Theophylline inhibits platelet aggregation, prostaglandin and thromboxane production by a mechanism which is independent of cyclic AMP

Theophylline (0.05-1.0 mM) greatly enhanced the inhibition of platelet aggregation by prostaglandin E1 and at higher concentrations (1.0-4.0 mM) theophylline alone inhibited platelet aggregation, by a mechanism which did not involve significant changes in platelet cAMP levels. These concentrations of theophylline and theophylline in combination with prostaglandin E1, inhibited the synthesis of thromboxane A2, prostaglandin E2 and F2a in stimulated platelets. Theophylline also inhibited arachidonic acid-stimulated platelet aggregation suggesting an effect on cyclooxygenase activity. The calcium ionophore A23187 prevented and reversed the inhibition of platelet aggregation by theophylline, suggesting that theophylline inhibits the rise in intracellular calcium levels which occurs in response to the synthesis of prostaglandin endoperoxides and thromboxane A2.     

Novel inhibitory actions on platelet thromboxane and inositolphosphate formation by xanthones and their glycosides

Xanthones and their glycosides were tested for their antiplatelet activities in washed rabbit platelets. Tripteroside acetate and norathyriol acetate were the most potent inhibitors. Tripteroside acetate inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, platelet-activating factor (PAF), collagen, ionophore A23187 and thrombin. The IC50 values of tripteroside acetate toward arachidonic acid- (100 microM) and collagen- (10 micrograms/ml) induced platelet aggregation were 10 and 30 micrograms/ml respectively. It inhibited thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, thrombin and ionophore A23187 and also that caused by the incubation of lysed platelet homogenate with arachidonic acid. Tripteroside acetate decreased the formation of inositolphosphate caused by thrombin, collagen and PAF, whereas it had no direct effect on fibrinogen-platelet interaction. It is concluded that xanthone derivatives inhibited platelet aggregation and release reaction by diminishing thromboxane formation and phosphoinositide breakdown.     

Biological activity and metabolism of 20-hydroxyeicosatetraenoic acid in the human platelet.

1. The cytochrome P-450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), was found to be a potent, dose-dependent inhibitor of platelet aggregation and inhibitor of thromboxane biosynthesis induced by arachidonic acid (IC50 5.2 +/- 1.5 microM), A23187 (IC50 16.2 +/- 5.4 microM), and U46619 (IC50 7.8 +/- 2.4 microM). 20-HETE did not inhibit thrombin-induced aggregation. 2. The human platelet metabolized 20-HETE to a series of novel metabolites formed by cyclo-oxygenase as well as lipoxygenase pathways. The structures of the metabolites were identified by mass spectrometry as 20-hydroxy-thromboxane B2, 12,17-dihydroxyheptadecatrienoic acid, 12,20-dihydroxyeicosatetraenoic acid, and 11,20-dihydroxyeicosatetraenoic acid. 3. The identification of the 11-hydroxy metabolite of 20-HETE suggests that 20-HETE is less efficiently cyclized to an endoperoxide intermediate by cyclo-oxygenase than is arachidonate. 4. Although some biological activity of 20-HETE may be related to competition with endogenous arachidonate for cyclo-oxygenase metabolism, the predominant mechanism of action of 20-HETE appears to be through antagonism of the prostaglandin H2/thromboxane A2 receptor.     

Pharmacology of L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpro pan oic acid); a potent, selective thromboxane/prostaglandin endoperoxide antagonist

