The mechanism of the inhibitory effect of proteinase inhibitors on platelet aggregation and cellular synthesis of prostaglandins. I. The effect on the release of arachidonic acid from phospholipids

The inhibitory effect of proteinase inhibitors on platelet aggregation was investigated. The proteinase inhibitors tested were SBTI, leupeptin and FOY (a synthetic proteinase inhibitor). Also, synthetic substrates for serine proteinases (TLME, ATEE) were tested. They completely inhibited the secondary aggregation of platelets induced by ADP or epinephrine. They also completely inhibited the platelet aggregation induced by collagen or thrombin. The aggregation induced by arachidonic acid was completely inhibited by all the proteinase inhibitors and synthetic substrates. The aggregation induced by Ca ionophore A 23187 was completely inhibited by leupeptin, FOY, TLME or ATEE but not by SBTI. It is generally accepted that platelet prostaglandin metabolism plays an important role in platelet aggregation. As the first step to elucidate the possible mechanism of the inhibitory effect of the proteinase inhibitors, their effect on the release of arachidonic acid from platelet phospholipids was investigated. The release of arachidonic acid from ¹⁴C-arachidonate incorporated gel filtered platelets (¹⁴C-AA-GFP) by thrombin or A 23187 was directly measured in the presence or absence of a proteinase inhibitor or synthetic substrate, utilizing thin layer chromatography (TLC) and a scintillation counting. The release was almost completely blocked when the aggregation of ¹⁴C-AA-GFP by thrombin or A 23187 was completely inhibited by a proteinase inhibitor or a synthetic substrate.     

An analysis of inhibitory effect of cyclic GMP on arachidonic acid- or collagen-induced aggregation in rabbit platelets

Aggregation of washed rabbit platelets induced by arachidonic acid (AA) or collagen was inhibited by nitroprusside (NP) and 8-bromo cyclic GMP in a concentration-dependent manner. Although NP and 8-bromo cyclic GMP inhibited the AA-induced aggregation, these agents did not affect the conversion of exogenous AA to PG endoperoxides and TXA2 (which were observed as TXB2). On the other hand, collagen caused release of AA from phospholipids and sequential formation of TXB2 in [14C]AA prelabeled platelets. In contrast with the case in which exogenous AA was used, NP and 8-bromo cyclic GMP inhibited the collagen-induced formation of TXB2 by preventing the liberation of endogenous AA. These results indicate that cyclic GMP has at least two different inhibitory actions in platelets; one is the inhibition of AA release from phospholipids and the other is the inhibition of the action of TXA2 in platelets.     

The effect of arachidonic acid metabolism on intravascular platelet aggregation in rats

The effects of BW755C, benoxaprofen, indomethacin and piroxicam were studied on intravascular platelet aggregation using continuous platelet counting. Plasma levels of thromboxane B₂(TxB₂) and 6-keto prostaglandin F₁(6-keto PGF_1α) were measured by radioimmunoassay. BW755C, a dual inhibitor of arachidonic acid metabolism, potentiated or inhibited aggregation depending on dose. BW755C increased TxB₂and 6-keto PGF_1α in plasma levels at low doses. At higher doses BW755C inhibited aggregation and reduced TxB₂plasma levels. At 16mg/kg BW755C, 6-keto PGF_1α was detected? Penoxaprofen also potentiated collagen-induced aggregation. 8 mg/kg indomethacin was shown to have a short lasting increased inhibitory action on collagen-induced aggregation when compared with the more specific cyclooxygenase inhibitor, piroxicam. 6-keto PGF_1α was detected in plasma fro rats treated with indomethacin and piroxicam. The results obtained using low doses of BW755C suggest that the lipoxygenase pathway is involved in platelet aggregation. Interpretation of the results obtained using higher doses of drugs also suggests this involvement though some nonspecific actions of the drugs must be taken into consideration. Further work is required to detail the role of lipoxygenase products in collagen-induced intravascular platelet aggregation.     

