R68070, a combined thromboxane/endoperoxide receptor antagonist and thromboxane synthase inhibitor, inhibits human platelet activation in vitro and in vivo: a comparison with aspirin

We have investigated the effects of R68070 on platelet function in vitro and in vivo. The drug inhibits U46619-induced aggregation (IC50 = 1.2 x 10(-6) mol/L), blocks serum thromboxane formation (IC50 = 1 x 10(- 7) mol/L), and increases serum prostaglandin (PG)E2 and 6-keto-PGF1 alpha levels, indicating that it combines thromboxane receptor blocking and thromboxane synthase inhibiting properties. The thromboxane- dependent aggregation of blood platelets is blocked by R68070, whereas no inhibition of thromboxane independent pathways occurs. A double- blind, randomized, cross-over study was performed on nine volunteers, comparing 400 mg placebo, 400 mg aspirin, and 400 mg R68070. Thromboxane-dependent aggregations were significantly inhibited by R68070 and by aspirin, the latter still having the most pronounced action. However, R68070 was clearly more powerful than aspirin (P less than .0005) in prolonging the bleeding time. Serum TxB2 formation was completely inhibited with both treatments, whereas serum 6-keto-PGF1 alpha and PGE2 and intralesional 6-keto-PGF1 alpha were inhibited after aspirin and stimulated after R68070. We conclude that R68070 inhibits platelet thromboxane synthase and its thromboxane receptor both in vitro and in vivo; local reorientation of cyclic endoperoxide metabolism toward prostacyclin induces a stronger inhibition of hemostasis than that produced by aspirin.

R68070, a combined thromboxane/endoperoxide receptor antagonist and thromboxane synthase inhibitor, inhibits human platelet activation in vitro and in vivo: a comparison with aspirin

We have investigated the effects of R68070 on platelet function in vitro and in vivo. The drug inhibits U46619-induced aggregation (IC50 = 1.2 x 10(-6) mol/L), blocks serum thromboxane formation (IC50 = 1 x 10(- 7) mol/L), and increases serum prostaglandin (PG)E2 and 6-keto-PGF1 alpha levels, indicating that it combines thromboxane receptor blocking and thromboxane synthase inhibiting properties. The thromboxane- dependent aggregation of blood platelets is blocked by R68070, whereas no inhibition of thromboxane independent pathways occurs. A double- blind, randomized, cross-over study was performed on nine volunteers, comparing 400 mg placebo, 400 mg aspirin, and 400 mg R68070. Thromboxane-dependent aggregations were significantly inhibited by R68070 and by aspirin, the latter still having the most pronounced action. However, R68070 was clearly more powerful than aspirin (P less than .0005) in prolonging the bleeding time. Serum TxB2 formation was completely inhibited with both treatments, whereas serum 6-keto-PGF1 alpha and PGE2 and intralesional 6-keto-PGF1 alpha were inhibited after aspirin and stimulated after R68070. We conclude that R68070 inhibits platelet thromboxane synthase and its thromboxane receptor both in vitro and in vivo; local reorientation of cyclic endoperoxide metabolism toward prostacyclin induces a stronger inhibition of hemostasis than that produced by aspirin.

Increased Response to Arachidonic Acid and U-46619 and Resistance to Inhibitory Prostaglandins in Patients with Chronic Myeloproliferative Disorders

SummaryIn patients with myeloproliferative disorders (MPD) a group of related diseases of the bone marrow stem cell and recurrent haemorrhagic and/or thrombotic complications, the production of aggregating prostaglandins (PGs) may be normal or slightly reduced, while PGI2 production is normal. However, MPD platelet sensitivity to antiaggregatory PGs is still unknown.We studied the potency of PGD2, PGI2 and PGEi as inhibitors of platelet aggregation induced by threshold aggregating concentrations of arachidonic acid and U-46619-analogue of the cyclic endoperoxide PGH2 in 20 patients with MPD in comparison with healthy controls, with the aim of evaluating the sensitivity of MPD platelets to antiaggregatory PGs. In these patients platelet prostanoid metabolism was normal. However, the functional response of platelets to aggregating and antiaggregating prostanoids was shifted towards potentially increased platelet aggregation response. These findings could have a clinical relevance in view of the haemostatic and thrombotic complications so frequent in MPD.

Regulation of Arachidonic Acid-Dependent Ca++ Influx in Human Platelets

SummaryQuin2 was used to study the rise in cytoplasmic free calcium ([Ca++]i) and the role of prostaglandin (PG) endoperoxides/thromboxane A2 (TxA2), reduced glutathione (GSH), ADP and the glycoprotein (GP) Ilb IIIa complex in mediating [Ca++]i rise during àiachidonic acid(AA)induced platelet aggregation. Ca++mobilization, mostly due to an influx across the plasma membrane, is completely inhibited by aspirin and persists after selective blockade of TxA2 synthase by dazoxiben. GSH total depletion causes a complete aggregation block and 90% inhibition of the transient: U-46619, a stable analog of cyclic endoperoxide PGH2, stimulates [Ca++]i transient in aspirintreated or in GSH depleted platelets. ADPscavengers, ATP (which competes for the ADP receptor), and monoclonal antibodies against the GP Ilb IIIa complex reduce AAinduced Ca++ influx. Therefore, PG endoperoxides alone or a PGH2/TxA2 mimetic stimulate Ca++ influx. Synthesis of PGH2 and TxA2 depends on the availability of GSH, which acts as the reducing cofactor for the PG peroxidase activity. ADP and GP II b ill a are regulating factors of AA mediated Ca++ influx during platelet activation.

