Effect of platelet depletion on lung vasoconstriction in heparin-protamine reactions

In six awake sheep the control heparin-protamine reaction was associated with a 150-fold rise in arterial plasma thromboxane B2 (TxB2) levels, a 4.5-fold increase in pulmonary vascular resistance, a 20% decrease in cardiac output, a 30% decrease in arterial PO2, and a 30% reduction in arterial white blood cell concentrations. Depletion of 99% of circulating platelets by antibodies did not prevent either acute and severe pulmonary hypertension or increased plasma TxB2 levels induced by heparin-protamine administration. We produced sheep platelet aggregation in vitro with bovine thrombin and measured marked TxB2 release (36.3 +/- 16.3 ng/10(9) platelets). In contrast, neither heparin, protamine, nor heparin-protamine complexes over a 10,000-fold range of concentrations induced platelet aggregation and release of thromboxane in vitro. Therefore sheep platelets are not the source of thromboxane production associated with acute pulmonary hypertension during the heparin-protamine reaction, and other cells must produce the thromboxane.     

Inhibitory effects of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) on the ADP-stimulated aggregation of gel-filtered bovine blood platelets

The effect of DIDS, a specific inhibitor of anion transport in the erythrocyte membrane, on the ADP-stimulated aggregation of gel-filtered bovine blood platelets was examined. Marked inhibition of aggregation was observed at concentrations of more than 5 x 10(-5)M DIDS. On preincubation with platelets for 30 min, DIDS was more potent and significant inhibition was observed at concentrations of over 2 x 10(-7)M. Since ADP-stimulated aggregation of bovine gel-filtered platelets precedes the release reaction, these results suggest that an anion transport system in the plasma membrane is involved in platelet aggregation.     

Native and modified low-density-lipoprotein interaction with human platelets in normal and homozygous familial-hypercholesterolaemic subjects.

The binding of low-density lipoproteins (LDL) as well as LDL modified by cyclohexanedione (CHD-LDL) to gel-filtered platelets (GFP) and its effect on platelet function were studied in normal and in homozygous familial hypercholesterolaemic (HFH) subjects. Only normal-derived LDL could significantly compete with normal 125I-labelled LDL for binding to normal platelets. When GFP from normal subjects were incubated with normal LDL at concentrations of 25-200 micrograms of protein/ml, platelet aggregation in the presence of thrombin (0.5 i.u./ml) was increased by 65-186%. CHD-LDL, at similar concentrations, caused the opposite effect and decreased platelet aggregation by 26-47%. Both LDL and CHD-LDL (100 micrograms/ml) from HFH patients, when incubated with normal GFP, caused a significant reduction in platelet aggregation (33 and 50% respectively). When HFH-derived platelets were used, both patient LDL and CHD-LDL (but not the normal lipoprotein) could markedly compete with the patient 125I-labelled LDL for binding to the platelets. LDL and CHD-LDL (100 micrograms/ml) from normal subjects decreased aggregation of HFH-platelets by 52 and 85% respectively, while corresponding concentrations of LDL derived from HFH subjects (HFH-LDL) and CHD-LDL derived from HFH subjects (CHD-HFH-LDL) increased platelet aggregation by 165 and 65% respectively. The present results support the following conclusions: platelet activation by LDL in normal subjects is through the arginine-rich apoprotein-binding site; more than one binding site for LDL exists on platelets; under certain circumstances, LDL binding can cause a reduction in platelet activity; specificity for LDL binding to the platelets resides in different regions of the lipoprotein in HFH and in normal subjects. We have thus suggested a model for LDL-platelet interaction in normal and in HFH subjects.     

A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release

Collagen-induced platelet aggregation and thromboxane release is inhibited, in a concentration response relationship, by preincubation of gel-filtered platelets with melatonin in the concentration range 430 nM – 4.3 mM. Inhibition of platelet aggregation and thromboxane release also occurs in the presence of indomethacin (4.3 nM – 4.3 mM), a known potent inhibitor of prostaglandin synthesis. Arachidonic acid-induced platelet aggregation and thromboxane release was inhibited in the presence of 4.0 mM melatonin. We therefore propose that inhibition of prostaglandin synthesis maybe the mechanism by which melatonin expresses its activity. Its antigonadotropic activity may result from inhibition of PGE₂ synthesis in the hypothalamus and median eminence.     