L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpropa noic acid) has been studied in vitro on the guinea-pig tracheal chain, pulmonary artery and thoracic aorta ring and shown to be a potent, competitive antagonist of contractions induced by the prostaglandin endoperoxide analogue, U-44069 (pA2 values 8.0, 8.4 and 8.0 respectively). Selectivity on the guinea-pig trachea was indicated by non-competitive antagonism of contractions induced by prostaglandin D2 and minimal activity against contractions induced by leukotriene D4, prostaglandin F2 alpha, serotonin, histamine and acetylcholine. L-655,240 was a potent inhibitor of the aggregation of washed human platelets induced by U-44069 (IC50 value 7 X 10(-9) M) and inhibited aggregation of human platelet rich plasma induced by U-44069, U-46619, thromboxane A2 and collagen but not ADP or platelet activating factor. In vivo i.v. L-655,240 administered to guinea-pigs inhibited bronchoconstriction induced by i.v. U-44069 and arachidonic acid (ED50 values 0.09 and 0.23 mg kg-1) but not histamine, acetylcholine or serotonin. When administered to rhesus monkeys (3 and 10 mg/kg p.o.), L-655,240 inhibited ex vivo platelet aggregation induced by U-44069 but not ADP. It is concluded that L-655,240 is a potent, selective, orally active thromboxane/prostaglandin endoperoxide antagonist.     

BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells

A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.     

Effect of a potent cyclooxygenase inhibitor, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), on human platelets

Because the metabolites of arachidonic acid participate in many physiopathological responses, including inflammation and platelet aggregation, cyclooxygenase inhibitors are important in the treatment of associated diseases. A biologically active compound, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), selectively and concentration dependently inhibited aggregation of platelets from man and ATP release caused by arachidonic acid (200 microM) and collagen (10 microg mL(-1)) without affecting the aggregation caused by thrombin (0.1 U mL(-1)) and U46619 (2 microM). The IC50 value (drug concentration inhibiting maximum response by 50%) of KTC-5 for aggregation induced by arachidonic acid and collagen was 0.11+/-0.04 microM and 0.20+/-0.03 microM, respectively. This inhibitory effect of KTC-5 was reversible and time dependent. KTC-5 specifically inhibited intracellular calcium mobilization initiated by arachidonic acid or collagen without affecting that caused by thrombin or U46619 in human platelets. Furthermore, KTC-5 inhibited thromboxane B2 and prostaglandin D2 formation provoked by arachidonic acid. The IC50 value of KTC-5 for arachidonic-acid-induced thromboxane B2 formation was 0.07+/-0.02 microM. Based on these observations, the data indicated that KTC-5 potently inhibited human platelet aggregation and ATP release mainly via the inhibition of the cyclooxygenase-1 activity. Moreover, KTC-5 inhibited lipopolysaccharide-induced prostaglandin E2 formation in RAW264.7 cells in the presence of external arachidonic acid with an IC50 value of 0.17+/-0.06 microM. Immunoblot analysis showed that KTC-5 did not affect the cyclooxygenase-2 expression in the presence of lipopolysaccharide on RAW264.7 cells. This result indicated that KTC-5 affects the activity of cyclooxygenase-2. According to these data, we concluded that KTC-5 is a cyclooxygenase inhibitor for both subtypes.     

An antithrombotic agent, NQ301, inhibits thromboxane A2 receptor and synthase activity in rabbit platelets

In previous studies we have reported that NQ301, a synthetic 1,4-naphthoquinone derivative, displays a potent antithrombotic activity, and that this might be due to antiplatelet effect, which was mediated by the inhibition of cytosolic Ca(2+) mobilization in activated platelets. In the present study, the effect of NQ301 on arachidonic acid cascade in activated platelets has been examined. NQ301 concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen (10 microg/ml), arachidonic acid (100 microM) and U46619 (1 microM), a thromboxane A2 receptor agonist, with IC50 values of 0.60+/-0.02, 0.78+/-0.04 and 0.58+/-0.04 microM, respectively. NQ301 also produced a shift to the right of the concentration-effect curve of U46619, indicating a competitive type of antagonism on thromboxane A2/prostaglandin H2 receptor. NQ301 slightly inhibited collagen-induced arachidonic acid liberation. In addition, NQ301 potently suppressed thromboxane B2 formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner, but had no effect on the production of prostaglandin D2, indicating an inhibitory effect on thromboxane A2 synthase. This was supported by thromboxane A2 synthase activity assay that NQ301 concentration-dependently inhibited thromboxane B2 formation converted from prostaglandin H2. Moreover, NQ301 concentration-dependently inhibited 12-hydroxy-5,8,10,14-eicosatetraenoic acid formation by platelets that were exposed to arachidonic acid. Taken together, these results suggest that NQ301 has a potential to inhibit thromboxane A2 synthase activity with thromboxane A2/prostaglandin H2 receptor blockade, and modulate arachidonic acid liberation as well as 12-hydroxy-5,8,10,14-eicosatetraenoic acid formation in platelets. This may also be a convincing mechanism for the antithrombotic action of NQ301.     

