Ticlopidine increases nitric oxide generation in heart-transplant recipients: a possible novel property of ticlopidine.

The objective of this study was to evaluate the effects of ticlopidine on the generation of eicosanoids and nitric oxide in heart-transplant recipients. In a randomized double-blind study, we studied the urinary excretion of the stable metabolites of thromboxane, prostacyclin, and nitric oxide before and after ticlopidine (250 mg/day). Platelet aggregation was significantly reduced in ticlopidine-treated patients [from 40.

Effect of various antiplatelet agents on acute arterial thrombosis in the rat.

Electrical stimulation of the rat carotid artery causes a deep medial injury and the formation of a platelet-rich thrombus. Occlusive thrombosis at sites of vessel wall injury was significantly reduced after the oral administration of clopidogrel, a potent analogue of ticlopidine, which showed dose-dependent inhibition of the thrombus formation (ED50 = 1.0 +/- 0.

Inhibitory effect of clopidogrel on platelet adhesion and intimal proliferation after arterial injury in rabbits.

The possible activity of ticlopidine and its analogue clopidogrel in early atherogenesis was investigated. Incubation of rabbit platelets with the extracellular matrix produced by endothelial cells in culture induced massive platelet adherence in vitro. This phenomenon was strongly reduced when platelets were isolated from rabbits that had been treated with a single dose of clopidogrel (10 mg/kg PO) or three doses of ticlopidine (each 200 mg/kg PO) (94% and 56% inhibition of platelet adhesion, respectively).

Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel.

The thienopyridine clopidogrel is not active in vitro and must be administered i.v. or orally, suggesting that metabolism is necessary for activity.

Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase.

The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1.

After oral administration, ticlopidine specifically inhibits ADP-induced platelet aggregation, prolongs the bleeding time and prevents thrombosis in man. Its mechanism of action is not well known. Ticlopidine inhibits ADP-induced binding of fibrinogen to platelet glycoprotein GP IIb-IIIa but not shape change and increases deaggregation.

Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats.

Antithrombotic potency of recombinant hirudins rHV2, rHV2-Lys47 and rHV2-Arg47 was studied in a model of experimental thrombosis induced by tissue factor in the rat. Venous thrombosis was induced by i.v.

Production and evaluation of recombinant hirudin.

Hirudin, a 65 amino acid polypeptide form the medicinal leech, is an extremely efficient and specific thrombin inhibitor whose therapeutic potential has been demonstrated in a number of animal models. We have developed protocols for the production of recombinant hirudin by secretion from S. cerevisiae and carried out a full biologic evaluation of the purified product.

Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin.

The hirudin variant HV2 was modified by in vitro site-specific mutagenesis of HV2 cDNA to generate HV2(Asn-47----Lys), HV2(Asn-47----Arg) and HV2(Lys-35----Thr, Asn-47----Lys). Residues 35 and 47 are positioned respectively within the finger and prothrombin-like domains of hirudin, both of which have been suggested as thrombin binding sites. The modified polypeptides were synthesized in Saccharomyces cerevisiae using a secretion vector and purified from culture supernatants.

Effect of PCR 4099 on ADP-induced calcium movements and phosphatidic acid production in rat platelets.

Antiplatelet activity of PCR 4099, an analogue of ticlopidine, resides in its specific effect against exogenous as well as released ADP. This study investigated in rat platelets the effects of the drug on ADP-induced shape change, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Shape change and influx of Ca2+ ions across the plasma membrane were not modified after PCR 4099 administration using aspirin-treated platelets.

ADP plays a key role in thrombogenesis in rats.

The relative importance of ADP, arachidonic acid metabolites and serotonin as thrombogenic factors was evaluated in rats by comparing, after oral administration, the effects of two inhibitors of ADP-induced platelet aggregation (ticlopidine and PCR 4099), three cyclo-oxygenase inhibitors (aspirin, triflusal and indobufen) and a selective serotonin 5HT2 receptor antagonist (ketanserin) on platelet aggregation, in four platelet-dependent thrombosis models and on bleeding time. Platelet aggregation induced by ADP and collagen was completely inhibited by ticlopidine and PCR 4099 whereas only the collagen aggregation was reduced by the cyclo-oxygenase inhibitors. Ketanserin or a depletion of platelet serotonin by reserpine did not affect platelet aggregation.

Broad spectrum anti-platelet activity of ticlopidine and PCR 4099 involves the suppression of the effects of released ADP.

