Prostacyclin potentiates 13-azaprostanoic acid-induced platelet deaggregation

The specific thromboxane A₂/prostaglandin H₂ (TXA₂/PGH₂) antagonist 13-azaprostanoic acid (13-APA) reverses platelet aggregation stimulated by TXA₂/PGH₂ and the prostaglandin endoperoxide analog U46619. The present report demonstrates that the deaggregatory properties of 13-APA are potentiated by prostacyclin (PGI₂). Human platelet-rich plasma was aggregated with U46619. Deaggregation was induced 2 min subsequent to the addition of the aggregating agent. Concentrations of 13-APA or PGI₂ which induced 20 percent deaggregation were determined. Simultaneous addition of half of these concentrations resulted in 60 percent deaggregation, demonstrating that the observed response was supraadditive. Measurement of cyclic adenosine 3′:5′ monophosphate (cAMP) in resting or deaggregating platelets demonstrated that 13-APA itself did not stimulate cAMP production nor did 13-APA facilitate PGI₂-induced increases in cAMP. In separate studies PGI₂ and 13-APA were added to PRP prior to the induction of aggregation by U46619. Under these conditions, additive inhibition of aggregation was observed. Thus, it is clear that the pharmacological interaction between PGI₂ and 13-APA depends upon the relative state of platelet activation.     

effects of picotamide on human platelet aggregation, the release reaction and thromboxane B2 production

We studied the effects of picotamide (N,N′ bis 3 picolyl-4-methoxy-isophthalamide) on human platelet aggregation, the release reaction and the production of thromboxane B₂ (TxB₂) induced by several platelet agonists. The effects of picotamide were compared to those of acetylsalicylic acid (ASA). Picotamide (0.5 mmol/1) inhibited platelet aggregation, the release of ATP and TxB₂ production induced by ADP, arachidonic acid (AA), collagen or the prostaglandin endoperoxide (PE) analogue U46619. ASA (0.5 mmol/1) did not affect platelet aggregation and the release of ATP induced by U46619. Picotamide and ASA inhibited the AA-induced platelet TxB₂ production both under stirring and non-stirring conditions, whereas the pure thromboxane A₂ receptor antagonist BM13177 (0.5 mmol/1) was inhibitory only under stirring conditions. Since under non-stirring conditions platelet aggregation does not occur, picotamide directly inhibits TxB₂ production, whereas BM13177 inhibits the potentiation of TxB₂ production due to TxA₂/PE-dependent platelet aggregation. Malondialdehyde (MDA) production by unstirred platelets stimulated with AA was not significantly inhibited by picotamide. In conclusion, picotamide inhibits the TxA2/PE-dependent platelet responses to agonists by a double mechanism: (i), TxA₂/PE antagonism; (ii) inhibition of thromboxane synthase.     

Purification and characterisation of a snake venom phospholipase A2: A potent inhibitor of platelet aggregation

An inhibitor of human platelet aggregation was identified from the venom of an Australian Copperhead snake, Austrelaps superba, as a novel phospholipase A₂. The inhibitor was purified to homogeneity by chromatography on Q-Sepharose, S-Sepharose and C8 reverse phase HPLC. The purified phospholipase A₂ has a molecular weight of 15 kDa as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence analysis of the platelet inhibitor revealed 70–80% sequence identity to other previously described secretory phospholipase A₂. Phospholipase activity of the purified protein was confirmed by the ability of the enzyme to hydrolyse lecithin. Pretreatment of the purified protein with the specific phospholipase A₂ inhibitor p-bromophenacyl bromide, resulted in abrogation of both its enzyme and platelet inhibitory activity. The phospholipase A₂ inhibited platelet aggregation and serotonin release, induced by a variety of platelet agonists, in a time and dose dependent manner.     