Effect of Indomethacin and Dazoxiben on Intravascular Platelet Aggregation in the Anaesthetized Rabbit

Summary Collagen (10-40 μg kg−1), thrombin (1-10 units kg−1), adenosine diphosphate (ADP; 3-300 pμ kg−1), 1-0-hexadecyl Paf-acether and 1-0-octadecyl Paf-acether (1-300 ng kg−1) administered by bolus intravenous injection each caused dose-dependent thrombocytopoenia accompanied by marked hypotension in anaesthetized rabbits. Responses to ADP and the Paf-acether derivatives were transient in nature (3-8 min) whereas those induced by collagen and thrombin were always of longer duration (5-20 min) and frequently fatal at high doses. Responses to collagen, thrombin, and the Paf-acether derivatives were invariably accompanied by substantial, dose-related increases in plasma levels of thromboxane B2 in samples obtained 30 s after agonist administration, whereas following ADP, no change in plasma thromboxane B2 was detected at any dose level. Indomethacin (3.0 mg kg−1 by infusion) had no effect on responses to thrombin or Paf-acether, partially inhibited collagen-induced thrombocytopenia, and potentiated responses to ADP. In contrast, dazoxiben (10 mg kg−1 by infusion) partially but significantly inhibited responses to thrombin, whereas those induced by collagen, Paf-acether or ADP were unchanged. These results indicate that in this model of intravascular aggregation, whilst platelet responses to collagen and thrombin appear partially dependent on intact cyclic endoperoxide and thromboxane A2 synthetic capacity respectively, responses to ADP and Paf-acether are independent of arachidonate metabolism via cyclo-oxygenase despite measurably increased TXB2 formation in the latter case.

Inhibition of breathing movements in fetal sheep by prostaglandins

We studied the effects of infusions (duration 13.4 +/- 2.9 h) of prostaglandins (PG) on fetal breathing movements (FBM) in 12 fetal sheep at 122–141 days gestation. We gave similar doses (1.1 +/- 0.7 microgram . kg-1 . min-1) of PGE2 (8 studies), PGF2 alpha (5 studies), and cyclic endoperoxide analogues (6 studies). During control periods (304 h), incidence of FBM was 41%; this decreased during every infusion. With PGE2, incidence of FBM markedly decreased to 9.8% of control (P less than 0.001). With the other agents the decrease was less profound; incidence of FBM with PGF2 alpha was 63.7% of control and with endoperoxide analogues 69.4% of control (P less than 0.05 for both). During infusions there were no changes in fetal heart rate, arterial blood pressure, pH, or blood gas tensions. In three fetuses (5 infusions) with electrocorticogram recordings, incidence of low-voltage fast activity was unchanged from control values. Inhibition of FBM by PGE2, combined with previous results showing stimulation of FBM by PG synthetase inhibitors, suggests that endogenous PG may inhibit breathing movements in utero and that a change in PG metabolism may contribute to the change in control of breathing at birth.

Effects of an Epoxymethano Stable Analogue of Prostaglandin Endoperoxides (U-46619) on Human Platelets

SummaryU-46619 is a stable epoxymethano analogue of cyclic endoperoxide PGH2. We studied platelet aggregation, 14C-5HT release, LDH extrusion and prostaglandin and thromboxane production induced by this compound in platelet-rich plasma samples from 15 healthy volunteers. Each subject was tested both before and 90 min after aspirin (500 mg) ingestion. The threshold aggregating concentration (TAC) of U-46619 ranged between 0.18 and 0.90 µM. Aggregation was maximal between 40 and 60 min after venipuncture and was concentration-dependent. At concentrations below the TAC, U-46619 induced primary reversible aggregation with minimal 14C-5HT release. At TAC or higher concentrations aggregation and release proceded as parallel events. Neither prostaglandin or thromboxane production nor LDH loss could be detected in any of the situations tested. Aspirin ingestion did not modify the pattern of platelet responses. In unstirred, not aggregated platelet samples 14C-5HT release by U-46619 occurred to a similar extent as in stirred, aggregated platelet samples. Addition to citrated PRP of 0.3 mM Na2 EDTA blocked both aggregation and release induced by U-46619. This compound, however, aggregated washed platelets resuspended in Ca++-free-tyrode-albumin containing fibrinogen. The mechanism by which U-46619 activates platelets differs from that of all other common aggregating agents.

Platelet Aggregation Induced by a Synthetase-Like Substance and Phospholipase-A.

Platelets contain arachidonic acid-bearing phospholipids and phospholipase-A (Phl-A); it has been suggested that exposure of these substances to each other by stimuli, such as thrombin or adenosine diphosphate (ADP), would result in liberation of arachidonic acid (AA), which is the precursor of cyclic endoperoxide; a factor causing irreversible aggregation of platelets.By using a saline eluate of a polyurethane (SPU, -polyester elastomer of the thermoplastic type-, secured by exposing saline for two minutes to the inner surface of a polyurethane bag) we observed that platelet (human PRP) aggregation (second phase type, irreversible) could be induced by Phl-A plus SPU or AA plus SPU. These mixtures failed to aggregate aspirinized platelets. Aspirin, however, incubated for 10 minutes with SPU, Phl-A or AA did not inhibit platelet aggregation induced by the addition of the missing factor. In control experiments no aggregation was produced by SPU-saline, saline-Phl-A or saline-AA. SPU plus suboptimal concentrations of ADP or epinephrine induced an immediate in onset aggregation. Phl-A added in reversibly, by ADP, aggregated platelets produced a second phase of aggregation.The results show that AA which is the precursor of cyclic endoperoxide could be liberated by Phl-A. The results also provide evidence that at least two enzymes (synthetase-α and synthetase-β; α is inactive whereas β is active and can be blocked by apsirin) are involved in the process of AA activation and biosynthesis of cyclic endoperoxide.Supported by a grant from the U.S. Public Servie HL-15425.