CGS 12970: a novel, long acting thromboxane synthetase inhibitor.

CGS 12970 is a potent selective inhibitor of human platelet thromboxane synthetase in vitro (IC50 = 12 nM). It is four orders of magnitude less potent as an inhibitor of sheep seminal vesicle cyclooxygenase, bovine aorta prostacyclin synthetase and human leucocyte 15-lipoxygenase. The compound inhibited collagen-induced thromboxane B2 production by human platelets in vitro without an effect on the accompanying platelet aggregation induced by collagen, ADP, platelet activating factor, thrombin, arachidonic acid or the prostaglandin mimetic, U 46619. Administration of CGS 12970 to rats inhibited collagen-induced thromboxane B2 production but had no effect on platelet aggregation ex vivo. It also had no effect on platelet aggregation induced by thrombin or on plasma clotting times. A single oral dose of 1 or 3 mg kg-1 to rabbits inhibited thromboxane B2 production in clotting blood ex vivo for 12 or 24 h respectively. CGS 12970 inhibited thromboxane B2 production in vivo induced by intravenous administration of collagen to rats or calcium ionophore to guinea-pigs. In both cases there was a concomitant elevation of immunoreactive 6-keto-prostaglandin F1 alpha but no effect on the induced thrombocytopenia. As with other thromboxane synthetase inhibitors, CGS 12970 prolonged tail bleeding time in the rat. However, CGS 12970 was not as potent as other thromboxane synthetase inhibitors in this test. In addition to these usual effects of thromboxane synthetase inhibitors, CGS 12970 inhibited the thrombocytopenia induced by the Forssman reaction or ADP administration. In these tests the effect of the compound was of short duration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution and metabolism of arachidonic acid in rabbit platelets during aggregation and its modification by drugs.

1 Gas chromatographic and radio-isotope labelling techniques have been used to establish the origin of the arachindonic acid used by the platelet cyclo-oxygenase for the synthesis of pro-aggregatory prostaglandin endoperoxide derivatives. 2 Measurements of total platelet arachidonate content indicated that more than 95% is esterified in the phosphatide fraction of the cells. 3 During aggregation by collagen or thrombin as much as 80% of the total platelet arachidonate may be liberated and transformed by the platelet enzymes into hydroxyacids and other more polar compounds. 4 The phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol fractions are major sources of the arachidonate thus used. 5 Indomethacin, which prevents platelet aggregation by inhibiting the cyclo-oxygenase, did not affect this release of arachidonate from the phosphatides but did prevent the transformation of arachidonate to endoperoxide derivatives. 6 Mepacrine, a drug which possesses weak anti-phospholipase activity in platelets, also prevents aggregation by collagen or thrombin, but seems to do so by preventing substrate release from the phosphatide fraction. 7 It is suggested that phospholipase A2 plays a key role in the initial events during platelet aggregation induced by collagen.     

Diversion of prostaglandin endoperoxide metabolism by selective inhibition of thromboxane A2 biosynthesis in lung, spleen or platelets.

Infusion of arachidonic acid through the guinea pig lung or the cat spleen causes a release of thromboxane A2 and prostaglandins, as measured by bioassay. After incubation of human platelets with arachidonate similar metabolites are formed, as demonstrated chromatographically. Infusion of imidazole (50-75 microgram/ml) through the lung or spleen specifically inhibits thromboxane A2 production and diverts the pathway to the prostaglandins, mainly prostaglandin F2alpha. In human platelets imidazole causes a dose-dependent inhibition of thromboxane A2 formation (ID50 5.5 X 10(-4) M). This inhibition is accompanied by a dose-dependent increase in prostaglandin F2alpha. Since thromboxane A2 induces platelet aggregation and is a potent vasoconstrictor, diversion of pathways to prostaglandins with opposite or less potent action might be of relevance in the treatment of cardiovascular diseases.     