Effect of op 1206, a prostaglandin E1 derivative, on guinea-pig platelet functions

OP 1206, 17(S)-methyl-ω-homo-trans-Δ²-prostaglandin E₁, inhibited guinea-pig platelet aggregation induced by ADP, collagen, A 23187, arachidonic acid, labile aggregation stimulating substances (LASS) and thromboxane A₂ (TXA₂)-like substance, and platelet adhesiveness to a glass bead column. The potency of inhibition was 10–16 times stronger than that of prostaglandin E₁ (PGE₁). ADP-induced platelet aggregation was notably disaggregated by the addition of OP 1206 after induction of aggregation. The release of ATP and ADP from guinea-pig platelets induced by collagen was suppressed by OP 1206, of which potency was 9–10 times stronger than that of PGE₁. OP 1206 and PGE₁ increased guinea-pig platelet cyclic AMP levels, and the increased levels were augmented by the pretreatment with theophylline. OP 1206 and PGE₁ inhibited synthesis of guinea-pig platelet malondialdehyde (MDA) induced by thrombin but not by arachidonic acid. This inhibition was released by exogenous calcium. OP 1206 and PGE₁ showed no influence on synthesis of radioactive TXA₂ (measured as a stable form, TXB₂) from [¹⁴C] arachidonic acid. From these results, increased levels of platelet cyclic AMP by OP 1206 as well as PGE₁ may exert their action on platelet functions.     

Aspirin insensitive eicosanoid biosynthesis in cardiovascular disease

Inhibition of platelet cyclooxygenase (COX)-1 is involved in aspirin cardioprotection observed in clinical trials, but, in some patients, aspirin is unable to protect from thrombotic complications. An incomplete suppression of platelet thromboxane (TX) A₂ biosynthesis has been assumed to participate in the phenomenon of aspirin resistance, as a consequence of the following possible mechanisms: (i) COX-2 expression in newly formed platelets; (ii) pharmacodynamic interactions between aspirin and coadministered nonsteroidal antiinflammatory drugs (e.g. ibuprofen); (iii) expression of variant isoforms of COX-1 with reduced sensitivity to irreversible inactivation at Ser529. Furthermore, aspirin failure may be due to enhanced formation of vasoactive and/or proaggregatory eicosanoids despite an almost complete suppression of platelet TXA₂ biosynthesis by aspirin. Thus, in a subset of patients with unstable angina treated with low-dose aspirin, to almost completely block platelet COX-1 activity, enhanced TXA₂ biosynthesis in vivo has been demonstrated, presumably through an increased generation of COX-2-dependent PGH₂ in plaque monocytes/macrophages or activated vascular cells. The concomitant increased formation of 8-iso-PGF₂, one of the most abundant F₂-isoprostanes in humans, generated by free-radical catalyzed arachidonate peroxidation, may be involved in aspirin resistance because of its capacity to induce platelet and vascular smooth muscle cell activation through the interaction with thromboxane receptors (TPs). Finally, enhanced production of vasoactive cysteinyl leukotrienes (cys-LTs) occurs in unstable angina despite conventional antithrombotic and antianginal treatment. The use of selective pharmacological tools (i.e. highly selective COX-2 inhibitors, TP antagonists, cys-LT inhibitors and antagonists) will allow to ascertain the contribution of aspirin insensitive eicosanoid biosynthesis in aspirin cardioprotective failure.     

Discrimination between platelet prostaglandin receptors with a specific antagonist of bisenoic prostaglandins

Platelet aggregation and secretion induced by PGG₂ and by analogues of PGH₂ and PGE₂ were inhibited by NO164. Inhibition was apparently competitive (K_i 20 μM). Responses to ADP, vasopressin and arachidonic acid were unaffected. Inhibition of ADP-induced platelet aggregation by PGD₂ and PGE₂ (but not by PGE₁) was antagonised by NO164.     

