Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

High concentrations of arachidonic acid induce platelet aggregation and serotonin release independent of prostagladin endoperoxides and thromboxane A2

We examined platelet aggregation and serotonin release, induced by less than 60 μM arachidonic acid, using washed platelet suspensions in the absense of albumin. The concentration of arachidonic acid use did not cause platelet lysis. Platelet responses induced by less than 20 μM arachidonic acid were inhibited by aspirin, whereas those induced by above 30 μM arachidonic acid were not inhibited, even by both aspirin and 5,8,11,14-eicosatetraynoic acid. Although phosphatidic acid and 1,2-diacylglcerol increased after the addition of arachidonic acid in aspirin-treated platelets, the amounts were not parallel to platelet aggregation. Oleic, linoleic and linolenic acids also induced platelet responses, while palmitic, stearic and arachidic acids did not. EDTA, dibutyryl cyclic AMP, apyrase and creatine phosphate / creatin phosphokinase brought about almost the same effects in platelet responses induced by the unsaturated fatty acids, other than arachodinic acid, as those induced by 40 μM arachodonic acid. These results suggest that the mechanism of the actions of more than 30 μM arachodinic acid on platelets is the same as that of the other unsaturated fatty acids and is independent of prostaglandin endoperoxides, thromboxane A₂ and, perhaps, phosphatidic acid and 1,2-diacylglycerol.     

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.     

Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine

The inhibitory effects of saturated fatty acids with 4 to 18 carbon atoms on ADP-induced aggregation of bovine platelets were investigated. The inhibitory effects of the acids increased with increase of their alkyl chain length up to C14. On the other hand, from C16 the inhibitory effects tended to decrease with increase of chain length, and stearic acid (C18) was not inhibitory. There was a linear relationship between the inhibitory effects and alkyl chain lengths up to C12. This linear relation and the slope of the linear regression line suggested that the inhibitory effects of the acids depended on their partition into the membrane. The fatty acids decreased the fluorescence of the surface charge probe 2-p-toluidinylnaphthalene-6-sulfonate, indicating that they increased the negative charge on the membrane surface. The relative effects of the acids on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that the inhibition of platelet aggregation by saturated fatty acids is due to a change in the membrane surface charge of the platelet plasma membrane.     

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.     

Effects of dietary polyunsaturated fatty acid on platelet aggregation in rats

In the present study, we changed the fatty acid profile in blood and platelet membranes by dietary manipulation, and examined the effect on platelet aggregation in rats. Fifty-five rats were divided into five groups and fed for 56 days with 1% cholesterol and different types of fatty acid-rich diets: basal, lard, lard + fish oil, soybean oil, and soybean oil + fish oil. a decrease in serum arachidonic acid (20:4, omega-6, AA) and an increase in serum eicosapentaenoic acid (20:5, omega-3, EPA) were found in all experimental dietary groups fed with refined fish oil. Similar changes in the polyunsaturated fatty acids were also found in the platelet membrane phospholipids. Platelet aggregation, quantitated by the slope and height of the aggregation curve induced by different concentrations of ADP in a platelet aggregometer, was inhibited in all groups fed with refined fish oil. This inhibition of platelet aggregation may be related to an increase in the ratio of EPA and AA in the platelet membrane phospholipids after dietary manipulation. The differences in the platelet aggregation and thromboxane B2 (TXB2) concentration between the lard and the lard + fish oil groups were more profound than that between the soybean oil and the soybean oil + fish oil group. However, the malondialdehyde (MDA) concentration revealed no significant differences between the five groups.     

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.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.     

ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationship between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation

Four spin-labeled probes (5-doxylstearic acid (5-NS), its methyl ester (5-NMS), 16-doxylmethylstearate (16-NMS) and 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl (CAT-15)) were used to monitor membrane fluidity change in bovine platelets induced by three alkyl alcohols, benzyl alcohol and two phenolic compounds. The relationship between the increase in membrane fluidity induced by these compounds and their inhibitory effects on platelet aggregation was observed. Experiments with the four probes showed that n-hexyl alcohol induced decreases in the order parameter of 5-NS and apparent rotational correlation times of the other probes at the same minimal alcohol concentration. The decreases were observed in the concentration range that inhibited aggregation. n-Amyl alcohol and n-butyl alcohol decreased the values of the parameters of the above mentioned only at higher concentrations that were dependent on their hydrophobicities. Like alkyl alcohols, benzyl alcohol and phenolic compounds decreased the values of the parameters in the concentration ranges in which these compounds inhibited platelet aggregation. The concentration of these compounds causing 50% inhibition of platelet aggregation, the IC50 values, and data on 5-NS-labeled platelets indicated that they inhibited aggregation and decreased the value of the order parameter at lower concentrations relative to their Poct values in comparison to the effective concentrations of alcohols. Phenolic compounds also decreased the values of the apparent rotational correlation times of 5-NMS and 16-NMS. These results indicate that the inhibition of platelet aggregation by alcohols and phenolic compounds is due to membrane perturbation in wide range in depths within the lipid bilayer.     

