP binding. The inhibitor constants of ATP, adenyl-5'-yl-(beta,gamma-methylene)diphosphate, IDP, adenosine-5'(2-O-thio)diphosphate, for aggregation and high affinity binding were in good correlation with each other. Exceptions formed fluorosulfonylbenzoyl adenosine and AMP. The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Q10 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism. All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.     

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin

Background

We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.

Principal Findings

Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE₁ induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg⁻¹) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.

Conclusions

These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.     

In vitro effect of a thymic epithelial culture supernate or thymosin fraction 5 on rabbit platelet aggregation and intracellular cyclic AMP levels

Supernates of thymic epithelial cell culture (STEC) strongly inhibit aggregation induced by addition of adenosine diphosphate (ADP: 1 microM) or thrombin (0.5 unit per ml) to washed platelet suspensions and accelerated the restoration from ADP-triggered aggregation. At the same time, STEC increased the level of platelet adenosine 3',5'-cyclic monophosphate (cyclic AMP) in a dose-dependent manner. Depending on the concentration used, thymosin fraction 5 increased the level of intracellular cyclic AMP ranging between 5 and 100 micrograms per ml, as well as inhibiting ADP-induced platelet aggregation. The activities of both STEC and thymosin fraction 5 were found to act exclusively on cyclic AMP phosphodiesterase activity in platelets. In contrast the supernates from Chang, HeLa, or HCC-M cells did not affect platelet aggregation induced by ADP, but slightly increased the cyclic AMP level (Chang, HeLa). Within 2 min after the treatment with STEC, more than 50% of the maximum inhibitory activity on platelet aggregation and increases in intracellular cyclic AMP were observed. These activities disappeared following STEC treatment with pronase E. STEC activity was found predominantly in the 1,000-50,000-dalton fractions. These activities were not altered when STEC was treated by adenosine deaminase. The level of prostaglandin E (PGE) derivatives in STEC was about two times that found in the control culture medium. These data suggest that the biological activity of STEC in the platelets might be attributed to thymosinlike polypeptides and PGE1.     

Interralation of prostaglandin endoperoxide (prostaglandin G2) and cyclic 3′,5′-adenosine monophosphate in human blood platelets

The prostaglandin endoperoxide, prostaglandin G₂, in platelet-rich plasma may produce reversible platelet aggregation without secretion, irreversible aggregation with secretion of platelet constituents inhibited by indomethacin, or the latter effects despite indomethacin, depending on the concentration of the endoperoxide. Irreversible aggregation and platelet secretion induced by prostaglandin G₂ apparently result from the action of ADP, since these responses are inhibited by 2-n-amylthio-5′-AMP (an inhibitor of the actions of ADP on platelets) and they do not occur in heparinized platelet-rich plasma. Prostaglandin G₂ lowers the platelet level of cyclic 3′,5′-AMP. Its actions are inhibited by elevation of cyclic AMP levels by prostaglandin E₁ or dibutyryl cyclic AMP or adenosine. Like malondialdehyde production induced by thrombin, ADP, or arachidonic acid, prostaglandin G₂-induced malondialdehyde production is reduced by dibutyryl cyclic AMP and prosraglandin E₁. Platelet activation by prostaglandin G₂ is enhanced by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)-adenine.The action of prostaglandin G₂ on platelets is more complex then previously reported.     

Antiplatelet Effect of Catechol Is Related to Inhibition of Cyclooxygenase,Reactive Oxygen Species,ERK/p38 Signaling and Thromboxane A2 Production