Thromboxane synthase inhibition causes re-direction of prostaglandin endoperoxides to prostaglandin D2 during collagen stimulated aggregation of human platelet rich plasma

Prostanoid synthesis and release during collagen-induced aggregation of human platelet rich plasma (PRP) was studied using a novel gas chromatography/mass spectrometry assay technique. Aggregation was associated with the production of mainly thromboxane A₂ (TXA₂), measured as TXB₂, and smaller amounts of the prostaglandins (PGs) D₂, E₂ and F₂. UK 37,248 inhibited T₁XB₂ formation by >95% and increased the production of PGD₂, PGE₂ and PGF₂ twenty-fold. The relative amounts of these three prostanoids were not changed by UK 37,248. Even though high concentrations of PGD₂ were formed, aggregation was not inhibited. In contrast, flurbiprofen inhibited aggregation, demonstrating that platelet aggregation produced by this concentration of collagen is cyclooxygenase dependent. These results support the proposal that the prostaglandin endoperoxides can induce aggregation alone, irrespective of the amount of PGD₂ that is produced.     

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.     

Epinephrine—Via Activation of P38-Mapk—Abolishes the Effect of Aspirin on Platelet Deposition to Collagen

The mechanism by which epinephrine enhances experimental thrombosis in the presence of aspirin is poorly understood. In this study, we set to explore, in aspirinised platelet-rich plasma (PRP), the effect of epinephrine (100 nmol/l) on platelet deposition to immobilised collagen and the subsequent involvement of several intracellular pathways. Under these experimental conditions, which allow platelet aggregation on top of the collagen-adherent platelets, epinephrine increased platelet deposition by 55–86%. This enhancement could be specifically prohibited by the _2A-adrenoceptor antagonist, atipamezole, the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580, and the cytosolic phospholipase A₂ (cPLA₂) inhibitor, mepacrine. The effect of epinephrine coincided with increased phosphorylation of p38MAPK and cPLA₂ and with arachidonic acid (AA) release from platelet membrane. We conclude that epinephrine enhanced platelet deposition on collagen in aspirinised PRP via a mechanism dependent on both free AA in platelet cytosol (released by cPLA₂) and p38MAPK.     

Some properties of the human platelet vasopressin receptor

Aggregation of human platelets by vasopressin is potently inhibited by 1-[βmercapto-(β,β′-cyclopentamethylene propionic acid)]-L-arginine vasopressin, a selective vasopressor (V₁) antagonist. 1-Desamino-8-D-arginine-vasopressin, a selective anti-diuretic (V₂) agonist failed to induce aggregation and acted as a weak antagonist. Vasopressin analogues which lacked the N-terminal amino group or which contained an uncharged amino acid residue at position 8 acted as partial agonists for the human platelet. The response to such partial agonists could be enhanced by increasing the cytosolic Ca²⁺ concentration but not by altering the level of cyclic-3′,5′-AMP. These observations provide further evidence indicating that the platelet vasopressin receptor is of the V₁ sub-type.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

Activation of protein kinase C by the action of 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, in human platelets

Incubation of human washed platelets with 9,11-epithio-11,12-methano-thromboxane A₂ (STA₂), a stable analogue of thromboxane A₂, caused the activation of protein kinase C and myosin light chain (MLC) kinase to the same extents as those induced by thrombin as judged by measuring the phosphorylation of a 40-kilodalton protein and MLC, respectively. However, STA₂ stimulated much less phosphoinositide turnover than thrombin. Furthermore, the doses of STA₂ necessary for protein kinase C activation and phosphoinositide turnover were higher than those necessary for MLC kinase activation, although the doses of thrombin necessary for these three reactions were nearly the same. These results suggest that protein kinase C may be activated at the Ca²⁺ concentrations higher than those required for MLC kinase activation by the action of STA₂, presumably due to the inability of this agonist to produce diacylglycerol in an amount enough to increase the affinity of the enzyme for Ca²⁺.     

