Carnitine inhibits arachidonic acid turnover,platelet function,and oxidative stress

Carnitine is a physiological cellular constituent that favors intracellular fatty acid transport, whose role on platelet function and O(2) free radicals has not been fully investigated. The aim of this study was to seek whether carnitine interferes with arachidonic acid metabolism and platelet function. Carnitine (10-50 microM) was able to dose dependently inhibit arachidonic acid incorporation into platelet phospholipids and agonist-induced arachidonic acid release. Incubation of platelets with carnitine dose dependently inhibited collagen-induced platelet aggregation, thromboxane A(2) formation, and Ca(2+) mobilization, without affecting phospholipase A(2) activation. Furthermore, carnitine inhibited platelet superoxide anion (O(2)(-)) formation elicited by arachidonic acid and collagen. To explore the underlying mechanism, arachidonic acid-stimulated platelets were incubated with NADPH. This study showed an enhanced platelet O(2)(-) formation, suggesting a role for NADPH oxidase in arachidonic acid-mediated platelet O(2)(-) production. Incubation of platelets with carnitine significantly reduced arachidonic acid-mediated NADPH oxidase activation. Moreover, the activation of protein kinase C was inhibited by 50 microM carnitine. This study shows that carnitine inhibits arachidonic acid accumulation into platelet phospholipids and in turn platelet function and arachidonic acid release elicited by platelet agonists.     

Beta-adrenergic inhibition of AGEPC-stimulated Na+/Ca2+ exchange and AGEPC-induced platelet activation

Recently, AGEPC (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was found to initiate contraction of ileal smooth muscle strips and to enhance Na+/Ca2+ exchange in ileal plasmalemmal vesicles. In the present study, the effects of the smooth muscle relaxant, isoproterenol, on Na+/Ca2+ exchange in rat ileal plasmalemmal vesicles was examined. In this preparation, Na+/Ca2+ exchange was stimulated 131 +/- 8% and 264 +/- 19% by addition of 50 nM and 100 nM AGEPC, respectively. Isoproterenol, a beta-adrenergic agonist, inhibited AGEPC stimulation of Na+/Ca2+ exchange in a dose- and time-dependent manner but had no effect on basal rates of Na+/Ca2+ antiport. At 1 microM, isoproterenol inhibited 86% of the Na+/Ca2+ exchange stimulated by 50 nM AGEPC. Vesicular cAMP levels were increased over 100% following the addition of 1 microM isoproterenol for 30 s. Inhibition of AGEPC-stimulated vesicular Na+/Ca2+ exchange and elevation of vesicular cAMP levels by isoproterenol was prevented by the beta-receptor antagonist propranolol (5 microM), demonstrating that these effects of isoproterenol were mediated by interaction with vesicular beta-adrenergic receptors. Additional studies with washed rabbit platelets demonstrated that isoproterenol inhibited AGEPC-induced aggregation and serotonin release. These effects of isoproterenol were dose- and time-dependent and were antagonized by propranolol. Isoproterenol had no effect on thrombin-induced aggregation and did not change appreciably platelet cAMP levels. Moreover, dibutyryl cAMP could not mimic the effect of isoproterenol to inhibit an AGEPC-induced aggregation. On a molar basis, the inhibitory effects of isoproterenol toward AGEPC action were greater in the ileal preparation than in the platelets. It is suggested that beta-adrenergic agonists may modulate AGEPC-induced ileal Na+/Ca2+ exchange and AGEPC-induced platelet aggregation through cAMP-dependent and-independent mechanisms, respectively.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C

Thrombin and trypsin induce serotonin release and aggregation in human platelets. Both proteases induce activation of phospholipase C as reflected by formation of inositol phosphates and phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Also, thrombin and trypsin activate protein kinase C and myosin light chain kinase as indicated, respectively, by phosphorylation of the 40,000 and 20,000 dalton proteins. Leupeptin, a known inhibitor of serine proteases, blocks all the observed responses of human platelets to trypsin and thrombin. Leupeptin does not inhibit serotonin release and aggregation induced by other platelet stimuli such as collagen, platelet-activating factor, ionophore A23187, and arachidonic acid. The implication of a proteolytic-mediated pathway in the transmembrane signalling involved in platelet activation is discussed.     