Plasma lipoprotein and platelet function after heparin injection: studies in normal fasted and postprandial and in type V hyperlipoproteinemic subjects

The effect of heparin injection (50 IU/kg body weight) on plasma lipoprotein concentration and composition as well as on platelet aggregation and 14C-serotonin release was studied in normal fasted subjects, normal subjects 4 hr after a fatty meal (postprandial state), and in primary type V hyperlipoproteinemic patients. Heparin injection resulted in a reduction in plasma triglyceride, cholesterol, and phospholipids as well as in the inhibition of platelet function in either the presence or the absence of the plasma environment. Heparin injection resulted in catabolism of triglyceride-rich lipoproteins and increment of cholesterol and protein in the high-density lipoprotein (HDL) density range. In fasted normal subjects, very-low-density lipoprotein (VLDL) was reduced by 50%; in the postprandial state, both VLDL and chylomicrons decreased similarly; but in phenotype V hyperlipoproteinemia, only chylomicrons (but not VLDL) degraded. Heparin injection also caused increased electrophoretic mobility of plasma lipoprotein. Upon incubation of similar lipoprotein concentration, derived before and after heparin injection, with normal washed platelets, we found that in all the groups all the lipoproteins (except HDL) derived after heparin injection caused reduction in platelet activity. High-density lipoproteins derived after heparin injection, especially from type V hyperlipoproteinemic subjects, increased normal platelet activity, and this probably represents an effect of chylomicron remnant particles in the HDL density range. Our study thus demonstrates altered composition and concentration of plasma lipoprotein after heparin injection and may suggest the appearance of remnant particles with atherogenic properties.     

Vinca alkaloids inhibit conversion of arachidonic acid to thromboxane by human platelet microsomes: comparison with other microtubule-active drugs

The Vinca alkaloid vinblastine causes dose-dependent inhibition of malondialdehyde formation and aggregation in activated human platelets as a result of inhibition of arachidonic acid metabolism via the thromboxane pathway (Brammer, J.P., Kerecsen, L. and Maguire, M.H. (1982) Eur. J. Pharmacol. 81, 577). The nature of the inhibition by vinblastine has been investigated with human platelet microsomes, measuring conversion of arachidonic acid to malondialdehyde and thromboxane B2 via spectrophotometric assay and RIA, respectively, determining arachidonate oxygenation by monitoring oxygen consumption, and identifying metabolites formed from [1-14C]arachidonic acid. Vinblastine was compared with other Vinca alkaloids and with structurally unrelated microtubule-active drugs. Vinca alkaloids were unique in causing dose-dependent inhibition of both malondialdehyde and thromboxane B2. Order of potency was vinblastine = vincristine = vindesine greater than leurosine greater than vinepidine. Inhibition of malondialdehyde and thromboxane B2 by 50 microM vinblastine was at least 60%. Microsomal cyclooxygenase was not inhibited by 200 microM vinblastine. Inhibition by vinblastine of [1-14C]arachidonic acid conversion to thromboxane B2 was associated with a 4-fold increase in prostaglandin E2 formation. Thromboxane B2, but not malondialdehyde, formation was inhibited by colchicine less than nocodazole much less than vinblastine. Results indicate that microsomal thromboxane synthetase is inhibited by Vinca alkaloids and other tubulin-binding drugs, and suggest that the action of vinblastine in inhibiting thromboxane synthesis, aggregation and release in intact platelets is not dependent upon its antimicrotubular actions.     

On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP

Concanavalin A aggregated gel-filtered platetes in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0–4 °C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0–4°C was similar to that of untreated platelets at 37°C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0–4°C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [³H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma.Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.     

