Comparison of protein and lipid composition of the human platelet α-granule membranes and glycerol lysis membranes

Platelet glycerol lysis membranes and α-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to β₂-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the α-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of ¹²⁵I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000–135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the α-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.     

PKCθ is required for hemostasis and positive regulation of thrombin-induced platelet aggregation and α-granule secretion

Platelet activation due to vascular injury is essential for hemostatic plug formation, and is mediated by agonists, such as thrombin, which trigger distinct receptor-coupled signaling pathways. Thrombin is a coagulation protease, which activates G protein-coupled protease-activated receptors (PARs) on the surface of platelets. We found that C57BL/6J and BALB/C mice that are deficient in protein kinase C θ (PKCθ), exhibit an impaired hemostasis, and prolonged bleeding following vascular injury. In addition, murine platelets deficient in PKCθ displayed an impaired thrombin-induced platelet activation and aggregation response. Lack of PKCθ also resulted in impaired α-granule secretion, as demonstrated by the low surface expression of CD62P, in thrombin-stimulated platelets. Since PAR4 is the only mouse PAR receptor that delivers thrombin-induced activation signals in platelets, our results suggest that PKCθ is a critical effector molecule in the PAR4-linked signaling pathways and in the regulation of normal hemostasis in mice.     

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.     

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca²⁺ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca²⁺/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca²⁺ titration of intact platelets using A23187 and Ca²⁺/HEDTA buffers, revealed half-maximal response at about 15 μM free Ca²⁺ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

The saliva of the medicinal leech hirudo medicinalis—II. inhibition of platelet aggregation and of leukocyte activity and examination of reputed anaesthetic effects

1. Leech saliva inhibits platelet aggregation induced by collagen, ADP and epinephrine.2. Leech saliva inhibits Superoxide production by neutrophils stimulated by tetradecanoyl phorbol acetate or polyhistidine. The effect is due in part at least to eglin.3. Reputed anaesthetic effects of leech saliva were not detected.     

Effect of platelet activating factor on leukocytes II. Enhancement of eosinophil chemotactic factor and β-glucuronidase release

Synthetic 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) and 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF) have previously been shown to induce chemotaxis and chemokinesis of human neutrophils. We present here data showing that these agents are inactive by themselves, but that they enhance neutrophil secretion once it has been initiated by a calcium ionosphore or by zymosan. Two substances, the lipid eosinophil chemotactic factor (ECF) and the lysosomal enzyme β-glucuronidase, are used as markers for neutrophil release. PAF augments secretion of both substances in a dose-dependent fashion, with lyso-PAF being less potent. The kinetics of enhancement are very rapid (<2 min) and are not reversible by washing of the cells. A pyrazoline derivative that inhibits arachidonate cyclo-oxygenation and lipoxygenation, reduces the enhancing effect of PAF and lyso-PAF. PAF, and less so lyso-PAF, are thus potentially important modulators of neutrophil secretion during inflammatory processes.     

Molecular comparison of α2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelet

Summary We have previously described a simple two-step purification technique to isolate ₂-adrenergic receptors from the rat adrenocortical carcinoma (Jaiswal, R. K. and Sharma, R. K. (1985) Biochem. Biophys. Res. Commun. 130, 58–64). Utilizing this technique we have now achieved 77 000-fold purification to apparent homogeneity of ₂-adrenergic receptors from human platelets. We have compared the biochemical characteristics of these receptors with those from the rat, which were purified 40000-fold to homogeneity.The [¹²⁵I] receptor proteins from two sources showed: (a) a single radioactive band with a Mr of 64000 as evidenced by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE); and (b) a single symmetrical peak with a pl of 4.2 by isoelectric focusing polyacrylamide gel electrophoresis. Both proteins showed typical ₂-adrenergic binding characteristics with specific binding activities of 13.85 nmol/mg and 14.17 nmol/mg protein. These values are close to the theoretical binding activity of 15.6 nmol/mg protein for 1 mol of the ligand binding 1 mol of the receptor protein. These results attest to the purity of the receptors, to its Mr of 64000, and to its acidic nature. However, the peptide maps of the radioiodinated ₂-adrenergic receptors from rat adrenocortical carcinoma and human blood platelets reveal some distinct differences which may relate to the differences in the pharmacological specificities between rodent and nonrodent ₂-adrenergic receptors.Abbreviations PAC p-aminoclonidine - PMSF Phenylmethyl-sulfonylfluoride - DTT Dithiothreitol - HPLC High Performance Liquid Chromatography     

Effect of ovariectomy in humans on serum 6-KETO-PGF1α and TXB2 concentrations and platelet fatty acids

The effects of ovariectomy on serum 6-keto-PGFI, and TXB2 concentrations and platelet fatty acids were investigated. One month after ovariectomy the levels of 6-keto-PGFI, were unaltered, whereas those of TXB2 were significantly increased. Ovariectomy had no effect on the fatty acid composition of platelets. Thus, the present study suggests that the hormonal changes at the time of menopause may modify the formation of metabolites of arachidonic acid.     