Effect of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine on calcium fluxes by human platelet microsomes

Under conditions where optimal concentrations of arachidonic acid, phosphatidic acid, or the calcium ionophore A23187 caused release of 50-95% of calcium from preloaded platelet microsomes, basophil platelet activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) did not cause the release of calcium at concentrations as high as 2 X 10(-5) M. The failure to stimulate calcium release was not due to metabolism or inactivation of AGEPC. These results show that AGEPC is not a calcium ionophore and is unable to directly effect the release of calcium from microsomes by mechanisms other than ionophoric action. The increase in intracellular levels that occurs during AGEPC-induced platelet aggregation must be an indirect effect of the AGEPC.     

A preferential inhibition by Zn2+ on platelet activating factor- and thrombin-induced serotonin secretion from washed rabbit platelets

Zinc ions at micromolar levels exhibited a significant inhibitory activity toward platelet activating factor (AGEPC)- and thrombin-induced serotonin release from washed rabbit platelets. In the ranges from 25 to 30 microM and 10 to 50 microM, respectively, zinc essentially prevented any serotonin release from 1.25 X 10(8) cells/microliter by 1 X 10(-10) M AGEPC and by 0.2 unit thrombin/ml. This inhibition by zinc ions, in micromolar range, occurred in the presence of 1.0 mM Ca2+. The amount of zinc needed for inhibition was inversely proportional to the amount of AGEPC present and further zinc must be added prior to or at the same time as the AGEPC to be effective. Introduction of zinc ions after the AGEPC essentially abolished the inhibitory properties of this divalent cation. Other cations such as Cu2+, La3+, Cd2+, and Mg2+ were ineffective as inhibitors at concentrations where zinc showed its maximal effects. Under conditions similar to those noted above, aggregation induced by AGEPC was blocked only to the extent of 25% of a control. No inhibitory action by zinc on thrombin-induced aggregation was noted. It is apparent that zinc ions influence a site(s) on the rabbit platelet of considerable importance to the activation (or signaling) process by AGEPC and thrombin in these cells, as expressed by serotonin release. Zinc should provide a suitable probe to explore the mechanism of action of these agonists in their interaction with sensitive cells and to define in more specific biochemical terms the putative receptor for these molecules.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

The activation by Ca2+ of platelet phospholipase A2. Effects of dibutyryl cyclic adenosine monophosphate and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate.

Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt2 cyclic AMP) or with 0.6 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt2 cyclic AMP. The addition of external Ca2+ to thrombin-treated platelets incubated with Bt2 cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt2 cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca2+. Inhibition of arachidonic acid liberation by Bt2 cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg2+ is substituted for Ca2+, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt2 cyclic AMP is slightly enhanced. The phospholipase A2 activity of platelet lysates is increased by the presence of added Ca2+, however, the addition of either A23187 or Bt2 cyclic AMP is without effect on this activity. We suggest that Bt2 cyclic AMP may promote a compartmentalization of Ca2+, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca2+ stores in situ or restrict access of Ca2+ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore.     

Human platelet electrorotation change induced by activation: inducer specificity and correlation to serotonin release

Electrorotation of single platelets was compared with [14C]serotonin release, aggregation and electron microscopy. Activation of washed and degranulated platelets was induced by thrombin, arachidonic acid, collagen, adrenaline, platelet activation factor (PAF), ADP and A23187. A strong correlation between electrorotation decrease and serotonin release was found. Electrorotation did not correlate with aggregation. It was concluded that an increase of the specific conductivity of the platelet membrane by three orders of magnitude (approx. 1.0.10(-7) S.m-1 to 1.0.10(-4) S.m-1) upon activation was responsible for the observed decrease of anti-field rotation and the shift of the first characteristic frequency towards higher values. Electrorotation allowed for time-dependent measurements of activation. Characteristic activation times in the order of minutes were found. There was the following sequence of activators classified by increasing activation time constants: A23187 was the fastest followed by thrombin, collagen, PAF, arachidonic acid, adrenaline, and ADP.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Modulation of cytoplasmic calcium in human platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine

