CV-3988 - a specific antagonist of platelet activating factor (PAF)

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.     

Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists

The platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and its analogs were examined to determine their effects on guinea pig peritoneal macrophages. PAF activated macrophages, but its effect on macrophages was much weaker than that observed on platelets: the concentration required for 50% maximum activation was 8.5 X 10(-6) M for macrophages and 2.9 X 10(-10) M for platelets. Three PAF agonists, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-glycero-3-phosphocholine (Compound I), 1-O-octadecyl-2-acetamido-2-deoxy-glycero-3-phosphocholine (Compound II), and 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Compound III), showed higher activity in stimulating macrophage function than PAF. The abilities of these non-metabolizable PAF agonists to activate macrophage paralleled their relative potency to induce platelet activation. The sn-3 enantiomers of PAF and Compound III exhibited activity, while the sn-1 did not. By comparing the activities of derivatives of Compound III, it was shown that the long-chain alkyl-ether group in the glycerol-1 position, a relatively small size of the substituent on the hydroxy group at the sn-2 position, and the choline moiety in the glycerol-3 position must play critical roles in the process of macrophage activation. A specific PAF antagonist, CV3988, which inhibits PAF-induced platelet activation and hypotension, inhibited the activation of macrophages caused by PAF and its agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Effect of prostacyclin on platelet-activating factor induced rabbit and platelet aggregation

In this paper, the effect of prostacyclin (PGI2) on the aggregation induced by Platelet-activating factor (PAF), a phospholipid mediator of anaphylaxis, was studied. Synthetic PGI2 and PGI2-like activity generated from rabbit aorta were demonstrated to be effective inhibitors of PAF-induced rabbit platelet aggregation and release of 3H-serotonin (3H-5HT).     

Dual effects of oleic acid on Ca2+ mobilization and protein phosphorylation in human platelets in presence or absence of platelet activating factor.

This laboratory demonstrated earlier that oleic acid inhibited platelet activating factor (PAF)-induced aggregation and serotonin release of rabbit platelets (M. Miwa, C. Hill, R. Kumar, J. Sugatani, M. S. Olson, and D. J. Hanahan, 1987, J. Biol. Chem. 262, 527-530). More recently, we reported that oleic acid caused a decrease in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), but did not affect the level of inositol-1,4,5-trisphosphate (IP3), in rabbit platelets (D. Nunez, J. Randon, C. Gandhi, A. Siafaka-Kapadai, M. S. Olson, and D. J. Hanahan, 1990, J. Biol. Chem. 265, 18330-18838). These results suggested that oleic acid did not stimulate phospholipase C. In contrast, PAF induced a decrease in PIP2 and an increase in PIP level and IP3. These effects were shown to be attenuated by oleic acid. In this current study, our experiments show that (a) oleic acid blocked PAF-induced rise in intracellular [Ca2+] (to provide a mechanism in agreement with our previous experiments which showed that oleic acid inhibited PAF-induced IP3 rise in platelets) and (b) oleic acid itself induced a gradual rise in [Ca2+]i, which would provide a mechanism for oleic acid-induced aggregation despite the fact that oleic acid did not cause the production of IP3 (Nunez et al., 1990). Oleic acid, in a dose-dependent manner, was shown to inhibit PAF-induced Ca2+ mobilization from intra- and extracellular sources. The inhibition was closely related to the suppressive effect of oleic acid on PAF-induced aggregation. Furthermore, oleic acid inhibited the PAF-stimulated phosphorylation of the 20- and 40-kDa proteins. At concentrations above 20 microM, oleic acid itself could induce platelet aggregation and Ca2+ mobilization, but the time sequence of these two responses in human platelets was significantly different from those obtained with PAF. Oleic acid alone, at 20 microM, caused a 1.4-fold increase in the cAMP level in platelets which was followed by a decline to a basal value at higher concentrations of this fatty acid. It seemed clear that elevation of adenylate cyclase activity was not associated with free fatty acid inhibition of platelet activation. Interestingly, both PAF and oleic acid added separately to human platelets induced protein-tyrosine phosphorylation, but oleic acid did not cause any inhibition of PAF-induced protein-tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanisms of platelet activating factor-induced aggregation and secretion in human platelets

The role of TXA2 in PAF-induced aggregation and secretion of human platelets is unclear. We have studied the relationship between aggregation, synthesis of TXA2 and release of 5-HT during the time course of aggregation induced by PAF and collagen. For PAF-induced aggregation there was strong aggregation and secretion with minimal production of TXA2 in contrast to collagen in which a surge in TXA2 synthesis preceded both aggregation and secretion. To determine the role of calcium flux in PAF-induced aggregation we have similarly studied the temporal relationships between aggregation, secretion and TXA2 synthesis for calcium ionophore A23187 induced aggregation but found these to be distinctly different from those determined for PAF. A method for measuring absolute amounts of 5HT released from platelets in small volumes of plasma is described. We conclude that TXA2 is not important in the mechanism of PAF induced aggregation and that an increase in the level of intraplatelet calcium per se is not sufficient to explain the mediation of PAF-induced aggregation.     

