Regulatory role of cyclic adenosine 3′,5′-monophosphate on the plattelet cyclooxygenase and platelet function

Thromboxane A₂ plays and important role in arachidonic acid- and prostaglandin H₂-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I₂, prostaglandin I₁, and prostaglandin E₁) and dibutyryl cyclic AMP inhibit both thromboxane A₂ formation and arachidonate-induced aggregation platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A₂ is still formed. Prostaglandin H₂-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A₂ production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cycyooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate mode for studying relationship between the platelet cyclooxygenase and platelet function.     

Acetyl glycerylphosphorylcholine inhibition of prostaglandin I2-stimulated adenosine 3',5'-cyclic monophosphate levels in human platelets. Evidence for thromboxane A2 dependence

Previous studies with AGEPC (1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) stress the independence of the proaggregatory activity of AGEPC from the platelet cyclooxygenase. However, our dose response analyses in human platelet-rich plasma show distinct primary and secondary waves of aggregation in response to AGEPC. Second wave aggregation is inhibited completely by either 10 micro M indomethacin, a cyclooxygenase inhibitor, or 5.6 micro M 9,11-azoprosta-5,13-dienoic acid, a thromboxane A2 synthetase inhibitor. Simultaneous addition of AGEPC and prostaglandin I2 to platelet-rich plasma results in a marked increase in platelet cyclic AMP, which is not different from the prostaglandin I2 response alone. However, if prostaglandin I2 is added to AGEPC-stimulated platelets at a point where secondary aggregation is just beginning, AGEPC can attenuate prostaglandin I2-stimulated cyclic AMP accumulation. The inhibition by AGEPC is blocked by either cyclooxygenase or thromboxane A2 synthetase inhibitors, and radioimmunoassay of thromboxane B2 confirmed that the inhibition of prostaglandin I2-stimulated cyclic AMP accumulation is due to thromboxane A2 synthesis, and that AGEPC-stimulated secondary aggregation does not start until thromboxane A2 is synthesized. These data suggest that much of the bioactivity of AGEPC is attributable to thromboxane A2.     

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 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.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to suppress aggregation, through the action of their respective non-enzymatic breakdown products PGE1, PGD2, or PGD3 all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cyclooxygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A2, and enhance formation of the anti-aggregatory metabolite, prostacyclin. Whereas EPA is not detectably metabolized by platelets, dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid) is primarily converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH1 and DLL which was partially compromised by the PGE1 formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH1. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A2.     

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation,on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function.Both analogues (Compound I, (15S)-hydroxy-9α,11α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid, and Compound II, (15S)-hydroxy-11α,9α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E₁ but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B₂ and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A₂ and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B₂ levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E₁. Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels.These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A₂ production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E₁.     

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.     

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase

It is generally agreed that arachidonic acid (20: 4 omega 6) can stimulate platelet aggregation after conversion to prostaglandin G2 and H2 and thence to thromboxane A2. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca2+-free buffer. Their stimulation by C 20: 4 6 was followed by 14C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca2+ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [14C]-5HT release. Other C 20 polyunsaturated fatty acids could not substitute for C 20: 4 omega 6 in inducing aggregation, and Ca2+ was found to be a prerequisite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use of the lipoxygenase inhibitor BW 755 C did not prevent C 20: 4 omega 6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these conditions, C 20: 4 omega 6-induced platelet aggregation might be due to an increased membrane permeability to Ca2+ induced by this fatty acid in the absence of oxidation.     

Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP.

It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2-diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+-calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i.     

9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogues: specific stable agonist (SQ 26, 538) and antagonist (SQ 26, 536) of the human platelet thromboxane receptor

A newly synthesized 9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26, 536, (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I50 value of 1.7 microM. SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH2 and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A50 value of 2.5 microM. SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a Ki value of 3 microM. Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH2-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.     

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid. Two selective thromboxane synthetase inhibitors

Two selective thromboxane A2 synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B2 and prostaglandin E2 accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B2 formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H2. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A2 formation with an accompanying, parallel, suppression of the platelet aggregation. Imidazole selectively suppressed thromboxane A2 formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H2 on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A2 formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A2 in arachidonate mediated platelet aggregation.     

Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: Pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to supress aggregation, through the action of their respective non-enzymatic breakdown products PGE₁, PGD₂, or PGD₃ all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cycloogygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A₂, and enhance formation of the anti-aggregatory metabolite, prostacyclin.Whereas EPA is not detectably metabolized by platelets, dihomo-γ-linolenic acid (8,11,14,-eicosatrienoic acid) is primariley converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH₁ and DLL which was partially compromised by the PGE₁ formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH₁. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A₂.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2 alpha

The inhibition of human platelet aggregation produced by PGF2 alpha is not specific for thromboxane A2 mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF2 alpha (8 microM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH2). In contrast the thromboxane receptor antagonist EP 045 (up to 20 microM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC50 = 0.5 microM), but not PGF2 alpha (28 microM), displaces the specific binding of [3H] 9,11-epoxymethano PGH2 to washed human platelets. PGF2 alpha produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF2 alpha is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF2 alpha. We suggest that the very weak effect of PGF2 alpha on cyclic AMP production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Prostaglandin E1 and forskolin antagonize C-kinase activation in the human platelet.

In human platelets, prostaglandin E1 and forskolin inhibit 5-hydroxytryptamine-induced phospholipase C, C-kinase and myosin light-chain kinase activity in a concentration-dependent way. Phospholipase C activation, however, was only partly inhibited, and this at higher concentrations than the protein kinases. Direct activation of the C kinase either by exogenous synthetic diacylglycerol or by 12-O-tetradecanoylphorbol 13-acetate was antagonized by prostaglandin E1 and forskolin. Since C-kinase activation is one of the key events in excitatory signal transduction in the platelets, we suggest that the inhibitory effect of agents that increase platelet cyclic AMP on platelet secretion and aggregation might reside in their capacity to antagonize C-kinase activity.     

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.     

Platelet aggregation. I. Regulation by cyclic AMP and prostaglandin E1

Platelet aggregation plays a major role in thrombogenesis. This study was undertaken to examine the inhibition of platelet aggregation induced by adenosine diphosphate. It is known that cyclic AMP (adenosine monophosphate) and its dibutyryl derivative inhibit platelet aggregation. This study showed that prostaglandin E1 (PGE1) also inhibits platelet aggregation and stimulates cyclic AMP synthesis by stimulation of adenyl cyclose. Caffeine, on the other hand, inhibits platelet phosphodiesterase, and increases cyclic AMP levels. PGA1 and PGF1 alpha can also inhibit platelet aggregation but only at very high concentrations.     

Prostaglandin I2 synthesis and elevation of cyclic AMP levels in 3T3 fibroblasts

Arachidonic acid and prostaglandin H2 elevate the levels of adenosine 3':5'-monophosphate (cyclic AMP) in Balb/c 3T3 fibroblasts. This effect was inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, an inhibitor of prostaglandin I2 synthase (Claesson, H.-E., Lindgren, J.A. and Hammarstr!om, S. (1977) FEBS Lett. 81, 415-418). After addition of arachidonic acid to 3T3 cultures, cellular cyclic AMP levels and growth medium concentrations of 6-ketoprostaglandin F1 alpha (degradation product of prostaglandin I2) were quantitatively determined. The stimulatory effect of exogenously-added prostaglandin I2 on cellular cyclic AMP levels was also determined. The results indicate that the endogenous production of prostaglandin I2 is sufficient to explain the stimulatory action of arachidonic acid on cyclic AMP formation in 3T3 fibroblasts.     

Lanthanum stimulates the accumulation of cyclic AMP and inhibits secretion and thromboxane B2 formation in human platelets

La3+ was found to inhibit the secretion of 5-hydroxytryptamine and the production of thromboxane B2 by washed platelets exposed to collagen or thrombin. In addition, La3+ inhibited secretion in response to sodium arachidonate, although the conversion of arachidonate to thromboxane B2 was not affected. La3+ was also found to enhance the accumulation of cyclic AMP under basal conditions and in response to prostaglandin E1, in washed platelets. The inhibition of cyclic AMP accumulation by ADP was prevented by La3+, suggesting that the effect of ADP on cyclic AMP metabolism was dependent upon the presence or flux of calcium at the platelet membrane. La3+ inhibited the activity of adenylate cyclase in platelet lysates both in response to prostaglandin E1 and to F-, indicating a possible effect at the catalytic subunit of the enzyme. None of the observed effects of La3+ could be reversed by the addition of Ca2+ up to 10 mM. The stimulation of cyclic AMP production by La3+ may largely explain the inhibitory effect of La3+ upon platelet secretion and thromboxane B2 production. These results also suggest that Ca2+ localised at the platelet plasma membrane may be important in the regulation of cyclic AMP metabolism.     

9α-homo-9,11-epoxy-5,13-prostadienoic acid analogues: Specific stable agonist (SQ 26,538) and antagonist (SQ 26,536) of the human platelet thromboxane receptor

A newly synthesized 9α-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26,536, (8(R)9(S)11(R)12(S)-9α-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I₅₀ value of 1.7 μ . SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH₂ and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9α-homo-9-, 11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A₅₀ value of 2.5 μ . SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a K_i value of 3 μ . Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH₂-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.