Differences among betaadrenoceptor blocking drugs in modifying platelet aggregation and arachidonic acid liberation under thrombin stimulation

The dose-dependent inhibition of thrombin stimulated platelet aggregation due to betaadrenoceptor blocking drugs followed the rank order of potency:propranolol >alprenolol > metipranolol and correlated with arachidonic acid (³H-AA) liberation. Atenolol which slightly potentiated stimulated aggregation increased also the liberation of ³H-AA from membrane phospholipids of isolated platelets. Stimulation of platelets resulted in decreased ³H-AA incorporation into phosphatidylcholine, phosphatidylinositol and phosphatidic acid and increased incorporation into phosphatidylethanolamine and phosphatidylserine. A₁-prenolol, metipranolol and propranolol enhanced the incorporation of ³H-AA into phosphatidylcholine of stimulated platelets.     

The effects of the oral administration of fish oil concentrate on the release and the metabolism of [14C]arachidonic acid and [14C]eicosapentaenoic acid by human platelets

It has been suggested by several investigators that eicosapentaenoic acid (C20:5 omega 3, EPA) might have anti-thrombotic effects. In this experiment, the effect of the oral administration of EPA rich fish oil concentrate on platelet aggregation and the release and the metabolism of [1-14C]arachidonic acid and [(U)-14C]eicosapentaenoic acid by human platelets was studied. Eight healthy male subjects ingested 18 capsules of fish oil concentrate (EPA 1.4 g) per day for 4 weeks. Plasma and platelet concentrations of EPA markedly increased, while those of arachidonic acid (C20:4 omega 6, AA) and docosahexaenoic acid (C22:6 omega 3, DHA) did not change. Platelet aggregation induced by collagen and ADP was reduced. Collagen induced [14C]thromboxane B2 (TXB2) formation from [14C]AA prelabeled platelets decreased. There was no detectable formation of [14C]TXB3 from [14C]EPA prelabeled platelets, and the conversion of exogenous [14C]EPA to [14C]TXB3 was lower than that of [14C]AA to [14C]TXB2. The release of [14C]AA from [14C]AA prelabeled platelets by collagen was significantly decreased. These observations raise the possibility that the release of arachidonic acid from platelet lipids might be affected by the alteration of EPA content in platelets.     

Effects of 9, 12, 15-octadecatrien-6-ynoic acid on the metabolism of arachidonic acid in platelets and on platelet aggregation

An acetylenic fatty acid: 9,12,15-octadecatrien-6-ynoic acid (dicranin) was extracted from and preincubated with platelets which were then stimulated by exogenous arachidonic acid (20:4 n-6). This molecule at 10⁻⁴ M weakly inhibited the cyclooxygenase activity as assessed by measurement of 12-hydroxy-heptadecatrienoic acid (HHT) In contrast, the 12-hydroxy-eicosatetraenoic acid (12-HETE) synthesized by the 12-lipoxygenase was strongly increased by about 650%. The same effects were observed with 10⁻⁵ M and with 10⁻⁵ M of dicranin but to a lesser extent.

Platelet hydroxylated dicranin metabolites were also found and the structure of the main compound determined by GC-MS was a 13-hydroxy derivative. Its origin has not yet been elucidated. Platelet aggregation induced by 1 μg/ml of U46619, a structural PGH₂ analogue was completely abolished in the presence of dicranin.

Platelet aggregation induced either by thrombin or by arachidonic acid was inhibited by 10⁻⁴ M of dicranin only after preincubation. This observation indicates that the formation of metabolites of dicranin are necessary to effect this inhibition.

Dicranin is thus a new inhibitor of platelet aggregation and may prove to be useful for elucidating the effects of 12-HETE in biological systems.     

Platelet adenine nucleotides and arachidonic acid metabolism in the myeloproliferative disorders

Platelet aggregation to collagen, adenosine diphosphate and arachidonic acid has been investigated in 17 patients with various myeloproliferative states. Ten patients who had abnormalities of aggregation to collagen and/or ADP were also all found to have diminished intracellular and releasable adenine nucleotides but aggregation to arachidonic acid was normal. Seven other patients who had normal aggregation responses had normal platelet adenine nucleotides. In the ten patients with abnormal platelet function platelet cyclo-oxygenase activity was normal but in two patients platelet lipoxygenase activity was reduced. Thromboxane B₂ production during collagen stimulation was found to be normal suggesting normal release of endogenous arachidonic acid. These findings suggest that the platelet defect in myeloproliferative states is due to an acquired storage pool disease.     