Catechol (benzenediol) is present in plant-derived products, such as vegetables, fruits, coffee, tea, wine, areca nut and cigarette smoke. Because platelet dysfunction is a risk factor of cardiovascular diseases, including stroke, atherosclerosis and myocardial infarction, the purpose of this study was to evaluate the anti-platelet and anti-inflammatory effect of catechol and its mechanisms. The effects of catechol on cyclooxygenase (COX) activity, arachidonic acid (AA)-induced aggregation, thromboxane B₂ (TXB₂) production, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK)/p38 phosphorylation were determined in rabbit platelets. In addition, its effect on IL-1β-induced prostaglandin E₂ (PGE₂) production by fibroblasts was determined. The ex vivo effect of catechol on platelet aggregation was also measured. Catechol (5-25 µM) suppressed AA-induced platelet aggregation and inhibited TXB₂ production at concentrations of 0.5–5 µM; however, it showed little cytotoxicity and did not alter U46619-induced platelet aggregation. Catechol (10–50 µM) suppressed COX-1 activity by 29–44% and COX-2 activity by 29–50%. It also inhibited IL-1β-induced PGE₂ production, but not COX-2 expression of fibroblasts. Moreover, catechol (1–10 µM) attenuated AA-induced ROS production in platelets and phorbol myristate acetate (PMA)-induced ROS production in human polymorphonuclear leukocytes. Exposure of platelets to catechol decreased AA-induced ERK and p38 phosphorylation. Finally, intravenous administration of catechol (2.5–5 µmole/mouse) attenuated ex vivo AA-induced platelet aggregation. These results suggest that catechol exhibited anti-platelet and anti-inflammatory effects, which were mediated by inhibition of COX, ROS and TXA₂ production as well as ERK/p38 phosphorylation. The anti-platelet effect of catechol was confirmed by ex vivo analysis. Exposure to catechol may affect platelet function and thus cardiovascular health.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [³²P]cyclic AMP (N₃ cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5′-AMP. Binding was unaffected by 5′-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.     

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+.     

Potentiation of anti-aggregating activity of PGI2 by 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in rabbit platelets in vitro

Anti-aggregating activity of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was tested using rabbit platelets in vitro. EG-626 alone, when added before, prevented platelet aggregation induced by ADP, as did PGI₂, papaverine and dipyridamole. Spontaneous disaggregation was also accelerated when EG-626 was added after the maximal aggregation induced by ADP. EG-626 alone also inhibited platelet aggregation induced by collagen and arachidonic acid. ID₅₀s of these agents in ADP-induced aggregation were 7–9 nM for PGI₂, 223 μM for EG-626, 266 μM for papaverine and 957 μM for dipyridamole. When EG-626 was used in combination with PGI₂, a threshold dose (50 μM) of EG-626 potentiated the anti-aggregating effect of subthreshold dose (3 nM) of PGI₂ upto 100% inhibition in collagen-induced platelet aggregation. The marked potentiating effect of EG-626 was accompanied by an accumulation of cyclic AMP in the platelets. These effects might be due to inhibition of phosphodiesterase. Papaverine and dipyridamole, other phosphodiesterase inhibitors, also potentiated the anti-aggregating activity of PGI₂. The activity of papaverine, however, was one eighth of EG-626 and that of dipyridamole was much less. The most effective combination of PGI₂ and EG-626 to induce 50% inhibition was obtained with 20% of ID₅₀ of each agent, whereas that of PGI₂ and papaverine or dipyridamole was 39 or 41%, respectively.     

Increased Platelet Reactivity in Idiopathic Pulmonary Fibrosis Is Mediated by a Plasma Factor

Introduction

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, incurable fibrotic interstitial lung disease with a prognosis worse than many cancers. Its pathogenesis is poorly understood. Activated platelets can release pro-fibrotic mediators that have the potential to contribute to lung fibrosis. We determine platelet reactivity in subjects with IPF compared to age-matched controls.

Methods

Whole blood flow cytometry was used to measure platelet-monocyte aggregate formation, platelet P-selectin expression and platelet fibrinogen binding at basal levels and following stimulation with platelet agonists. A plasma swap approach was used to assess the effect of IPF plasma on control platelets.

Results

Subjects with IPF showed greater platelet reactivity than controls. Platelet P-selectin expression was significantly greater in IPF patients than controls following stimulation with 0.1 µM ADP (1.9% positive ±0.5 (mean ± SEM) versus 0.7%±0.1; p = 0.03), 1 µM ADP (9.8%±1.3 versus 3.3%±0.8; p<0.01) and 10 µM ADP (41.3%±4.2 versus 22.5%±2.6; p<0.01). Platelet fibrinogen binding was also increased, and platelet activation resulted in increased platelet-monocyte aggregate formation in IPF patients. Re-suspension of control platelets in plasma taken from subjects with IPF resulted in increased platelet activation compared to control plasma.

Conclusions

IPF patients exhibit increased platelet reactivity compared with controls. This hyperactivity may result from the plasma environment since control platelets exhibit increased activation when exposed to IPF plasma.     

The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin.