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.     

trans-2,5-Bis-(3,4,5-trimethoxyphenyl)tetrahydrofuran. An orally active specific and competitive receptor antagonist of platelet activating factor

trans-2,5-Bis(3,4,5-trimethoxyphenyl)tetrahydrofuran (L-652,731) is found to be a potent and orally active platelet activating factor (PAF)-specific and competitive receptor antagonist. It potently inhibits [3H]PAF (1 nM) binding to receptor sites on rabbit platelet membranes with an ED50 of 2 X 10(-8) M under the assay condition without the addition of mono- or divalent cations. In a comparative study, it is more potent than CV-3988, kadsurenone, and ginkgolide B as a receptor antagonist. The equilibrium dissociation constants (KB) of L-652,731 obtained either from the inhibition of receptor binding or from the inhibition of PAF-induced aggregation of gel-filtered rabbit platelet are 2.7 X 10(-8) and 2.1 X 10(-8) M, respectively. The agreement of these KB determinations based on receptor and cellular function suggests that L-652,731 does not inhibit other steps following PAF-receptor binding. L-652,731 does not antagonize the binding of several radioligands to their respective receptor. It shows no inhibitory effect on platelet aggregation induced by other aggregating agents including thrombin, collagen, A-23187, arachidonic acid, epinephrine, and ADP. L-652,731 is orally active; it inhibits PAF-induced rat cutaneous vascular permeability with an ED50 of 30 mg/kg orally. Significant inhibitory results of L-652,731 suggest that PAF may be partially involved in cutaneous vascular permeability induced by histamine and bradykinin.     

The inhibition of platelet-activating factor-induced platelet activation by oleic acid is associated with a decrease in polyphosphoinositide metabolism

In an earlier study (Miwa, M., Hill, C., Kumar, R., Sugatani, J., Olson, M. S., and Hanahan, D. J. (1987) J. Biol. Chem. 262, 527-530) it was shown that an inhibitor of platelet-activating factor (PAF), a powerful endogenous mediator of platelet aggregation, was present in freeze-clamped perfused livers. Subsequently, we determined that this substance was a mixture of unsaturated free fatty acids (FFA). Among these FFA, oleic acid between 10 and 100 microM was found to be a potent inhibitor of PAF-induced platelet aggregation and serotonin secretion. Consequently, in order to understand the molecular mechanism of oleic acid action, we investigated the effects of this FFA on several biochemical events associated with platelet aggregation induced by PAF. The effect of oleic acid and/or PAF on the level of [32P]phosphatidylinositol 4-phosphate (PIP) and [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) was examined by using platelets labeled with [32P]phosphate. Oleic acid induced a dose-dependent decrease in the levels of [32P]PIP and [32P]PIP2; a maximal decrease in [32P]PIP and [32P]PIP2 of approximately 50 and 25%, respectively, was observed within seconds after the addition of 20 microM oleic acid and persisted for at least 15 min. Oleic acid did not induce the formation of [3H]inositol phosphates in platelets prelabeled with [3H]inositol, suggesting that the decrease in [32P]PIP and [32P]PIP2 was not due to a stimulation of phospholipase C. In contrast to oleic acid, PAF induced a dose-dependent increase in the [32P]PIP level, reaching a maximum of approximately 200% 3 min after the addition of 1 nM PAF to the platelets. This increase in [32P]PIP was accompanied by platelet aggregation and secretion, and a close correlation was established between the [32P]PIP level and the degree of aggregation. Oleic acid and PAF, when added together to the platelets, interacted by affecting the level of [32P]PIP and [32P]PIP2 in an opposite way since the decrease in the level of [32P]PIP and [32P] PIP2 induced by oleic acid was partially reversed by an excess of PAF. The decrease in the levels of [32P] PIP and [32P]PIP2 caused by oleic acid was associated with an inhibition of platelet aggregation induced by PAF. Interestingly, oleic acid did not block [3H]PAF binding to platelets but inhibited the PAF-induced phosphorylation of platelet proteins of 20 kDa and 40 kDa. These results suggest that inhibition of the PAF response by oleic acid may be at one of the steps in the signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)     

The benzodiazepine receptor ligands RO 5-4864 and RO 15-1788 do not block the inhibition of PAF-induced platelet aggregation seen with the hetrazepine WEB2086

The hypothesis was tested that the hetrazepine WEB 2086 acts as an inhibitor of PAF-induced platelet aggregation via interaction with the platelet benzodiazepine receptor(BDZR). WEB 2086 is a potent inhibitor of rabbit platelet aggregation and ATP secretion induced by 370 nM PAF. The two BDZR ligands RO 5-4864 and RO 15-1788 (7-96 microM) are inactive as PAF antagonists. When platelets were pretreated with either BDZR ligand, and then exposed to various concentrations of WEB 2086, there was no alteration of the dose-response relationship of the hetrazepine on PAF-induced aggregation, as reflected by threshold concentration, ED50, or maximum inhibition seen with WEB 2086. Pretreatment of platelets with the BDZR ligands also failed to block the inhibitory action of WEB 2086 on PAF-induced ATP release. The data are consistent with the notion that WEB 2086 acts as a PAF antagonist through its action at a specific PAF receptor, and is dissociated from, and independent of, interaction with the benzodiazepine receptor.     

