Comparison of the effects of prostacyclin (PGI2), prostaglandin E1 and D2 on platelet aggregation in different species.

The activity of prostacyclin (PGI2), PGE1 or PGD2 as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD2 was a weak inhibitor in dog, rabbit and rat plasma whereas PGE1 and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE1 or PGD2. Compound N-0164 abolished the inhibition by PGD2 of human platelet aggregation but did not inhibit the effects of PGE1 or prostacyclin. The results suggest that prostacyclin and PGE1 act on similar sites on platelets which are distinct from those for PGD2.     

Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2.

The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.     

Platelet aggregation. II. Adenyl cyclase, prostaglandin E1, and calcium

In exploration of the proposal that prostaglandin E1 (PGE1) inhibits platelet aggregation via stimulation of adenyl cyclase, the temporal relationship of adenosine cyclic 3',5' monophosphate (cyclic AMP) synthesis and inhibition of ADP-induced aggregation in response to PGE1 was studied. The requirement for calcium in aggregation led to the investigation of the effects of calcium ions on platelet adenyl cyclase activity. PGE1 stimulated the synthesis of cyclic AMP from adenosine-5'-triphosphate-8-14-C by platelet membrane fractions and also increased cyclic AMP synthesis in intact platelets previously incubated for 2 hours with adenosine-14-C. The accumulation of cyclic AMP increased signficiantly at low concentrations of PGE1 and reached a maximum at about 1 mug. Regardless of the inducing agent, calcium ions are an absolute requirement for the aggregation of platelets.     

Postprandial hyperlipemia inhibits platelet aggregation without affecting prostanoid metabolism

The objective of this work was to characterize changes in platelet aggregability during postprandial hypertriglyceridemia with special emphasis on arachidonic acid metabolism. Ten healthy young men consumed 100 g fat after a fasting period of 12 hr. In-vitro platelet aggregation induced by ADP and collagen was measured at 0, 3, 5, and 9 hours after the fat intake. The major arachidonic acid metabolites, 12-hydroxyeicosatetraenoic acid (12-HETE), thromboxane A2 (TXA2), prostaglandin F2 alpha (PGF2a), and prostaglandin E2 (PGE2) produced during collagen-induced platelet activation were quantified by gas chromatography/mass spectrometry. A significant decrease in platelet aggregability induced by both ADP and collagen was detected during the postprandial hyperlipemia. No significant changes could be found in the prostanoid pattern of collagen activated platelets. There was no correlation between the degree of the inhibition of platelet aggregation and the relative or absolute increase of triglyceride-levels in the plasma during the postprandial hyperlipemia.     

1-Arachidonyl-monoglyceride causes platelet aggregation: Indirect evidence for an acylglycerol acylhydrolase involvement in the release of arachidonic acid for prostaglandin synthesis

Phosphatidylcholine liposomes containing 1-arachidonylmonoglyceride were found to cause aggregation of human platelets. In contrast, addition of phosphatidylcholine liposomes, 1-arachidonyl-monoglyceride, or phosphatidylcholine liposomes containing 1-oleoyl-monoglyceride to a similar platelet preparation had no effect. Aggregation stimulated by 1-arachidonyl-monoglyceride was inhibited by 100 μM aspirin or 1 μM indomethacin, suggesting that the arachidonic acid is first released by a platelet acylglycerol acylhydrolase and then converted to PGG₂ and thromboxane AZ which initiate the platelet aggregation. Changes in platelet morphology in response to 1-arachidonyl-monoglyceride were similar to those reported previously to occur following stimulation of platelets by arachidonic acid or PGGZ providing further support for this concept. EDTA inhibited aggregation of platelets but not shape change or granule centralization in response to 1-arachidonyl-monoglyceride. PGE, and theophylline inhibited both aggregation and morphological changes. These results with inhibitors are similar to the effects of these inhibitors on PGGZ and provide further evidence for similarity between the action of 1-arachidonyl-monoglyceride and PGG₂. The results provide important evidence to support the concept that an acylglycerol acylhydrolase may be involved in arachidonic acid release and platelet aggregation.     