Effects of Ethanol on Platelet Responses Associated with Adhesion to Collagen

Adhesion of platelets to collagen in damaged blood vessels or ruptured atherosclerotic plaques is important in hemostasis and arterial thrombosis. Adhesion to collagen results in secretion of granule contents and formation of thromboxane A₂; thromboxane A₂ and released ADP synergistically promote aggregation around platelets adherent to collagen. Ethanol inhibits collagen-induced platelet aggregation, secretion, arachidonate mobilization, and thromboxane A₂ formation but does not inhibit platelet adhesion to de-endothelialized rabbit aortae. We investigated whether ethanol affects the initial signalling events and responses of platelets adherent to collagen, independent of the actions of secondary agonists. Suspensions of washed human platelets, labelled by incorporation of [³H]oleate into phospholipids, were used to measure platelet adhesion to collagen by a filtration method; studies were done in the presence of an ADP-removing system and blockers of receptors for thromboxane A₂, platelet-activating factor, serotonin, and fibrinogen. Ethanol (87 mM) did not affect the rate or extent of platelet adhesion to collagen or secretion of [¹⁴C]serotonin from prelabelled platelets adherent to collagen, but ethanol did inhibit thromboxane A₂ formation. Previous studies showed that ethanol does not affect platelet stimulation by arachidonate, leading to the suggestion that reduced mobilization of arachidonate, rather than inhibition of its conversion to thromboxane A₂, is responsible for inhibition by ethanol of thromboxane A₂ formation. Here, we show by a gel mobility shift assay and immunoblotting, that ethanol delays the collagen-induced increase in the phosphorylation of cytosolic phospholipase A₂, the enzyme responsible for arachidonate mobilization. However, ethanol has no effect on collagen-induced tyrosine phosphorylation of phospholipase Cγ2, determined by immunoprecipitation and immunoblotting. Thus, ethanol's effect on signal transduction in collagen-adherent platelets occurs distal to phosphorylation of phospholipase Cγ2 but proximal to phosphorylation of cytosolic phospholipase A₂.     

Effect of platelet activity of inhibition of adenylate cyclase

SQ22536 (9-[tetrahydro-2-furyl]-adenine), an inhibitor of adenylate cyclase, blocks the inhibition of platelet function by substances that stimulate this enzyme, including PGE₁, PGI₂, PGD₂, and adenosine. SQ22536 enhances platelet aggregation and serotonin release induced by prostaglandin endoperoxides and their analogs but does not consistently affect the platelet response to ADP or other agonists. SQ22536 reduced platelet cyclic AMP and inhibits the effect on this nucleotide of PGE₁ and adenosine. The elevation of cyclic GMP induced by ADP is prevented by SQ22536.     

Antiplatelet effect of capsaicin

Capsaicin was found to be a potent inhibitor of platelet aggregation and release reaction. It inhibited the aggregation of rat platelet induced by collagen and thrombin, but only slightly reduced those of AA and A23187. The IC₅₀ on collagen-induced platelet aggregation was about 85 μg/ml. Less inhibition was observed in the aggregation of platelet-rich plasma. Increase of the calcium concentration could not overcome the inhibitory effect. Washing of the capsaicin-pretreated platelets only partially reversed the inhibition. Capsaicin also inhibited ATP release induced by thrombin and A23187 in the presence of EDTA. MDA and TXB₂ formation were markedly inhibited by capsaicin in platelets challenged by collagen, thrombin and A23187. In AA-stimulated platelets, MDA formation was slightly decreased and TXB₂ formation was not affected. Capsaicin showed more marked inhibition in the presence of CP/CPK, indomethacin or a combination of both. Capsaicin reduced the hemolysis of RBCs induced by hydrogen peroxide or hypotonicity. It was concluded that capsaicin had some membrane stabilizing property and this might lead to the interferance of the activation of phospholipase A₂.     