Inhibitory effect of various traditional Chinese medicines on rabbit platelet phospholipase A2 in vitro and suppressive effect of t?ki-syakuyaku-san on increased aggregability in hypercholesterolemic rabbit ex vivo

The inhibitory mechanism of 6 traditional Chinese medicines on rabbit platelet aggregation in vitro, and the suppressive effect of oral administration of T?ki-syakuyakusan on hyper-aggregability of the platelet from rabbit fed high cholesterol diet for 2 months, were investigated. Collagen-induced aggregation was inhibited by Keisi-bukury?gan, Kami-sy?y?-san, Dai-saiko-t?, T?ki-syakuyaku-san, Hatimi-zi?-gan and Sy?-saiko-t? in their lower concentrations than those inhibiting arachidonic acid- and thrombin-induced aggregation. These traditional Chinese medicines inhibited the release of [3H]arachidonic acid from membrane phospholipids by phospholipase A2, in [3H]arachidonic acid-labelled platelets under stimulation with collagen and thrombin in the concentration ranges that inhibited each aggregation. In their higher concentrations to inhibit arachidonic acid-induced aggregation, they suppressed the conversion of arachidonic acid to thromboxane A2 by about 50%. However, they had no effect on diacylglycerol formation induced by thrombin. The oral administration of T?ki-syakuyaku-san depressed the increased aggregability of platelets from rabbit fed high cholesterol diet by 20-40% at the period of 1-2 months of feeding, without affecting plasma and platelet cholesterol level. These results indicate that the traditional Chinese medicines used here have an inhibitory effect on platelet phospholipase A2 activation, rather than on cyclooxygenase, and therefore inhibit platelet activation in vitro and ex vivo.     

Inhibitory effect of 8-bromo cyclic GMP on an extracellular Ca2+-dependent arachidonic acid liberation in collagen-stimulated rabbit platelets

The inhibitory effect of cyclic GMP on collagen-induced platelet activation was studied using 8-bromo cyclic GMP (8brcGMP) in washed rabbit platelets. Addition of collagen (1 micrograms/ml) to platelet suspension caused shape change and aggregation associated with thromboxane (TX) A2 formation. 8brcGMP (10-1000 microM) inhibited collagen-induced platelet aggregation and TXA2 formation in a concentration-dependent manner. 8brcGMP did not affect platelet cyclooxygenase pathways, but markedly inhibited collagen-induced arachidonic acid (AA) liberation from membrane phospholipids in [3H]AA-prelabeled platelets, indicating that the inhibitory effect of 8brcGMP on collagen-induced aggregation is due to an inhibition of AA liberation. In [32P]orthophosphate-labeled platelets, collagen stimulated phosphorylation of a 20,000 dalton (20-kD) and 40-kD proteins. 8BrcGMP stimulated phosphorylation of a specific protein having molecular weight of 46-kD and inhibited collagen-induced both 20- and 40-kD protein phosphorylation. Collagen could stimulate the AA liberation without activation of phospholipase C or Na+-H+ exchange, but could not in the absence of extracellular Ca2+. These findings suggest that cyclic GMP inhibits collagen-induced AA liberation which is mediated by an extracellular Ca2+-dependent phospholipase A2. However, cyclic GMP seems to inhibit the Ca2+-activated phospholipase A2 indirectly, since 8brcGMP had no effect on Ca2+ ionophore A23187-induced platelet aggregation or AA liberation. It is therefore suggested that cyclic GMP may regulate collagen-induced increase in an availability of extracellular Ca2+ which is responsible for phospholipase A2 activation in rabbit platelets.     