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

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

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

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

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.     

Inhibitory effects of long-chain alkyltrimethylammonium ions on aggregation of bovine platelets and the relation of their effects to Ca2+ mobilization

The inhibitory efefcts of alkyltrimethylammonium ions on ADP-and thrombin-induced aggregation of bovine platelets were investigated. The ammonium cations inhibited the two aggregation reactions to similar extents. The relationship between their inhibitory efects on ADP-induced aggregation and their alkyl chain lengths from C₈ to C₁₈ was investigated. Results showed that the inhibitory effects of ammonium cations increased with increase of their alkyl chain lengths up to C₁₆, and that the increase was linear with chain lengths of up to C₁₄. This linear relation and slope of the linear regression line suggested that the inhibitory effects of the ammonium cations depended on their partitioning into the membrane. However, unlike long-chain unsaturated fatty acids, they did not affect the membrane fluidity of the platelets, Fluorescence analysis of fura-2 loaded platelets revealed that, in the concentration range where the alkyltrimethylammonium ions inhibited aggregation, they inhibited agonist-induced increase in cytosolic Ca²⁺ both in the presence and absence of extracellular Ca²⁺. These results suggest that inhibition of platelet aggregation by alkyltrimethylammonium ionsis mainly due to their inhibition of increase in cytoplasmic Ca²⁺ by inhibition of both intracellular Ca⁺ mobilization and Ca²⁺ uptake.     

Antiplatelet activity of caffeic acid phenethyl ester is mediated through a cyclic GMP-dependent pathway in human platelets

The aim of this study was to examine the inhibitory mechanisms of caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee, in platelet activation. In this study, CAPE (15 and 25 microM) markedly inhibited platelet aggregation stimulated by collagen (2 microg/ml). CAPE (15 and 25 microM) increased cyclic GMP level, and cyclic GMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser157 phosphorylation, but did not increase cyclic AMP in washed human platelets. Rapid phosphorylation of a platelet protein of Mw. 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (150 nM). This phosphorylation was markedly inhibited by CAPE (15 and 25 microM). The present study reports a novel and potent antiplatelet agent, CAPE, which involved in the following inhibitory pathways: CAPE increases cyclic GMP/VASP Ser157 phosphorylation, and subsequently inhibits protein kinase C activity, resulting in inhibition of P47 phosphorylation, which ultimately inhibits platelet aggregation. These results strongly indicate that CAPE appears to represent a novel and potent antiplatelet agent for treatment of arterial thromboembolism.     

Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells.

Rat fat cells incubated with lipolytic agents released substances to the medium which acted as feedback regulators of cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The feedback regulators were not removed by adenosine deaminase. Dialyzed medium that had previously been incubated with fat cells in the presence of norepinephrine markedly inhibited cyclic AMP accumulation by fresh cells, whereas dialyzed medium from control cells did not inhibit cyclic AMP accumulation. The effects of lipolytic agents could be mimicked by adding dialyzed medium previously incubated with fat cells in the presence of oleic acid. This suggested that free fatty acids were the nondialyzable and adenosine deaminase-insensitive inhibitors of cyclic AMP accumulation released to the medium by fat cells incubated with lipolytic agents. The regulatory function of free fatty acids was related to the molar ratio of fatty acid to albumin. Profound inhibition of both lipolysis and cyclic AMP accumulation was seen as the free fatty acid/albumin ratio exceeded 3. The inhibition of cyclic AMP accumulation by oleate was seen as soon as there was a detectable increase in cyclic AMP due to lipolytic agents. Protein kinase activity (in the presence of cyclic AMP) of the infranatant obtained after centrifugation of fat cell homogenates at 48,000 x g was inhibited by medium from cells incubated with lipolytic agents or added oleate. Adenylate cyclase activity of rat fat cell ghosts was also inhibited by dialyzed or nondialyzed medium that previously had been incubated with lipolytic agents or added fatty acids. The direct addition of oleate markedly inhibited adenylate cyclase activity as the free fatty acid/albumin ratio exceeded 2. These data suggest that the prolonged drop in cyclic AMP accumulation seen during the incubation of rat fat cells with lipolytic agents is due to the inhibition of adenylate cyclase. This occurs when the free fatty acid/albumin ratio exceeds 3.     