Obligatory role of lipid mediators in platelet-neutrophil adhesion

Platelet–neutrophil interactions play an important role in thrombotic and inflammatory responses. Although it is well known that adhesion of platelets to neutrophils requires interactions of adhesion molecules on platelets such as P-selectin, or GPIIb/IIIa with their counterparts on neutrophils, little is known on the role of lipid mediators in this response. Here we studied involvement of thromboxane (TX) A₂, platelet activating factor (PAF) and cysteinyl leukotrienes (cysLTs) in the mechanisms of platelet–neutrophil adhesion that was induced by thrombin (10–100 mU/ml), fMLP (0.01–1 μM) or LPS (0.001–100 μg/ml). All three stimulators in a concentration- and time-dependent manner induced platelet–neutrophil adhesion as quantified by the method of Jungi et al. [Blood 67(3) (1986) 629]. Platelet–neutrophil adhesion induced by each of the three activators was inhibited by blocking antibodies towards P-selectin, GPIIb/IIIa or CD18, but it was not affected by anti-E selectin antibody. Moreover, platelet–neutrophil adhesion induced by thrombin, fMPL or LPS was inhibited by the inhibitor of cyclooxygenase (aspirin), by TXA₂ synthase inhibitor (camonagrel), by PAF receptor antagonist (WEB 2170), by the inhibitor of FLAP (MK 886) and by cysLTs receptors antagonist (MK 571). On the other hand, the selective inhibitor of COX-2 (rofecoxib) as well as the inhibitor of cytochrome P₄₅₀-dependent monoxygenase (17-ODYA) were ineffective.

In summary, adhesion of platelets to neutrophils is regulated not only by specific interaction between adhesion molecules on platelets and neutrophils, but also by lipid mediators such as TXA₂, PAF and cysLTs released upon activation of platelets or/and neutrophils.     

Mechanisms of vascular graft thrombosis: Role of altered canine platelet sensitivity to thromboxane

Using the standard turbidimetric method of platelet aggregation and quantitation of platelet secretion with ¹⁴C-Serotonin, we have examined the responsiveness of the platelets of mongrel dogs to arachidonic acid (AA), and the thromboxane agonist U46619 in the presence and absence of a subthreshold concentration of epinephrine. In response to stimulation with 750 μM AA, the platelets of 18 dogs produced irreversible aggregation (Group I), the platelets of 22 dogs showed, at most, reversible aggregation (Group II), while the platelets of 8 dogs demonstrated no aggregatory response (Group III). In the presence of AA and a subthreshold concentration of epinephrine (0.5 μM), the platelets of all three groups demonstrated enhanced aggregatory and secretory responses although the extent of ¹⁴C-Serotonin secretion differed significantly between all three groups. These differences in platelet aggregation correlate with the deposition of platelets onto synthetic vascular grafts and the maintenance of graft patency. When stimulated with 0.5 μM U46619 and a subthreshold concentration of epinephrine, the platelets of 97% Group I dogs and 75% of Group II dogs exhibited irreversible aggregation, while the platelets of all Group III dogs showed only reversible aggregation. In addition, significant differences in the extent of ¹⁴C-Serotonin secretion to this combination of agonists were observed between groups. Further examination of the specific effects of U46619 on canine platelets revealed that although the aggregatory and secretory responses to U46619 vary between the different canine platelet populations, the threshold concentration of U46619 required to produce platelet shape change is identical among all groups. Quantitation of the stable metabolite of AA produced via the cyclooxygenase pathway, thromboxane B₂(TxB₂), revealed no significant differences in the production of TxB₂ by the platelets of these different populations upon stimulation with AA. Our results suggest that the mechanisms underlying the differences in responsiveness of canine platelets to AA, are likely due to differences in sensitivity of canine platelets to TxA₂, and may be localized to the mechanism responsible for mediating platelet aggregation and secretion in response to TxA₂.     