The calcium-sensitive, fluorescent dye Quin 2 was used to quantitate changes in free intracellular calcium [( Ca2+]i) induced in platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). The Ca2+]i of unstimulated platelets was 91 +/- 18 nM (mean +/- SD, n = 8), and treatment with 1 to 16 nM AGEPC increased [Ca2+]i in a dose-related manner, with 16 nM AGEPC increasing [Ca2+]i by 102 +/- 20 nM. [Ca2+]i was not increased by analogs of AGEPC which do not activate platelets including the lysophospholipid precursor of AGEPC, the optical isomer, and a C-2 benzoyl analog. The capacity of AGEPC to increase [Ca2+]i exceeded that required to induce maximal platelet aggregation. In four experiments, 100% platelet aggregation was induced by 4.5 +/- 2.4 nM AGEPC (mean +/- SD) and was associated with a submaximal increase in [Ca2+]i of 56 +/- 22 nM. Pretreatment of platelets with AGEPC rendered the platelets specifically unresponsive to repeat stimulation with AGEPC in terms of both platelet aggregation and increased [Ca2+]i, whereas the platelet response to thrombin was undiminished by pretreatment with AGEPC. In contrast, the platelet response to 0.5 microM calcium ionophore A23187 was undiminished by pretreatment with the same concentration of ionophore, suggesting that AGEPC does not activate platelets by an ionophore-like mechanism. IgG aggregates and AGEPC in combination activate platelets synergistically, as shown by the observation that a 1-min exposure of platelets to 60 micrograms/ml of IgG aggregates increased the platelet aggregation response to 2 nM AGEPC from 44 to 100%. In contrast, sequential exposure of platelets to IgG aggregates and AGEPC increased [Ca2+]i additively, suggesting that increased [Ca2+]i contributes to but does not fully mediate synergistic platelet activation by IgG aggregates and AGEPC. Quantitation of free intracellular calcium with the fluorescent dye Quin 2 is a highly sensitive technique for delineating the role of calcium in mediating platelet activation.     

Inhibiting action of anti-arrhythmia agents of the phenothiazine series--etmozin and its diethylamino analog--on platelet aggregation and metabolism of endogenous arachidonic acid

Effect of the cardiotropic drugs of the phenothiazine series ethmozine, and its diethylamine analogue (DAAE), on platelet aggregation and formation of arachidonic acid metabolites has been studied. Both drugs inhibit the ADP-induced aggregation in the platelet-rich plasma. Ethmozine inhibits only the second (irreversible) wave of aggregation, while DAAE inhibits both the first (reversible) and the second one. 50% inhibition (ID50) of the second wave of aggregation is observed at the following concentrations of the two agents: 300-500 micrograms/ml (ethmozine) and 20 micrograms/ml (DAAE). DAAE completely inhibits the irreversible aggregation of platelets washed off plasma, induced by arachidonic acid (ID50 approximately 30 micrograms/ml) and Ca2+-ionophore A23187 (ID approximately 55 micrograms/ml); the aggregation, induced by thrombin is inhibited by 80-90% (ID approximately 130 micrograms/ml). Formation of arachidonic acid metabolites in platelets effected by these inducers was measured by the accumulation of malondialdehyde (MDA). DAAE fails to inhibit MDA formation induced by exogenous arachidonic acid, but completely prevents the synthesis of MDA induced by A23187 and thrombin. These data suggest that DAAE inhibits the release of endogenous arachidonic acid from membrane phospholipids catalysed by phospholipase A2, but does not affect its subsequent metabolic transformations. In all probability, ethmozine and DAAE, just as other phenothiazines, affect platelets via the inhibition of Ca2+-calmodulin-dependent reactions and processes.     

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.     

Platelet activation by tetraprenol via stimulation of phospholipase A2 action

Only tetraprenol (n = 4), among the (n)-polyprenols studied, induced activation of rabbit platelets. Tetraprenol-induced responses, including platelet aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release, were greatly inhibited by a thromboxane A2 (TXA2) receptor antagonist and a cyclooxygenase inhibitor, indicating an essential role for endogenously produced TXA2. The TXA2-mimetic agonist U46619 induced platelet aggregation, Ca2+ mobilization and phospholipase C action but did not induce arachidonic acid release. These results suggest that arachidonic acid is not released via phospholipase C but by phospholipase A2, and this is also supported by the finding that phospholipase C action was inhibited by depletion of extracellular Ca2+, while arachidonic acid release was not. Full arachidonic acid release was found to be induced by the synergistic action of U46619 and tetraprenol. Therefore, the initial, most essential response induced by tetraprenol is a small arachidonic acid release by phospholipase A2, which results in initial TXA2 formation. Further action of phospholipase C as well as Ca2+ mobilization and aggregation were induced by the initially formed TXA2 while further activation of phospholipase A2 required the synergistic action of tetraprenol and TXA2.     