Aggregation and serotonin secretion were studied in washed rat platelets after oral administration of ticlopidine or its more potent analog PCR 4099. Besides a complete suppression of the ADP-induced aggregation, the two drugs significantly inhibited aggregation and secretion induced by three protein kinase C activators (1-oleoyl-2-acetyl-sn-glycerol, OAG; 12-0-tetradecanoyl phorbol-13-acetate, TPA; phospholipase C), by the calcium ionophore A 23187 and by thrombin. The highest inhibition was observed at low stimuli concentrations but could be partly or almost completely overcome by increasing their concentrations.

Arterial reactivity, blood pressure, and plasma levels of atrial natriuretic peptides in normotensive and hypertensive rats: effects of acute and chronic administration of atriopeptin III.

We compared acute and chronic effects of atriopeptin III in normotensive and spontaneously hypertensive rats. Atriopeptin III relaxed isolated aortae and intrarenal microarteries but not coronary and mesenteric microarteries of normotensive rats. Effects on arterial smooth muscle were comparable in hypertensive and normotensive rats and were not affected by long-term treatment of the animals with the peptide.

Low prostacyclin synthetase activity of fetal rat aorta. Progressive increase after birth.

Aortae from fetal or 3 weeks old rats produced very small amounts of PGI2, prostacyclin. This production increased from 4 weeks on, reaching adult values at about ten weeks. This maturation seemed to be predominantly determined by a change in the PGI2 synthetase system, rather than in arachidonic acid availability, phospholipase or cyclo-oxygenase activity.

Abnormal prostacyclin metabolism in the hemolytic uremic syndrome: equivocal effect of prostacyclin infusions.

In a child with the hemolytic uremic syndrome, plasma 6 keto-prostaglandin F1 alpha levels remained undetectable throughout the acute phase of the disease. The patient's plasma failed to stimulate prostacyclin production by "exhausted" rat aorta rings. In vitro study of the patient's vessels indicated that they retained the capacity to synthesize prostacyclin from exogenous arachidonic acid but that their endogenous arachidonic acid stores were either depleted or non-available.

A thromboxane synthetase inhibitor reorients endoperoxide metabolism in whole blood towards prostacyclin and prostaglandin E2.

During incubation of citrated blood at 37 degrees C the levels of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and prostaglandin E2 (PGE2) remain constant, but rise markedly within one minute after the addition of collagen, particularly when thromboxane synthetase is blocked. The amount of 6-keto PGF1 alpha formed is dose-dependent for both collagen and the thromboxane synthetase inhibitor (UK-37,248). Moreover, the number of platelets will determine the extent of the 6-keto PGF1 alpha jump, that does not occur when blood is drawn after aspirin ingestion.

Familial bleeding tendency with partial platelet thromboxane synthetase deficiency: reorientation of cyclic endoperoxide metabolism.

Three family members from three successive generations presented with a moderate bleeding tendency and a functional platelet defect. They had absent aggregation with arachidonic acid (0.6--3 microM), reversible aggregation with ADP (4 microgram) and cyclic endoperoxide analogues, single wave aggregation only with adrenaline (5.

Thromboxane synthetase inhibition as antithrombotic strategy.

The imidazole derivative UK-37 248, a thromboxane synthetase inhibitor, reduces the in-vitro formation of thromboxane B2 and hydroxyheptadecatrienoic acid by washed platelets, and this is compensated for by an increased production of prostaglandins E2 and F2 alpha; arachidonic acid challenged platelets pretreated with UK-37 248 also stimulate the production of prostacyclin by aspirin pretreated cultured endothelial cells. In a double-blind placebo controlled study to examine the in vivo properties of UK-37 248, human volunteers ingested 200 mg of the compound. Their serum thromboxane B2 levels dropped and their plasma 6-keto-prostaglandin F1 alpha values rose.

The effect of clinical prostacyclin infusions in advanced arterial disease on platelet function and plasma 6-keto PGF1 alpha levels.

We have infused synthetic prostacyclin (PGI2) continuously for approximately 72 h at the maximum tolerated dose (ranging from 5 to 60 ng/kg/min) into nine patients with advanced arterial disease. Prior to the infusion seven out of nine patients had spontaneous platelet aggregation and five out of six patients tested had an abnormal circulating platelet aggregate ratio. During the infusion only one patient still had spontaneous aggregation and all the abnormal circulating platelet aggregate ratios returned to the normal range.