Plasma thromboxane B2 levels and thromboxane B2 production by platelets are increased in patients during spinal and epidural anesthesia

Concentrations of thromboxane (Tx) B2 in plasma and its production by platelets were measured in 20 spinal and 10 epidural anesthesia patients scheduled for small operations in the lower extremities. The main metabolite of prostacyclin, 6-keto-PGF1 alpha and prostaglandin (PG) E2 in plasma were also determined. Plasma TxB2 and TxB2 production by platelets increased during both spinal and epidural anesthesia. Plasma TxB2 levels also remained elevated 1 h after anesthesia. The plasma concentrations of 6-keto-PGF1 alpha and PGE2 did not change during spinal or epidural anesthesia. In in vitro studies, only low concentrations of lidocaine (0.5-1.0 micrograms/ml) and bupivacaine (0.5-3.0 micrograms/ml) increased platelet TxB2 production. In platelet rich plasma, neither lidocaine nor bupivacaine in concentrations of 0.5-3.0 micrograms/ml caused constant changes in ADP-induced platelet aggregation, but they inhibited it in toxic concentrations (12 micrograms/ml). The results suggest that the increased TxB2 plasma levels and platelet TxB2 production during regional anesthesia are not caused by local anesthetics itself but by other factors, e.g. tissue trauma. In clinically found concentrations, local anesthetics do not cause any constant changes in platelet aggregation.     

Metabolism of gammalinolenic acid by human blood platelet microsomes

The microsomal fraction was used to test the ability of human platelets to metabolize gammalinolenic acid. The microsomal delta 6 and delta 5 fatty acid desaturase activities were measured and the incorporation of [14C]malonyl CoA into prostaglandins was also determined. The results indicate that human platelets have the capacity to elongate gammalinolenic acid (18:3 n-6) to dihomogammalinolenic acid (20:3 n-6) precursor of PGE1. Labeled PGE1 could be detected when human platelets microsomes were incubated with [14C]malonyl CoA in the presence of gammalinolenic acid. The results also show that human platelet microsomes have little delta 6 or delta 5 desaturase enzyme activity.     

C1q (c1) receptor on human platelets: inhibition of collagen-induced platelet aggregation by C1q (C1) molecules.

Hemolytically active human C1q incubated with EA before the addition of complement inhibited the immune hemolysis. On the contrary, heat-inactivated preparation (30 min 56 degrees C) was ineffective. Preincubation of EA with bovine collagen also resulted in a decreased hemolysis. When aggregation was measured by a turbidimetric method in citrated human platelet-rich plasma, it was found that hemolytically active human C1q (C1) alone does not induce platelet aggregation. However, in its presence the platelets failed to aggregate or exhibited a significantly reduced aggregation response to bovine collagen. The inhibition by C1q depended on the preincubation time with platelets. Heat treatment (30 min 56 degrees C) destroyed the inhibitory action of C1q (C1). The effect of C1q proved to be highly specific because different C1q preparations at their inhibitory doses in collagen-induced platelet aggregation did not influence the response to other aggregating agents (bovine thrombin, ADP, horse anti-human thymocyte globulin, goat anti-baboon platelet antiserum). The results prove that collagen and C1q are capable of binding to the same site(s); namely, to those of EA and human platelets; furthermore, they suggest the presence of a receptor for C1q (C1) on human platelets.     

Cyclic AMP and platelet prostaglandin synthesis.

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3'5'-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Electronic particle size measurements of platelet aggregates formed in vitro.

The aggregation of human platelets induced by adenosine diphosphate (ADP) was used to evaluate electronic particle size analyzer measurements of platelet aggregates in plasma. As platelets began to clump in plasma, the total volume and the diameter of individual aggregates increased; after a time dependent on experimental conditions, the diameter increased but the total volume remained unchanged. Similar but opposite changes in size distribution occurred during platelet deaggregation. The total volume of aggregates formed in plasma varied (linear correlation coefficient = 0.99) with the total volume of platelets which were available to clump and with simultaneous changes in optical density. The diameter of the aggregates varied with the concentration of, and time of exposure to, ADP and with the total volume of platelets and aggregates in plasma was not different from that of control platelets in untreated plasma, the individual platelets aggregated without an accompanying increase in size. This study demonstrates that platelet aggregation can be characterized by electronic measurements of the size distribution of platelet aggregates.     