Addition of prostacyclin (PGI2) temporarily inhibits platelet aggregation and permits the isolation of platelets free from plasma proteins, which have the same sensitivity as those in plasma [Moncada, Radomski & Vargas (1982) Br. J. Pharmacol. 75, 165P]. By using a modification of this technique we have established that platelets isolated from normal subjects aggregate more readily in response to ADP and adrenaline when physiological concentrations of low-density lipoproteins (LDL) are present. At high LDL concentrations spontaneous aggregation occurs. High-density lipoproteins (HDL) and very-low-density lipoproteins (VLDL) had no effect on agonist-induced platelet aggregation at normal concentrations, but HDL sensitized at higher concentrations. These effects by lipoproteins are not accompanied by changes in platelet lipid content. Cyclohexanedione treatment of LDL to modify apolipoproteins appeared to abolish the sensitization effect, indicating that binding to receptors was essential for the effects of LDL. LDL, but not HDL, overcame the inhibitory effect of PGI2 on platelet aggregation, except at very high concentrations of PGI2. PGI2 raised the cyclic AMP content of isolated platelets, but LDL only partially prevented this rise. These results suggest that LDL may have a greater role in platelet aggregation than previously recognized and may also regulate effects of PGI2. These findings may be of relevance to an understanding of cardiovascular diseases.     

Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2.

The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.     

Activation of human platelets by N-substituted aminophospholipids

N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidylserine (NBD-PS), a fluorescent phospholipid synthesized from phosphatidylserine by reaction with NBD-chloride, caused platelet shape change and aggregation when added at micromolar concentrations to suspensions of washed human platelets in the absence of added fibrinogen. Platelet aggregation by NBD-PS was accompanied by thromboxane synthesis and secretion of contents from dense, alpha-, and lysosomal granules in the absence of appreciable platelet damage. Indomethacin completely inhibited NBD-PS-induced thromboxane synthesis, but platelet aggregation and [14C]serotonin secretion were only slightly inhibited. Neither inhibition of the ADP-dependent pathway with creatine phosphate/creatine kinase plus ATP, alone or in combination with indomethacin, nor maximum elevation of cyclic AMP by treatment with prostaglandin I2 and theophylline completely inhibited NBD-PS-induced platelet aggregation or [14C]serotonin secretion. Platelet effects of NBD-PS were specific in that neither phosphatidylserine nor lyso-NBD-PS were similarly active. The activation of platelets by NBD-PS is not attributable to the NBD moiety exclusively since acylation of the amino group with 5-dimethylaminonaphthalene-1-sulfonyl-chloride yielded a similarly active derivative. Dansylated phosphatidylethanolamine was also active. The findings indicate that NBD-PS and other N-substituted aminophospholipids can activate a central pathway of platelet secretion and aggregation that is independent of released ADP and thromboxane formation and is only partially controlled by platelet cyclic AMP.     

Factors influencing the response of human blood platelets to analogues of ADP which may act as partial agonists at the ADP receptor.

1. The prior addition of non-aggregating concentrations of the divalent cation ionophore, A-23187, causes human platelets to aggregate in response to a subsequent addition of the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ADP (oADP and or ADP). Previous studies [Pearce et al. (1978) Eur. J. Biochem. 88, 543--555] have shown that these derivatives act as partial agonists at the platelet ADP receptor inducing only the transition from discoid to globular morphology ('shape change'). A secretion response is also observed on addition of a low concentration of ionophore A-23187 prior to orADP. These responses are not observed if ionophore A-23187 is added prior to the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ATP (oATP and or ATP) and are markedly inhibited by prior addition of the ADP antagonist, adenosine 5'-[beta, gamma-methylene]triphosphate. 2. The aggregation response to oADP in the presence of ionophore A-23187 is reduced but not eliminated by addition of 3 mM EGTA when studies are performed in heparinised platelet-rich plasma. Additions of 3 mM EGTA in citrated platelet-rich plasma, or of 4 mM EDTA in either system completely inhibits this response. Inhibitors which are reported to elevate the intracellular concentration of adenosine 3':5'-monophosphate (cyclic AMP) or to prevent Ca2+ movement also inhibit the aggregation response to oADP which is observed in the presence of ionophore A-23187. 3. Prior addition of inhibitors of adenylate cyclase fails to cause an aggregation response to subsequent addition of oADP or orADP. Certain of these inhibitors enhance and prolong the shape change response to oADP or orADP but only at concentrations an order of magnitude in excess of those required to antagonise inhibition by agents such as prostaglandin E1, which act by increasing the concentration of cyclic AMP. 4. The concentration of prostaglandin E1, adenosine or papaverine required to inhibit shape change induced by oADP is one to two orders of magnitude lower than that required to inhibit shape change induced by ADP. 5. Prior addition of oADP decreases the lag phase in the response of human platelets to arachidonate while also increasing the concentration required to observe half-maximal response, and causing a decrease in the extent of the response. Prior addition of oATP also diminishes the extent of this response and increases the concentration of arachidonate required but has no effect on the lag phase. 6. The data suggest that oADP and orADP are capable only of acting as partial agonists at the ADP receptor because of a defective ability to increase cytosolic Ca2+ concentration. The defect is rectified by the presence of low concentrations of ionophore A-23187, which promotes mobilisation of Ca2+ from an intracellular store. The results do not appear consistent with the thesis that a decrease in platelet cyclic AMP is an initiating event in aggregation induced by ADP, but do support a model which implicates cyclic AMP in depletion of cytosolic Ca2+.     