Effect of crotapotin and heparin on the rat paw oedema induced by different secretory phospholipases A2.

The effects of crotapotin (a non-toxic and non-enzymatic acid polypeptide naturally complexed with phospholipase A2) and heparin on rat paw edema induced by different secretory phospholipases A2 (sPLA2) have been investigated. The ability of crotapotin to affect the enzymatic activity of the sPLA2(s) have also been evaluated. Secretory PLA2(s) obtained from both snake (Naja naja, Naja mocambique mocambique, Crotalus adamanteus and Crotalus durissus terrificus) and bee (Apis mellifera) venoms as well as that from bovine pancreas were used in this study. Rat paw oedema was induced by a single subplantar injection of the sPLA2s (5-30 microg/paw) in absence and presence of either crotapotin (10-100 microg/paw) or heparin (50 U/paw). Paw volume was measured using a hydroplethysmometer. Phospholipase A2 from Naja naja, Naja mocambique mocambique, Apis mellifera venoms and the basic component of Crotalus durissus terrificus venom all induced dose-dependent rat paw oedema whereas those from Crotalus adamanteus venom and bovine pancreas were ineffective. Paw oedema induced by PLA2(s) from both Naja naja and Apis mellifera venoms was significantly (P < 0.05) inhibited by crotapotin (0.1-100 microg/site) whereas the Naja mocambique mocambique venom PLA2-induced oedema was significantly potentiated (P < 0.05) by this polypeptide (40 microg/site). On the other hand, heparin (50 U/paw) had no effect on the paw oedema induced by PLA2 from Naja naja and Apis mellifera venoms but significantly inhibited the Naja mocambique mocambique venom PLA2-induced oedema. The measurement of the in vitro phospholipasic activity revealed that crotapotin inhibited by 60-70% the enzymatic activities of PLA2(s) from Crotalus adamanteus, Naja mocambique mocambique, Apis mellifera venoms and bovine pancreas. Our results suggest that despite the great homology between the various types of sPLA2 they interact with crotapotin on cell surfaces in different ways leading to either inhibition or potentiation of the paw oedema by a mechanism unrelated to their enzymatic activities. Since heparin reduced paw oedema induced by PLA2 from Naja mocambique mocambique venom it is likely that this sPLA2 is similar to the novel heparin-sensitive PLA2 found in mast cells.     

Antiplatelet activity of carnosic acid, a phenolic diterpene from Rosmarinus officinalis

Carnosic acid is a major phenolic diterpene derived from Rosmarinus officinalis and has been reported to have antioxidant, antibacterial, anticancer, antiobese and photoprotective activities. This study investigated the antiplatelet activity of carnosic acid. carnosic acid significantly inhibited collagen-, arachidonic acid-, U46619- and thrombin-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 39+/-0.3, 34+/-1.8, 29+/-0.8 and 48+/-2.9 microM, respectively, while it failed to inhibit PMA-(a direct PKC activator) and ADP-induced platelet aggregation. In agreement with its antiplatelet activity, carnosic acid blocked collagen-, arachidonic acid-, U46619- and thrombin-mediated cytosolic calcium mobilization. accordingly, serotonin secretion and arachidonic acid liberation were also inhibited in a similar concentration-dependent manner. However, in contrast to the inhibition of arachidonic acid-induced platelet aggregation, carnosic acid had no effect on the formation of arachidonic acid-mediated thromboxane A2 and prostaglandin D2, thus indicating that carnosic acid has no effect on the cyclooxygenase and thromboxane A2 synthase activity. Overall, these results suggest that the antiplatelet activity of carnosic acid is mediated by the inhibition of cytosolic calcium mobilization and that carnosic acid has the potential of being developed as a novel antiplatelet agent.     

Effects of salicylic and acetylsalicylic acid alone and in combination on platelet aggregation and prostanoid synthesis in man.