Effects of alcohols on fluorescence anisotropies of diphenylhexatriene and its derivatives in bovine blood platelets: relationships of the depth-dependent change in membrane fluidity by alcohols with their effects on platelet aggregation and adenylate cyclase activity.

The effects of three short-chain alkyl alcohols and benzyl alcohol on the membrane fluidity of bovine blood platelets were investigated by studies on the fluorescence anisotropies of diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and its anionic propionic acid derivative (DPH-PA). These alcohols decreased the fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane, in concentration ranges that inhibited platelet aggregation. On the other hand, they had little or no effects on the fluorescence anisotropy of DPH-PA which is thought to be located in the interfacial region of the lipid bilayer. Likewise, they had little or no effects on the fluorescence anisotropy of TMA-DPH, which is also thought to be located in the interfacial region of the lipid bilayer, either when the probe was located in the outer layer of the plasma membrane or when the probe was located in the inner membrane compartment. These results suggest that alcohols mainly increase the fluidity in the central region of the lipid bilayer. Consistent with their effects on the fluorescence anisotropy of DPH, these alcohols increased the intracellular cyclic AMP concentration. Thus alcohols may inhibit platelet function due to stimulation of adenylate cyclase, which is mediated by perturbation of the central region of the membrane lipid bilayer.     

Inhibitory effects of phenol derivatives on bovine platelet aggregation and their effects on Ca2+ mobilization

The effects of phenol derivatives on aggregation of bovine platelets induced by ADP, thrombin, platelet activating factor, collagen and A23187 were investigated. The phenol derivatives inhibited all these induced aggregations except that by the calcium ionophore. The derivatives each inhibited the aggregations induced by ADP, thrombin, platelet activating factor and collagen, respectively, within a similar concentration range. A linear relation was found between the inhibitory potencies of the phenol derivatives and their partition coefficients between n-octanol and water (Poct values), suggesting that their interaction with hydrophobic regions of the cell was important for inhibition. Fluorescence analyses with fura-2-loaded platelets showed that in the concentration ranges in which the phenol derivatives inhibited aggregation, they also inhibited agonist-induced increases in Ca2+ both in the presence and absence of extracellular Ca2+. Moreover, a high correlation was found between the inhibitory effects of the derivatives on aggregation and their effects on Ca2+ mobilization. These results suggest that inhibition of platelet aggregation by phenol derivatives is mainly due to inhibition of the increase in cytoplasmic Ca2+ by inhibition of both intracellular Ca2+ mobilization and Ca2+ uptake.     

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

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

Irreversible platelet aggregation does not depend on lipoxygenase metabolites

Previous investigations in our laboratory demonstrated the existence of an intrinsic mechanism, termed membrane modulation, capable of restoring sensitivity to aspirin treated platelets, resulting in irreversible aggregation in response to arachidonic acid (AA). The mechanism underlying correction of aspirin induced inhibition of platelet function, however, was not clear. In the present study we have evaluated the role of lipoxygenase (LO) metabolites of AA in securing irreversible aggregation of drug induced cyclooxygenase (CO) deficient platelets. Platelets treated with aspirin or Ibuprofen did not convert radiolabeled AA to thromboxane, but generated significant quantities of hydroxy acids via the LO pathway. However, drug exposed platelets, when stirred with epinephrine first and then challenged with AA, aggregated irreversibly. Eicosatetraynoic acid (ETYA 1, U53119) inhibited AA conversion by the LO pathway, whereas 5,8,11,14-eicosatetraynoic acid (ETYA 2) inhibited AA conversion by both CO and LO enzymes. Yet, at the inhibitory concentration these fatty acids failed to prevent AA induced irreversible aggregation of CO deficient, alpha adrenergic receptor stimulated platelets. Results of four studies show that the generation of LO metabolites of AA are not essential for securing irreversible aggregation of platelets.