Effect of cobra venom phospholipase A2 on platelet aggregation in comparison with those produced by arachidonic acid and lysophophatidylcholine

Cobra venom phospholipase A₂ induced a biphasic effect on washed rabbit platelets. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The aggregation and thromboxane B₂ formation were inhibited by indomethacin, mepacrine, tetracaine and imipramine, while PGE₁ and sodium nitroprusside inhibited only the aggregation, but not the thromboxane B₂ formation. The second phase was an inhibitory effect on platelet aggregation induced by arachidonic acid, PAF, ADP or collagen but not that by thrombin or ionophore A23187. The longer the incubation time of cobra venom phospholipase A₂ with platelets, the more the inhibitory effect. The aggregating and anti-aggregating effects could be overcome by bovine serum albumin. Lysophosphatidylcholine (Lyso-PC) and arachidonic acid showed synergistic inhibition in platelet aggregation. Lyso-PC decreased thromboxane B₂ formation in platelets formed by collagen. The inhibitory effect of Lyso-PC on platelet aggregation was more marked at lower calcium concentrations. It is concluded that the aggregating effect of exogenous addition of venom phospholipase A₂ is due to thromboxane formation and the antiplatelet effect is similar to those produced by arachidonic acid and lysophosphatidylcholine.     

Eicosapentaenoic acid potentiates the production of prostacyclin-like material in the arachidonic acid perfused human umbilical vein

The production of prostacyclin (PGI2)-like material in human umbilical veins perfused continuously at 37 degrees C with Hanks buffer solution with 1% human albumin (HBA) was studied by bioassay. Subsequent perfusion resulted in a time dependent significant decrease in production of PGI2-like material. After addition of 20 mumol/l arachidonic acid (HBA-AA) the production of PGI2-like material increased significantly. The production of PGI2-like material was significant greater when the vein was perfused with HBA-AA than when perfused with HBA with 20 mumol/l eicosapentaenoic acid (HBA-EPA). Examination of the HBA-EPA perfusate by thin layer chromatography showed that it contained a substance that comigrated with genuine 6-keto-PGF1 alpha and a substance that comigrated with delta 17-6-keto-PGF1 alpha. Finally, perfusion with HBA containing 10 mumol/l AA plus 10 mumol/l EPA resulted in a significant greater production of PGI2-like material than perfusion with HBA-AA alone. These results support the hypothesis that EPA has beneficial antithrombotic properties in human.     

Uptake of arachidonic acid,arachidic acid,oleic acid and their incorporation into phospholipids and triacylglycerols of isolated murine hepatocytes. Effect of thrombin-antithrombin III complex

Uptake and metabolism of arachidonic acid, arachidic acid and oleic acid were investigated in isolated hepatocytes prepared from mouse liver with the collagenase perfusion method. The rate of uptake of arachidonic acid was time— and concentration- dependent. 94–98% of the arachidonic acid was incorporated into the phospholipid and triacylglycers1 fractions following a 60 min incubation period at 37°C. In the presence of thrombin-anti-thrombin III complex a change in the distribution of arachidonic acid incorporated into lipid fractions was found, i.e. increased incorporation into phosphatidyl-serine and phosphatidylethanolamine, whereas the uptake was not altered. There was no change in the uptake and incorporation of arachidic acid and oleic acid.     

Glutamate signalling and secretory phospholipase A2 modulate the release of arachidonic acid from neuronal membranes