Platelet aggregation by group B streptococci

Forty-six strains of group B streptococci (GBS), including various serotypes and non-serotypable strains, were tested for their ability to induce platelet aggregation in human platelet-rich plasma; four strains, all belonging to type III, showed a positive reaction. The characteristics of the reaction were investigated in these four positive strains. Aggregation was dependent on the ratio of bacteria to platelets, being maximal at a ratio of 4.3. Platelet aggregation was inhibited by EDTA (100% inhibition at 3.1 mM), indomethacin (100% inhibition at 10 mM), acetylsalicylic acid (93-100% inhibition at 5.0 mM) and quinacrine (100% inhibition at 0.25 mM). Thus the reaction was cation-dependent and required cyclooxygenase activity. Assays for cytosolic lactate dehydrogenase did not indicate platelet lysis. GBS induced the release of [3H]serotonin, which was maximal (68-78%) at 10 min after the reaction was started. Experiments with gel-filtered platelets suggested that GBS-induced platelet aggregation required both fibrinogen and heat-resistant (56 degrees C, 30 min) serum factors. Type-specific antisera prevented the platelet aggregation activity of heat-killed bacteria, but not of live bacteria. Trypsin digestion of the bacterial cells caused an almost complete loss of the platelet aggregation activity.     

Exaggerated polymerisation of beta-amyloid 40 stimulated by plasma lipoproteins results in fibrillar Abeta preparations that are ineffective in promoting ADP-induced platelet aggregation

The cytotoxic beta-amyloid peptide (Abeta) of Alzheimer's disease (AD) occurs in both plasma and platelets and may modulate platelet function. Its biological activity may relate to its fibril content and factors that promote Abeta fibrillogenesis, e.g., plasma lipoproteins could, therefore, have implications for Abeta action. We undertook a study in which structure-activity relationships were considered with respect to the actions of Abeta(1-40) on platelet function. Thus, the influence of soluble Abeta and various fibrillar Abeta preparations (0.1-10 microM) on platelet aggregation and endogenous 5-hydroxytryptamine (5-HT) efflux was investigated. Soluble Abeta(1-40) only enhanced platelet aggregation (+30%, P<0.05) and 5-HT release (+28%) stimulated by ADP (1 microM) at the highest concentration tested (10 microM). By contrast, fibrillar Abeta(1-40) at 1, 5 and 10 microM potentiated aggregation by 17.4%, 68.8% (P<0.05) and 99.5% (P<0.0001), respectively, and 5-HT efflux by 17.4%, 65% and 208% (P<0.001). Abeta(1-40) fibrils generated in the presence of native and oxidised very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) yielded platelet responses that did not differ from those seen with the lipoproteins alone. These responses were markedly lower than those obtained with homogeneous Abeta fibrils. Our data indicate that homogeneous Abeta(1-40) fibrils are more potent than soluble Abeta(1-40) in promoting platelet reactivity and that interactions with plasma lipoproteins result in the formation of Abeta fibrils that are ineffective. We suggest that lipoproteins may interfere with the recognition of Abeta by appropriate platelet receptors and/or cause Abeta to assume an "overaggregated" biologically inert state.     

Enrichment of human platelet phospholipids with linoleic acid diminishes thromboxane release

We have investigated whether exposure of human platelets to elevated concentrations of linoleic acid, the principal dietary polyunsaturate, would influence platelet thromboxane A₂ release. Platelets were incubated with albumin-bound linoleic acid at 30°C for 24 h, with prostaglandin E₁ added to prevent aggregation. The linoleic acid supplemented platelets released, on averaged, 50% less thromboxane A₂ in response to stimulation with thrombin than corresponding control platelets. Other fatty acids were without appreciable effect. The inhibition of thrombin-stimulated thromboxane A₂ release was dependent on the time and temperature of incubation, as well as on the concentration of added linoleic acid. Supplementation increased the amount of linoleic acid in the platelet phospholipids, but the arachidonic acid content of the phospholipids was reduced. [1-¹⁴C]Linoleic acid was not converted to arachidonic acid by the platelets. Linoleic acid was released exclusively form the inositol phosphoglycerides when the enriched platelets were stimulated with thrombin. The linoleate-enriched platelets converted less [1-¹⁴C]arachidonic acid to all prostaglandin products, suggesting that the platelet cyclooxygenase was partially inhibited.     