Kinetics of merthiolate-induced aggregation of human platelets]

Incubation of human platelets (in the form of platelet rich plasma or washed platelet suspension) with sodium merthiolate (ethyl mercuric salicylate inhibiting the arachidonic acid incorporation into phospholipids) induces their irreversible aggregation, which is accompanied by TxB2 synthesis. The merthiolate-induced aggregation has a lag-period of 0.5-10 min, whose magnitude is inversely correlated with the merthiolate concentration. The concentration dependencies of the rate of the merthiolate-induced and arachidonate-induced aggregation are threshold ones; the Hill coefficients are more than 30. The merthiolate-induced aggregation occurs in two phases: a slow phase which is independent of the arachidonic acid cyclooxygenase metabolism and a fast phase which is fully blocked by indomethacin. This aggregation is inhibited by PGE1 and ajoene (an inhibitor of the fibrinogen interaction with the fibrinogen receptor, GPIIb/IIIa). Quantitative and qualitative analyses of the experimental data were performed, using a model which took account of: (a) increase in the concentration of free endogenous arachidonic acid resulting from the inhibition by merthiolate of the arachidonic acid re-incorporation into phospholipids, and (b) existence of a threshold intracellular arachidonic acid concentration needed for the irreversible aggregation of platelets.     

Inhibition of Fc-receptor dependent platelet aggregation by monoclonal antibodies against the glycoprotein IIb-IIIa complex]

A murine monoclonal antibody (MoAb) VM16a specifically binding to human platelets has been produced. Approximately 56,000 molecules of VM16a bound per platelet at saturation (Kd = 7.9 nM) but no binding to platelets from Glanzmann's thrombasthenia patients was detected. VM16a precipitated two proteins with molecular masses corresponding to those of glycoproteins (GP) IIb and IIIa from solubilized surface-labelled platelets. However, after dissociation of the GPIIb--IIIa complex with EDTA VM16a did not bind to platelets and precipitated nothing from their lysate, thus evidencing that its determinant is complex-dependent. VM16a had no effect on ADP-, thrombin- and ristocetin-induced platelet aggregation but inhibited the aggregation induced by collagen. This inhibitory effect was more pronounced in the presence of plasma. VM16a completely blocked the Fc-receptor-mediated aggregation induced by aggregated human IgG, aggregated murine IgG1 and the previously described MoAb VM58. F(ab')2 fragments of VM16a were also able to inhibit this aggregation by decreasing the rate of aggregation induced by aggregated IgG and by extending the lag phase of VM58-induced aggregation. These results suggest that the platelet Fc-receptor may be topographically associated with the GPIIb-IIIa complex.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to suppress aggregation, through the action of their respective non-enzymatic breakdown products PGE1, PGD2, or PGD3 all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cyclooxygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A2, and enhance formation of the anti-aggregatory metabolite, prostacyclin. Whereas EPA is not detectably metabolized by platelets, dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid) is primarily converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH1 and DLL which was partially compromised by the PGE1 formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH1. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A2.     

Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation.

The effects of aqueous extracts of raw and boiled garlic and onions were studied in vitro on the collagen-induced platelet aggregation using rabbit and human platelet-rich plasma. A dose dependant inhibition of rabbit platelet aggregation was observed with garlic. Onion also showed dose-dependent inhibitory effects on the collagen-induced platelet aggregation but this inhibition was of a lesser magnitude compared to garlic when related to dose. The concentration required for 50% inhibition of the platelet aggregation for garlic was calculated to be approximately 6.6 mg ml(-1) plasma, whereas the concentration for onion was 90 mg ml(-1) plasma. Boiled garlic and onion extracts showed a reduced inhibitory effect on platelet aggregation. Garlic but not onion significantly inhibits human platelet aggregation in a dose-dependent fashion. The potency of garlic in inhibiting the collagen-induced platelet aggregation is approximately similar to that of rabbit platelets (8.8 mg ml(-1) produced 50% inhibition of platelet aggregation). The results of this study show that garlic is about 13 times more potent than onion in inhibiting platelet aggregation and suggest that garlic and onion could be more potent inhibitors of blood platelet aggregation if consumed in raw than in cooked or boiled form.     