The present study was designed to investigate the effects of salicylate on the antiplatelet action of acetylsalicylic acid as well as on in vivo prostanoid formation and platelet function in healthy volunteers. In the first study six female volunteers received 350 mg acetylsalicylic acid intravenously, with and without previous oral administration of sodium salicylate (1200 mg daily for 3 days). Urinary prostanoid excretion as well as platelet aggregation and thromboxane formation were measured before and during salicylate and after acetylsalicylic acid. In the second study seven female volunteers received sodium salicylate (52.6 mg kg-1) or acetylsalicylic acid (60.7 mg kg-1) for 8 days in a randomized cross-over protocol. Urinary prostanoid excretion, platelet aggregation and thromboxane formation as well as salicylate plasma concentrations were determined before, during and after administration of each drug. Sodium salicylate did not impair the complete suppression of arachidonic acid-induced platelet thromboxane formation and aggregation obtained by the single intravenous dose of acetylsalicylic acid in the first study. Sodium salicylate in the second study did not affect urinary excretion of prostaglandin E2, its major urinary metabolite (7 alpha-hydroxy-5,11-diketo-tetranor-prostane-1,16-dioic acid), and 2,3-dinor-6-keto-prostaglandin F1 alpha, the main urinary metabolite of epoprostenol (prostacyclin, PGI2). In contrast, acetylsalicylic acid significantly decreased excretion rates of these prostanoids by 64, 59 and 61%, respectively. In both studies platelet aggregation and thromboxane formation induced by collagen, thrombin or arachidonic acid were not significantly affected by salicylate administration, whereas acetylsalicylic acid inhibited platelet aggregation induced by all three agents as well as thrombin- and arachidonic acid induced thromboxane formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization and role of platelet membrane phospholipids as studied with phospholipases A2 from various venoms and phospholipases C from bacterial origin

Phospholipases A₂ from various snake or bee venoms and phospholipases C secreted as exotoxins by several bacteria have been used to study the transverse distribution of phospholipids in the platelet plasma membrane and their role in platelet activation. An asymmetric distribution was described for phospholipids, characterized by a preferential localization of sphingomyelin and phosphatidylcholine in plasma membrane outer leaflet, whereas the inner half contains almost all of the anionic procoagulant phosphatidylserine and phosphatidylinositol. Such a distribution might explain the latency of procoagulant activity in resting platelets and implies an intracellular localization of arachidonic acid, the precursor of prostaglandins and thromboxanes. The external arachidonic acid is involved in phospholipase A₂-induced aggregation, whereas phospholipase C from Clostridium welchii stimulates platelets through a thromboxane-independent pathway. The latter one is directly linked to the formation of phosphatidic and lysophosphatidic acids, which are able to activate cells through calcium mobilization. So, phospholipase C represents an interesting tool for studying the biochemical processes accompanying stimulation, since it is shown that it mimicks the effects of an intracellular phospholipase C, the role of which in platelet activation is discussed.     

Mechanism of action of the platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

The platelet aggregation inhibitor purified from Agkistrodon halys snake venom inhibited rabbit platelet aggregations induced by thrombin, sodium arachidonate, collagen or ionophore A-23187. The ic₅₀ was about 11 μg/ml in platelet aggregation regardless of which aggregation inducer was used. β-Mercaptoethanol abolished both the phospholipase A enzymatic and platelet aggregation inhibitory activities of this venom inhibitor. p-Bromophenacyl bromide-treated venom inhibitor lost almost completely its phosphilipase A enzymatic activity, but retained its platelet aggregation inhibitory effect. In the presence of EGTA, the venom inhibitor still showed the same inhibitory activity on thrombin-, sodium arachidonate-, collagen- or ionophore A23187-induced platelet aggregations triggered by successive addition of Ca²⁺. The activation of platelet phospholipase A and the serotonin release reaction triggered by Ca²⁺ influx were unaffected by this venom inhibitor. It also inhibited the clot retraction of platelet-rich plasma. It is concluded that the inhibitory effect of the venom inhibitor on platelet aggregation is independent of its phospholipase A enzymatic activity. Its mode of action is different from those of other known platelet inhibitory drugs. This venom inhibitor possibly acts on a common step subsequent to platelet shape change, leading to inhibition of platelet aggregation.     