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.     

Determination of platelet adhesion to collagen and the associated formation of phosphatidic acid and calcium mobilization

A method was developed to study the adhesion of platelets to fibrillar collagen at 37 degrees C in the absence of aggregation. Human platelets were labeled with [3H]-oleic acid, gel-filtered, and incubated with collagen in the presence of receptor antagonists to thromboxane A2, 5-hydroxytryptamine, and platelet-activating factor, as well as a fibrinogen/fibronectin inhibitor and an ADP-removing system. Those platelets that adhered to collagen were separated from those that did not by filtration through a 10-microns nylon mesh and the extent of platelet adhesion was quantitated by determination of the radioactivity retained by the mesh. The extent of platelet adhesion was proportional to the amount of collagen added up to 100 micrograms/ml and was essentially complete by 1 min. At least 80-90% of the platelets were capable of adhering to collagen. Adhesion was potentiated by the presence of extracellular Mg2+ and this potentiation was inhibited by extracellular Ca2+. Phosphatidic acid increased markedly in those platelets that adhered to collagen and this was associated with increases in cytosolic free Ca2+ levels that could be detected using the fluorescent Ca2+ indicator fura-2.     

Effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced arachidonate release and 5-hydroxytryptamine secretion in human platelets. Dependence of effects on agonist type and time of incubation with PMA

We have previously demonstrated synergistic potentiation of secretion by phorbol 12-myristate 13-acetate (PMA) and platelet agonists such as thrombin and the thromboxane mimetic, U46619, with short (less than 2 min) pre-incubations of PMA, despite inhibition of agonist-induced [Ca2+]i mobilization and arachidonate/thromboxane release. In this study, the effect of PMA on 5-hydroxytryptamine secretion in relation to arachidonate/thromboxane B2 release induced by collagen as well as the 'weak agonists', ADP, adrenaline and platelet-activating factor (PAF), was investigated using human platelet-rich plasma. Short incubations (10-30 s) with PMA (400 nM) before agonist addition caused an inhibition (60-100%) of 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation in response to maximally effective doses of ADP (10 microM), adrenaline (10 microM) and PAF (0.5 microM) but potentiated collagen-induced 5-hydroxy[14C]tryptamine secretion and [3H]arachidonate/thromboxane release. However, a longer pre-incubation with PMA (5 min) caused a significant reduction (20-50%) in the extent of collagen-induced 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation as seen earlier with thrombin, although collagen-induced [3]arachidonate release was still unaffected. Pretreatment of platelets with the cyclo-oxygenase inhibitor, indomethacin (10 microM), abolished 5-hydroxy[14C]tryptamine secretion in response to the weak agonists and reduced collagen (2.5-10 micrograms/ml) -induced secretion by 50-90%, depending on the collagen concentration. Addition of PMA (400 nM) 10 s before these agonists in indomethacin-treated platelets resulted in synergistic interactions between agonist and PMA leading to enhanced 5-hydroxy[14C]tryptamine secretion, although this was notably less than the synergism observed previously between thrombin and PMA or U46619 and PMA. The results suggest that the effect of short incubations with PMA on 5-hydroxytryptamine secretion induced by 'thromboxane-dependent' agonists, such as those examined in this study, is determined by the effect on agonist-induced thromboxane synthesis. However, when endogenous thromboxane synthesis is blocked, weak agonists as well as collagen can synergize with PMA at potentiating 5-hydroxytryptamine secretion, albeit to a weaker extent than thrombin or U46619. The results also suggest that PMA has differential effects on arachidonate release induced by collagen and thrombin.     