The lipid mediators generated by phospholipases A(2) (PLA(2)), free arachidonic acid (AA), eicosanoids, and platelet-activating factor, modulate neuronal activity; when overproduced, some of them become potent neurotoxins. We have shown, using primary cortical neuron cultures, that glutamate and secretory PLA(2) (sPLA(2)) from bee venom (bv sPLA(2)) and Taipan snake venom (OS2) elicit synergy in inducing neuronal cell death. Low concentrations of sPLA(2) are selective ligands of cell-surface sPLA(2) receptors. We investigated which neuronal arachidonoyl phospholipids are targeted by glutamate-activated cytosolic calcium-dependent PLA(2) (cPLA(2)) and by sPLA(2). Treatment of (3)H-AA-labeled cortical neurons with mildly toxic concentrations of sPLA(2) (25 ng/ml, 1.78 nM) for 45 min resulted in a two- to threefold higher loss of (3)H-AA from phosphatidylcholine (PC) than from phosphatidylethanolamine (PE) and in minor changes in other phospholipids. A similar profile, although of greater magnitude, was observed 20 hr posttreatment. Glutamate (80 microM) induced much less mobilization of (3)H-AA than did sPLA(2) and resulted in a threefold greater degradation of (3)H-AA PE than of (3)H-AA PC by 20 hr posttreatment. Combining sPLA(2) and glutamate resulted in a greater degradation of PC and PE, and the N-methyl-D-aspartate receptor antagonist MK-801 only blocked glutamate effects. Thus, activation of the arachidonate cascade induced by glutamate and sPLA(2) under experimental conditions that lead to neuronal cell death involves the hydrolysis of different (perhaps partially overlapping) cellular phospholipid pools.     

Effects of the ratio of exogenous eicosapentaenoic acid to arachidonic acid on platelet aggregation and serotonin release

We added arachidonic acid (AA) and eicosapentaenoic acid (EPA) to washed platelet suspensions in the absence of albumin, holding the total amount of the fatty acids constant at 2 microM, and changing the ratio of EPA to AA. Platelet aggregation, serotonin release and the amount of thromboxane (TX) B2, a cyclooxygenase product synthesized from exogenous AA, decreased as the ratio was increased. The decreases were greater than the expected ones from the diminution of the amount of exogenous AA. On the other hand, 12-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product synthesized from exogenous AA, increased in the presence of EPA. Although EPA was reported to be a poor substrate for platelet cyclooxygenase, the amount of TXB3 synthesized from exogenous EPA increased markedly by the simultaneous addition of AA. These results suggest that the EPA/AA ratio-dependent decrease in platelet aggregation and serotonin release is caused at least by both the decrease in the absolute amount of AA and the inhibitory effect of EPA on AA-metabolism via the cyclooxygenase pathway. Further studies on effects of EPA-metabolites via the cyclooxygenase pathway on platelet responses will be needed.     

Effect of cetiedil on platelet aggregation and thromboxane synthesis

Cetiedil was found to inhibit platelet aggregation and thromboxane synthesis induced by thrombin and arachidonic acid. When platelets were activated by thrombin, half maximal inhibition (ED₅₀ effective dose of cetiedil necessary for 50% inhibition) for platelet aggregation was 100 μM while that for thromboxane B₂ (TXB₂) production was 50 pM. When arachidonic acid was used, the ED₅₀ for platelet aggregation was 100 μM while that for TXB₂ production was 150 μM. The presence of calcium ions did not affect on the inhibitory effects of cetiedil. The cAMP level in platelets did not increase after incubation with cetiedil. Cetiedil appears to inhibit the activation of platelets related to thromboxane synthesis.     

Chronic carbamazepine decreases the incorporation rate and turnover of arachidonic acid but not docosahexaenoic acid in brain phospholipids of the unanesthetized rat: relevance to bipolar disorder.

BACKGROUND: The basis for carbamazepine's efficacy in treating bipolar disorder is not agreed on. One hypothesis is that, similar to lithium and valproate (antibipolar drugs), carbamazepine might selectively decrease the kinetics of arachidonic acid (AA) in brain phospholipids. METHODS: To assess whether it targets brain AA kinetics, we administered carbamazepine (25 mg/kg/day, IP) to rats for 30 days and then determined its effect compared with that of vehicle on incorporation and turnover rates of AA and docosahexaenoic acid (DHA) in brain phospholipids. In unanesthetized rats that had received carbamazepine or vehicle, [1-14C]AA or [1-14C]DHA was infused intravenously, and arterial blood plasma was sampled until the animal was killed at 5 min and its brain, after being microwaved, was used for acyl-coenzyme A (acyl-CoA) and phospholipid fatty acid analysis. RESULTS: Chronic carbamazepine, compared with vehicle, decreased the rate of incorporation of AA-CoA (27%-29%) and turnover of AA (25%-27%) but not of DHA-CoA or DHA in brain phospholipids. CONCLUSIONS: The results, which are comparable to published findings after chronic administration of lithium and valproic acid to rats, support the hypothesis that drugs effective against mania in bipolar disorder act by selectively downregulating the incorporation rate of AA-CoA and turnover of AA in brain phospholipids.     