Effects of all CIS-5, 8,11,14,17 eicosapentaenoic acid and PGH3 on platelet aggregation

In human platelet-rich plasma (PRP) eicosapentaenoic acid (EPA) inhibited platelet aggregation induced by a stable analogue of PGH₂ (U46619), arachidonic acid, collagen or ADP. EPA was more potent than oleic, linoleic, α-linolenic or γ-linolenic acids. In aspirin-treated platelets, aggregation induced by U46619 was inhibited to a similar extent by arachidonic acid or by EPA over a range of concentrations of 0.05–0.3 mM. EPA incubated with PRP did not induce the generation of a thromboxane (TXA)-like activity; indeed it prevented the formation of TXA₂ induced by arachidonic acid or by collagen. The anti-aggregatory activity of EPA was not influenced by inhibitors of cyclo-oxygenase and lipoxygenase. The anti-aggregatory action of EPA may be caused by a rapid occupancy by EPA of TXA₂/PGH₂ “receptors” on platelet membrane as well as by a slower displacement of arachidonic acid from platelet phospholipids by chemically unchanged molecules of EPA.Not all samples of PRP were irreversibly aggregated by PGH₂, but in those that were, PGH₃ also induced an immediate dose-dependent but reversible aggregation. After a 4 min incubation of non-aggregating doses of PGH₂ or PGH₃ (100–300 nM) with PRP a stable anti-aggregatory compound was detected. The inhibitory activity produced from PGH₃ was apparently more potent (ca 10 times) than that obtained from PGH₂. The anti-aggregating compounds were identified by TLC and GLC-MS as PGD₂ and PGD₃. The apparent difference of potency between PGD₂ and PGD₃ was attributed to the concurrent production of PGE₂ and PGE₃. PGE₂ prevented the inhibitory effect of PGD₂ whereas PGE₃ did not affect the activity of PGD₃.It is concluded that one of the reasons for the low incidence of myocardial infarction in Eskimos could be that the pro-aggregatory arachidonic acid is replaced in their phospholipids by the anti-aggregatory EPA.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Cyclic adenosine 3',5'-monophosphate and prostacyclin inhibit membrane phospholipase activity in platelets.

[¹⁴C]-Arachidonic acid is incorporated mainly into phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of horse platelet membranes. Treatment of washed platelets with thrombin leads to a rapid loss of radioactivity from these phospholipids. The liberated [¹⁴C]-arachidonate is immediately transformed into hydroxyacids and thromboxanes. Treatment with dibutyryl cyclic AMP, cyclic AMP phosphodiesterase inhibitors or prostacyclin, a newly discovered prostaglandin that stimulates platelet adenylate cyclase, prevents the action of thrombin on phospholipid break-down as well as on platelet aggregation. Dibutyryl cyclic AMP does not affect the metabolism of exogenous [¹⁴C]-arachidonic acid. Cyclic AMP may thus play a crucial role in the regulation of platelet phospholipase acitivity, and this could explain at least in part the inhibition of aggregation caused by substances which, like prostacyclin, raise the levels of cyclic AMP.     

Cyclic AMP and platelet prostaglandin synthesis.

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3'5'-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

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.