The dopamine D(1) receptor agonist SKF-82958 serves as a discriminative stimulus in the rat

The antiplatelet effect of the pyridazinone analogue, 4, 5-dihydro-6-[4-[2-hydroxy-3-(3,4 dimethoxybenzylamino)propoxy]naphth-1-yl]-3(2H)-pyridazinone (HCL-31D), was investigated in vitro with rabbit platelets. HCL-31D dose-dependently inhibited the platelet aggregation and ATP release induced by collagen (10 microg/ml), arachidonic acid (100 microM) or thrombin (0.1 U/ml) with an IC(50) of about 0.95-5.41 microM. HCL-31D (0.5-5 microM) increased the platelet cyclic AMP level in a dose-dependent manner. Furthermore, HCL-31D potentiated cyclic AMP formation caused by prostaglandin E(1) but not that caused by 3-isobutyl-1-methylxanthine (IBMX). HCL-31D also attenuated phosphoinositide breakdown and intracellular Ca(2+) elevation induced by collagen, arachidonic acid or thrombin. HCL-31D inhibited the formation of thromboxane B(2) induced by collagen or thrombin but not by arachidonic acid. In addition, HCL-31D did not affect platelet cylooxygenase and thromboxane synthase activity. These data indicate that HCL-31D is an inhibitor of phosphodiesterase and that its antiplatelet effect is mainly mediated by elevation of cyclic AMP levels.     

Effect of R and S enantiomers of naproxen on aggregation and thromboxane production in human platelets

The effects of R and S enantiomers of naproxen [(+)-6-methoxy-alpha-methyl- 2-naphthaleneacetic acid] were studied on platelet aggregation and on the production of thromboxane B2 from collagen-stimulated human platelets in order to determine the effect of each enantiomer in terms of cyclooxygenase inhibition. S-Naproxen caused inhibition of platelet aggregation in platelet-rich plasma and washed human platelets in a concentration-related fashion in the range 1-80 micrograms/L. A similar concentration-related suppression was noted for R-naproxen, but this inhibition was significantly less than that induced by S-naproxen for all concentrations except 1 micrograms/L. Similarly, both R- and S-naproxen (1-80 micrograms/L) caused a concentration-dependent suppression of thromboxane B2 production from platelet-rich plasma. These values were significant at all concentrations of drug (10-80 micrograms/L) except at 1 micrograms/L. Significant differences in thromboxane B2 production from washed human platelets were noted at concentrations of 10 and 25 micrograms/L. The findings support previous studies reported in the literature that S-naproxen is more active than R-naproxen. Our findings that S-naproxen is more active than R-naproxen on collagen-stimulated platelet aggregation and prostaglandin production suggest that the findings of greater activity of S isomer over the R isomer in animal models of inflammation may be a direct expression of the differential action on prostaglandin synthesis.     

The coumarin derivative AD6 inhibits the release of arachidonic acid by interfering with phospholipase A2 activity in human platelets stimulated with thrombin

AD6 is a coumarin derivative which is able to inhibit platelet aggregation and release due to various agonists as adrenaline, PAF, Ca++ ionophore and others. It has been demonstrated that this compound reduces the production of free arachidonate and diglyceride from human platelets pulse-labeled with radioactive arachidonic acid thus suggesting a possible interference with the activity of phospholipase A2 and/or phospholipase C. The present report indicates that the drug has no effect on the increase of the labeling of phosphatidic acid which takes place when platelets pulse-labeled with arachidonic acid are stimulated with thrombin. Furthermore, AD6 is not able to cause changes on the metabolism of phosphoinositides monitored using platelets pre-labeled with [3H] inositol. These observations exclude the possibility that AD6 interferes with phospholipase C activity. Experiments with platelets pulse-labeled with arachidonate suggest that AD6 inhibits phospholipase(s) A2 activity or modulate negatively one or more processes involved in its activation.