Pharmacological actions of Y-24180, a new specific antagonist of Platelet Activating Factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of ³H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC₅₀ 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC₅₀ values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC₅₀ 0.84 nM) was more potent than WEB 2086 (IC₅₀ 4.21 nM) and etizolam (IC₅₀ 998 nM). Y-24180, WEB 2086 and etizolam displaced ³H-PAF binding from the washed-platelets of rabbits with an IC₅₀ value of 3.50, 9.35 and 29.5 nM, respective- ly. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180(1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced ³H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 μ M. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

A comparison of the inhibitory effects of prostacyclin and carbacyclin on platelet adhesion to collagen

The effect of carbacyclin, a chemically stable analogue of prostacyclin (PGI2), on the adhesion of platelets to collagen has been examined. The compound was compared to PGI2 which is unstable and rapidly hydrolysed to the inactive derivative, 6-oxo-PGF 1 alpha. The adhesion of 111Indium-labelled human platelets to collagen in the absence of platelet aggregation and secretion was measured. The cAMP level in the platelets was also monitored. Both PGI2 and carbacyclin inhibited platelet-collagen adhesion and caused a rise in the platelet cAMP level. Carbacyclin was approximately 15-fold less effective than PGI2, however, its effect was longer lasting, remaining constant for at least 30 minutes.     

Modulating effects of adenosine on the process of human platelet activation]

The inhibitory effect of adenosine on aggregation of human platelets activated by platelet activating factor (PAF), ADP and serotonin (5-HT) were examined using native platelets from blood of volunteers. Platelet aggregation was determined by Born's method. Effective adenosine concentrations (IC50) which had inhibited platelet aggregation were found to be 0.63 +/- 0.11, 1.47 +/- 0.31 and 0.64 +/- 0.18 microM, respectively. It was shown that 10 microM adenosine inhibited PAF-induced platelet aggregation completely. The same adenosine concentration blocked ADP- and 5-HT-induced aggregation only partially. Adenosine is physiological inhibitor of human platelet aggregation in administration of PAF, ADP and 5-HT. Specific characteristics of adenosine modulating effect on these ligands was elicited.     

Recycling of platelet phosphorylation and cytoskeletal assembly

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.     

Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets.

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Inhibition of Human Platelet Aggregation by Amides and Ester of Salicylic Acid with Platelet-Activating Factor Analogs

The influence of acetyl salicylic acid (ASA) derivatives with platelet-activating factor (PAF) lipid analogs on PAF-induced human platelet aggregation has been studied. It was found that the ASA amide with an ethanolamine plasmalogen PAF analog (1-0-alk-1"-enyl-2-acetyl-sn-glycero-3-phospho-(N-2"-acetoxybenzoyl)ethanolamine) and the ASA ester with a choline plasmalogen PAF analog (1-0-alk-1"-enyl-2-(2"-acetoxybenzoyl)-sn-glycero-3-phosphocholine) at concentrations of 10^–7-10^–6 M effectively inhibit PAF-induced aggregation of human platelets. In contrast to these compounds, the ASA amide with an alkyl PAF analog (1-0-alkyl-2-acetyl-sn-glycero-3-phospho-(N-2"-acetoxybenzoyl)ethanolamine) did not inhibit PAF-induced platelet aggregation. As possible mechanisms of action of the studied compounds, the blockade of PAF-receptor and cyclooxygenase inhibition are proposed.     

A role for phosphatidylinositol 3-kinase in platelet aggregation in response to low, but not high, concentrations of PAF or thrombin

In this study we show that platelet activating factor (PAF) activates PI 3-kinase over a rapid time course that correlates closely with the aggregation response. Tyrosine kinases are involved in this response, since there is increased PI 3-kinase activity associated with tyrosine-phosphorylated proteins. PI 3-kinase inhibitors were used to probe the dependence of PAF-induced aggregation on PI 3-kinase. Both wortmannin and LY-294002 inhibited PAF-induced aggregation that correlated with PI 3-kinase inhibition only when using lower concentrations of PAF giving reversible aggregation (primary phase). Similar results were obtained with human platelets using thrombin or thrombin receptor activating peptide. The same pattern of response was observed when activation of GPIIbIIIa was assessed by flow cytometry, i.e., PI 3-kinase inhibitors blocked integrin activation only when lower concentrations of agonist were used. We suggest that PI 3-kinase is important for reversible (primary) aggregation of platelets in response to PAF or thrombin, perhaps by contributing to the 'inside-out' activation of the platelet integrin GPIIbIIIa, only when submaximal concentrations of agonists are used. The lack of effect of PI 3-kinase inhibitors, when high concentrations of agonist are used, suggests that PI 3-kinase-independent pathways contribute to aggregation under these conditions.