The interaction of β2-glycoprotein I with lysophosphatidic acid in platelet aggregation and blood clotting

β₂-Glycoprotein I (β₂-GPI) is a plasma protein that binds to oxidized low-density lipoprotein (LDL) and negatively charged substances, and inhibits platelet activation and blood coagulation. In this study, we investigated the interaction of β₂-GPI with a negatively charged lysophosphatidic acid (LPA) in platelet aggregation and blood clotting. Two negatively charged lysophospholipids, LPA and lysophosphatidylserine, specifically inhibited the binding of β₂-GPI to oxidized LDL in a concentration-dependent manner. Intrinsic tryptophan fluorescence studies demonstrated that emission intensity of β₂-GPI decreases in an LPA-concentration-dependent manner without a shift in wavelength maxima. LPA specifically induced the aggregation of β₂-GPI in phosphate-buffered saline, and in incubated plasma and serum, both of which are known to accumulate LPA by the action of lecithin-cholesterol acyltransferase and lysophospholipase D/autotaxin. β₂-GPI aggregated by LPA did not inhibit activated von Willebrand factor-induced aggregation, and did not prolong the activated partial thromboplastin time in blood plasma, in contrast to non-aggregated β₂-GPI. These results suggest that β₂-GPI aggregated by the binding to LPA fails to inhibit platelet aggregation and blood clotting in contrast to non-aggregated β₂-GPI.     

Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3′:5′-cyclic monophosphate

1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-(14)C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3':5'-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3':5'-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3':5'-cyclic AMP occurred by 10-30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3':5'-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3':5'-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3':5'-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3':5'-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3':5'-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.     

Co-localization of CD9 and GPIIb-IIIa (·IIb ·3 integrin) on activated platelet pseudopods and ·-granule membranes

Summary CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of ·-granule membranes. These two antigens also co-localized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from ·-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets. This revised version was published online in November 2006 with corrections to the Cover Date.     

Alterations in the extracellular catabolism of nucleotides and platelet aggregation induced by high-fat diet in rats: effects of α-tocopherol

The aim of this study was to assess whether α-tocopherol administration prevented alterations in the ectonucleotidase activities and platelet aggregation induced by high-fat diet in rats. Thus, we examined four groups of male rats which received standard diet, high-fat diet (HFD), α-tocopherol (α-Toc), and high-fat diet plus α-tocopherol. HFD was administered ad libitum and α-Toc by gavage using a dose of 50 mg/kg. After 3 months of treatment, animals were submitted to euthanasia, and blood samples were collected for biochemical assays. Results demonstrate that NTPDase, ectonucleotide pyrophosphatase/phosphodiesterase, and 5′-nucleotidase activities were significantly decreased in platelets of HFD group, while that adenosine deaminase (ADA) activity was significantly increased in this group in comparison to the other groups (P?<?0.05). When rats that received HFD were treated with α-Toc, the activities of these enzymes were similar to the control, but ADA activity was significantly increased in relation to the control and α-Toc group (P?<?0.05). HFD group showed an increased in platelet aggregation in comparison to the other groups, and treatment with α-Toc significantly reduced platelet aggregation in this group. These findings demonstrated that HFD alters platelet aggregation and purinergic signaling in the platelets and that treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition.     

Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation

Extracorporeal circulation (ECC) and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K) p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2)Y(12) and P(2)Y(1) blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2)Y(12) antagonist 2-MeSAMP, the P(2)Y(1) antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls). Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2)Y blockers (p<0.05), while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2)Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P(2)Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05). Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2)Y and PI3K blockade (p<0.05). Combined blockade of P(2)Y(12), P(2)Y(1) and PI3K p110β completely inhibits hypothermic ECC-induced activation processes. This novel finding warrants further studies and the development of suitable pharmacological agents to decrease ECC- and hypothermia-associated complications in clinical applications.     

Involvement of nuclear factor κB in platelet CD40 signaling

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.     

Inhibition of platelet adhesion by peptidomimetics mimicking the interactive β-hairpin of glycoprotein Ibα

β-Hairpin peptidomimetics mimicking the interaction sites of the platelet receptor glycoprotein (GP)Ibα with von Willebrand factor (vWF) were synthesised and evaluated for their ability to increase platelet velocity under high shear conditions and to inhibit shear-induced platelet aggregation. A cyclic and bridged dodecapeptide 2e containing a heterochiral diproline motif was identified as a lead compound for the generation of a novel class of potential antiplatelet agents.     

Dissolution of pre-existing platelet thrombus by synergistic administration of low concentrations of bifunctional antibodies against β3 integrin

Most antithrombotic approaches target prevention rather than the more clinically relevant issue of resolution of an existing thrombus. In this study, we describe a novel and effective therapeutic strategy for ex vivo clearance of pre-existing platelet thrombus by the combination of two bifunctional platelet GPIIIa49-66 ligands that target different parts of the arterial thrombus. We produced an additional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like our previously described SLK (which targets newly deposited fibrin strands surrounding the platelet thrombus), APAC destroys platelet aggregates ex vivo in an identical fashion with 85% destruction of platelet aggregates at 2 hours. The combined application of APAC and SLK demonstrated a ~2 fold greater platelet thrombus dissolution than either agent alone at a low concentration (0.025 μM). Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T(50%)) by APAC (95 ± 6.1 min) or SLK (145 ± 7.1 min) was much longer than that by combined APAC + SLK (65 ± 7.6 min) at the final concentration of 0.025 μM (APAC + SLK vs APAC, p<0.05; APAC + SLK vs SLK, p<0.01). Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.