The role of phospholipase C in platelet responses

Degradation of inositides induced by phospholipase C in activated platelets leads to the formation of 1,2-diacylglycerol (1,2-DG) and its phosphorylated product, phosphatidic acid (PA). We have studied the relationship between activation of phospholipase C and the appearance of specific platelet responses, such as phosphorylation of proteins, shape change, release reaction and aggregation induced by different stimuli such as thrombin, platelet-activating factor, collagen, arachidonic acid (AA) and dihomogamma linolenic acid. A low degree of platelet activation induces only shape change which is associated with partial activation of phospholipase C (formation of phosphatidic acid), and phosphorylation of both a 40K molecular weight protein (protein kinase C activation) and a 20K molecular weight protein (myosin light chain). A higher degree of platelet activation induces aggregation, release of serotonin and a higher level of phospholipase C and protein kinase C activities. Metabolism of AA occurs concomitantly to aggregation and serotonin release, but AA metabolites are not related to the shape change of human platelets. Platelet shape change and the initial activation of phospholipase C induced by thrombin or platelet-activating factor is independent of the metabolites derived from cyclo-oxygenase activity. Further activation of phospholipase C which occurs during platelet aggregation and release reaction is, however, partly dependent on cyclo-oxygenase metabolites.     

KRDS, a new peptide derived from human lactotransferrin, inhibits platelet aggregation and release reaction

KRDS (Lys-Arg-Asp-Ser), a tetrapeptide from human lactotransferrin, was tested in vitro on human platelet function, and its effects were compared to those of RGDS, a tetrapeptide from human fibrinogen. Both peptides had a high probability of initiating a beta-turn and were highly hydrophilic. KRDS inhibited ADP-induced platelet aggregation [median inhibitory concentration (IC50) 350 microM] and fibrinogen binding (IC50 360 microM) to a lesser extent than RGDS (IC50 75 microM and 20 microM, respectively). Different from RGDS, thrombin-induced serotonin release was inhibited by KRDS (750 microM) on normal platelets (55 +/- 10%) and type I Glanzmann's thrombasthenia platelets (43% +/- 1). However, KRDS had no effect on cytoplasmic Ca2+ mobilization, inositol phospholipid metabolism or protein phosphorylation (myosin light chain P20 and P43). In contrast to RGDS, KRDS does not inhibit the binding of monoclonal antibody PAC-1 to activated platelets. KRDS and RGDS inhibited 4 beta-phorbol-12-myristate-13-acetate (PMA)-induced aggregation and fibrinogen binding, while proteins were normally phosphorylated. Thus, the tetrapeptide KRDS is (a) an inhibitor of serotonin release by a mechanism independent of protein phosphorylation and (b) an inhibitor of fibrinogen binding and, hence, aggregation by a mechanism that may not necessarily involve its direct binding to the glycoprotein IIb-IIIa-complex.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Shape change induced in human platelets by platelet-activating factor. Correlation with the formation of phosphatidic acid and phosphorylation of a 40,000-dalton protein

Washed human platelets that have been separated from plasma in the presence of prostacyclin are activated by the addition of platelet activating factor (PAF). Activation (shape change, serotonin release, and aggregation) correlates closely with the formation of phosphatidic acid and the phosphorylation of a 40,000-dalton protein. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation precede aggregation and are induced at lower concentrations of PAF than those required to induce release of serotonin and platelet aggregation. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation induced by PAF are not affected by trifluoperazine or indomethacin. This indicates that these responses are independent of the liberation of arachidonic acid from platelet phospholipids and the metabolism of arachidonic acid via cyclooxygenase and lipoxygenase. These responses are, however, inhibited by prostacyclin. Platelet shape change is the first measurable physiologic response to platelet agonists and may be associated with the stimulation of phospholipase C, inducing formation of 1,2-diacylglycerol and its phosphorylated product, phosphatidic acid. Transient formation of 1,2-diacylglycerol may also induce the specific activation of the protein kinase C that phosphorylates a 40,000-dalton protein.     

Defining the role of protein kinase c in calcium-ionophore-(A23187)-mediated activation of phospholipase A2 in pulmonary endothelium.

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.     

Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans

Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasma. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerythrine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-loaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by chelerythrine chloride was easily recovered after sedimenting the platelets by centrifugation and then the platelet pellets were resuspended. Chelerythrine chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inhibitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on thromboxane formation and phosphoinositides breakdown.