Evidence that activation of platelets and endothelium by thrombin involves distinct sites of interaction. Studies with the dysthrombin, Thrombin Quick I

Previous results indicate extensive similarity of the active site regions of thrombin (EC 3.4.21.5) and Thrombin Quick, a congenital dysthrombin. A binding defect of Thrombin Quick toward fibrinogen is indicated by an increased KI when fibrinogen is present as a competitive inhibitor in the hydrolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. In the present study, Thrombin Quick I is shown to have an activity of 1.3 and 34%, respectively, toward fibrinogen and prothrombin. Like the activity observed in prothrombin hydrolysis, Thrombin Quick I was 30% as effective as thrombin in stimulating release of thromboxane from platelets. Thrombin Quick was 1.7 and 2.4%, as effective as thrombin in stimulating platelet aggregation and prostacyclin production, respectively. Based on the activity of Thrombin Quick I in the reactions investigated, it is concluded that 1) the three cellular responses studied are initiated by proteolytic action of thrombin, 2) thrombin stimulation of aggregation and thromboxane release from platelets occurs via two different receptors, 3) the thrombin cellular interaction resulting in platelet aggregation and prostacyclin release must involve the thrombin active site as well as a secondary binding site required for optimal interaction with fibrinogen, and 4) the release of thromboxane from platelets does not involve the interaction of thrombin at the extrinsic binding site.     

Malonaldehyde formation in intact platelets is catalysed by thromboxane synthase.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.     

Rapid activation of human platelets by low concentrations of low-density lipoprotein via phosphatidylinositol cycle

The interaction of low-density lipoprotein (LDL) with the human platelet was investigated with regard to saturable high-affinity binding, shape change, cytosolic free Ca2+ concentration, phosphatidylinositol (PtdIns) turnover, and thromboxane B2 biosynthesis. The experiments show that LDL, at a concentration approximately 100 times lower than in plasma, causes platelet activation concomitantly with stimulation of the PtdIns cycle and thromboxane B2 formation, similarly to other activators of platelets. The effects of LDL were inhibited by high-density lipoprotein. The results suggest that activation of platelets by low concentrations of LDL may play a role in pathophysiological conditions and that platelet can serve as a model for studying the influence of LDL on various target cells.     

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.     

Binding of 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (thrombocyte activating factor) by plasma components. Exchange of the thrombocyte activating factor between lipoproteins and thrombocytes

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a platelet activating factor (PAF), to plasma components was studied. Gel filtration and lipoprotein fractionation revealed the presence in the plasma of PAF-binding fractions corresponding to plasma albumin as well as of low and high density lipoproteins. Incubation of PAF-containing lipoproteins with rabbit platelets resulted in a transfer of PAF to the platelets. PAF bound to plasma albumin is less exchangeable than PAF bound to lipoproteins. The PAF-transferring efficiency of high density lipoproteins (HDL) and of low density lipoproteins (LDL) correlates with the amounts of HDL- and LDL-receptors on the platelet surface. It may thus be assumed that PAF released by various cells interacts with lipoproteins which further transport the bound PAF to target cells carrying lipoprotein receptors.     

Effect of dietary lipid on collagen- and adenosine diphosphate-induced platelet aggregation and thromboxane A2 synthesis in the rat

Dietary lipids containing different proportions of long-chain polyunsaturated fatty acids can affect platelet thromboxane A(2) formation and aggregation. In the present work, the effects of dietary lipid, from animal and vegetable sources, on collagen- and adenosine diphosphate (ADP)-induced thromboxane A(2) (measured as thromboxane B(2)) production and aggregation in washed rat platelets were studied. In addition, plasma thromboxane B(2) levels in rats fed different dietary lipids were measured. Animals were fed 10% fat by weight as lard (LRD), corn oil, soy bean oil, canola oil (CAN), or cod liver oil (CLO) for a period of 7 weeks. Circulating thromboxane B(2) levels detected in platelet-poor plasma of the CLO-fed animals were significantly lower than those of rats fed all other dietary lipids. The platelets of CLO-fed animals synthesized significantly less thromboxane A(2) compared with those from other dietary groups following ex vivo stimulation of platelets with agonists such as collagen and ADP, with the exception of platelets from the LRD-fed animals. Ex vivo stimulation of platelets obtained from this group with collagen resulted in the synthesis of significantly greater levels of thromboxane A(2) compared with all other groups. However, aggregation responses to collagen and ADP were not significantly affected by dietary treatment, although relatively the lowest responses to these agonists were apparent in the CLO-fed and CAN-fed groups, respectively.     

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

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