缺血性脑血管病常用药物对血小板聚集功能的影响

应用比浊法测定了川芎嗪，刺五加，维脑路通，潘生丁，阿斯匹林对２０例脑梗塞病人和１２例健康人的血小板聚集功能的影响。结果显示：不论是正常人还是脑硬塞病人，这些药物在体外都能抑制血小板聚集。其中川芎嗪对血小板聚集抑制作用最强，维脑路通作用最弱。     

Mechanism of inhibitory effect of some amphiphilic drugs on platelet aggregation induced by collagen, thrombin or arachidonic acid

The effects of two representative groups of amphiphilic drugs, lysophosphatidylcholine species (myristoyl, palmitoyl and stearoyl) and phenothiazine neuroleptics (promethazine, chlorpromazine and trifluoperazine), on the morphology of intact rabbit platelets, arachidonate release from membrane phospholipids, pseudopod formation and the resulting aggregation of stimulated platelets were examined and compared with those of two known cycloxygenase inhibitors, aspirin and indomethacin.

Collagen-induced aggregation was inhibited by relatively low concentrations of amphiphilic drugs in parallel with the prevention of arachidonate release from the membrane phospholipids. Thrombin- and arachidonate-induced aggregations were inhibited by these drugs at higher concentrations, at which they transform intact platelets from discoid to spiny and spherical shape, respectively, and consequently suppress formation of native pseudopods induced by these stimuli. Washing the drug-treated platelets with BSA-Tyrode solution abolished all the effects of drugs. In contrast, the cycloxygenase inhibitors blocked both collagen- and arachidonate-induced aggregations without causing membrane shape change of the intact platelets, although they inhibited thrombin-induced aggregation at extremely high concentrations, at which they did elicit membrane shape change.

These observations suggest that these amphiphilic drugs act on the plasma membrane of platelets and impair membrane-linked functions. At lower concentrations they specifically inhibit the arachidonate     

The effect of lithium on platelet aggregation and platelet release reaction

Lithium increases the aggregation of human platelets in vivo as well as in vitro. One of the mechanisms presumed to underlie this aggregation-promoting effect, potentiation of ADP release, was investigated in relation to lithium uptake. It appeared that lithium is not taken up by human platelets against a concentration gradient, and equilibrium is achieved in about 120 min. When platelets are incubated for this fixed time with different lithium concentrations, the aggregation response is related to the intraplatelet lithium concentration. When platelets are incubated during increasing periods with a fixed lithium concentration, no direct correlation exists between intraplatelet lithium concentration and extent of aggregation. After blocking the release with aspirin, the stimulatory effect of lithium on platelet aggregation was still observed. Low ADP concentrations, causing no measurable release, still caused more marked aggregation of platelets preincubated with lithium chloride than of platelets incubated with sodium chloride. It is concluded that the increase in platelet aggregation which occurs after incubation of platelets with lithium, is not caused merely by potentiation of the release reaction.     

The tachyphylactic effect of arachidonic acid on in-vivo ADP induced arterial platelet thrombosis

The continuous superfusion of arachidonic acid over a branch of the mesenteric artery in an animal in-vivo model results in an initial increase followed by a decrease to control values of ADP induced platelet thrombogenesis. This phenomenon is observed in normal rats as well as in rats submitted to the intravenous administration of ASA three hours prior to the investigation. In the latter group the cyclooxygenase of the platelets is permanently affected while the cyclooxygenase of the endothelial cells is regenerated at the time the investigation is started. The tachyphylactic-like phenomenon observed with arachidonic acid is probably related to the dynamics of the enzymatic reactions of the arachidonate cascade involving the platelets and the endothelial cells surrounding the local de-endothelialized area.