Phospholipase C from clostridium perfringens induces human platelet aggregation in plasma

We studied the aggregating effect of different concentrations of phospholipase C (PLC) (extracted from Clostridium perfringens) on human platelet-rich plasma (PRP). PRP was preincubated with PLC for 3 min at 37 degrees C and the platelet aggregation was followed for 10 min. The threshold aggregating concentration (TAC) of PLC was 3-4 U/ml. We also studied the potentiation of PLC with other stimuli on platelet aggregation. Potentiating stimuli, such as arachidonic acid (AA), ADP. Platelet Activating Factor (PAF) and U-46619 (a stable analogue of cyclic endoperoxides) were all used at subthreshold concentrations. We also studied the possible inhibitory effect of aspirin, apyrase, TMQ, a prostaglandin endoperoxide/thromboxane receptor antagonist and BN-52021, a PAF receptor antagonist. Only aspirin and apyrase were able to reduce aggregation induced by PLC alone and PLC + AA and PLC + ADP respectively. TMQ and BN-52021 were inactive. In ex vivo experiments oral aspirin (500 mg) partially inhibited platelet aggregation induced by PLC alone, PLC + AA and PLC + ADP 2 and 24 h after administration. Aspirin 20 mg for 7 days also reduced aggregation induced by PLC + AA.     

Antagonism of platelet aggregation by 13-azaprostanoic acid in acute myocardial ischemia and sudden death

The effects of 13-azaprostanoic acid (13-APA) were studied during acute myocardial ischemia in cats and in rabbit sudden death induced by sodium arachidonate (Na-Ar). To more clearly define the mechanism of action of 13-APA, we also examined its effects on isolated cat and rabbit coronary arteries, in vitro aggregation of cat and rabbit platelet-rich plasma (PRP) and circulating rabbit platelet count measured in vivo. 13-APA provided minimal protection during myocardial ischemia in cats, partially reversing ischemia-induced ST segment elevations by 3-5 hours after coronary artery occlusion. However, 13-APA was ineffective in inhibiting the rise in plasma creatine kinase (CK) activity or the loss of CK from ischemic myocardial tissue. 13-APA (1.0 - 100 microM) did not inhibit contraction of cat coronary arteries produced by a stable thromboxane A2 analog. However, 13-APA (100 microM) inhibited aggregation of cat PRP induced by AA (1.0 microM). 13-APA also provided significant protection against sudden death induced by Na-Ar in rabbits. While this agent was ineffective in reducing vasoconstriction of rabbit coronary arteries or inhibiting platelet aggregation in response to 500 microM AA, aggregation of rabbit PRP by 250 microM AA was completely inhibited. AA injection produced a significant decrease in circulating platelet count in vehicle-treated rabbits. However, 13-APA reduced the decrease in circulating platelet count in rabbits which survived AA injection during the 13-APA infusion. These results indicate that antagonism of thromboxane A2 receptors in platelets may be an important feature in protecting against sudden death. The difference in sensitivities of vascular and platelet thromboxane receptors as well as the accessability of 13-APA to these receptors may explain the lack of protection of 13-APA in myocardial ischemia.     

Thromboxane A2 and the endoperoxides mediate canine platelet activation

With canine platelets, arachidonate consistently induces a shape change and potentiates the response to a submaximal ADP stimulus even though it does not generally induce aggregation. We have determined that TXA₂ and possibly the endoperoxides are responsible for these effects based on the following data: (i) indomethacin (14uM) blocks both actions of arachidonate, (ii) imidazole (5uM) partially blocks both, and (iii) the endoperoxide analog, U-46619, produces effects similar to arachidonate, whereas other stable metabolites of arachidonate (PGE₂, PGF₂, PGD₂, PGI₂, 6-keto-PGF₁ and TXB₂) do not. Specifically, PGI₂ and PGD₂ inhibit ADP-induced aggregation; low concentrations of PGE₂ and PGF₂ facilitate secondary aggregation; and the other metabolites are relatively inactive. With the exception of PGF₂ , these activities are qualitiatively identical to those observed with human platelets. A decreased sensitivity of canine platelets to TXA₂ and the endoperoxides is advanced as the explanation of their diminished responsiveness to arachidonate relative to other species.