Studies on platelet functions in hirudin plasma

Comparative studies on platelet responses in citrated, hirudin and heparin plasma were carried out. The adhesion of 111In-labelled rabbit platelets to the subendothelium of rabbit aorta was more pronounced in hirudin plasma than in heparin and citrated plasma. There were no significant differences in the collagen-induced aggregation and secretion of 14C-serotonin of human blood platelets in the three plasma samples. The extent of the ADP-induced aggregation was nearly the same in the three plasma samples, however, the aggregation was reversible in hirudin plasma. Adrenaline induced a small primary aggregation in hirudin plasma whereas in citrated and in heparin plasma the aggregation was a biphasic one. Secretion of 14C-serotonin induced by ADP or adrenaline occurred in citrated plasma only. Hirudin proved to be a suitable anticoagulant for studying platelet functions at physiological calcium concentrations.     

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.     

Effect of low doses of ethanol on platelet function in long-life abstainers and moderate-wine drinkers

In vitro, high concentrations of ethanol (EtOH) reduce platelet aggregation. Less is known about the effect of low EtOH doses on platelet function in a selected human population of long-life abstainers and low moderate-wine drinkers to avoid rebound effect of EtOH on platelet aggregation. Results of our experiments suggest that moderate-wine drinkers have higher levels of high density lipoprotein (HDL) than long-life abstainers while fibrinogen levels are unchanged. Furthermore, platelets obtained from these individuals do not differ in their response when stimulated by agonists such as AA and collagen. The effect of in vitro exposure of low doses of EtOH has been studied in PRP and in washed platelets. EtOH (0.1-10 mM) inhibits platelet aggregation induced by collagen at its ED50 while is ineffective when aggregation was triggered by U-46619 and by 1 microM adenosine diphosphate (ADP). 5-10 mM EtOH partially reduces the second wave of aggregation induced by 3 microM ADP. 0.1-10 mM EtOH dose-dependently lowers the aggregation induced by AA at its ED50 but it is less effective at ED75 of AA. The antiaggregating effect of EtOH on aggregation induced by AA is unchanged by inhibitor of nitric oxide synthase. In addition, 10 mM EtOH reduces thromboxane (Tx) formation. In washed platelets, 1-10 mM EtOH partially inhibits platelet aggregation induced by thrombin. In washed resting platelets, 10 mM EtOH does not change the resting [Ca++]i while significantly reduces the increase in [Ca++]i triggered by AA. The results of ex vivo experiments have demonstrated that wine increases the HDL. However, this observation may or may not influence the response of platelets to agonists. Results of our studies demonstrate that low doses of alcohol reduces platelet function.     

Beclobrinic acid--a new hypolipidemic agent--inhibits in vitro human platelet activation by blocking prostaglandin synthesis

Effects and the mechanism of the antiplatelet actions of beclobrinic acid, free acid form of a new hypolipidemic agent beclobrate [(+)-2-[d-(P-chlorophenyl)p-tolyl)oxy)-2-methyl-butyrate), were examined using human platelets. Platelet-rich plasma (PRP) which has been prelabeled with (14C)-serotonin was incubated with beclobrinic acid (BBA) for one minute before the addition of various agonists. BBA (0.1-1.5 mM) inhibited platelet aggregation and serotonin secretion induced by ADP, epinephrine, arachidonic acid and collagen in a concentration dependent manner. BBA also inhibited arachidonic acid-induced production of malondialdehyde (MDA), a byproduct of prostaglandins, in a concentration dependent manner. However, up to 1.0 mM BBA did not inhibit platelet aggregation induced by U46619, a stable analog of prostaglandin H2. In other experiments BBA also blocked thrombin-induced release of (3H)-arachidonic acid from platelet phospholipids. These findings suggest that: (a) BBA inhibits platelet aggregation and serotonin secretion by inhibiting prostaglandin synthesis at two steps. First by interfering in the release of arachidonic acid from platelet phospholipids and second by inhibiting its conversion into prostaglandins; and (b) BBA does not inhibit the action of prostaglandins on human platelets.     

Inhibition of PGE1-stimulated cAMP accumulation in human platelets by thromboxane a2.

The prostaglandin endoperoxide PGH2, HHT, HETE, thromboxane A2, and thromboxane B2, which are all products of arachidonic acid metabolism of human platelets, were tested for their ability to modulate platelet cyclic nucleotide levels. None of the compounds tested altered the basal level of cAMP or cGMP, and only PGH2 and thromboxane A2 inhibited PGE1-stimulated cAMP accumulation. Thromboxane A2 was found to be a more potent inhibitor of PGE1-stimulated cAMP accumulation and inducer of platelet aggregation than PGH2.