Inhibition of ADP-induced platelet aggregation by reduced factor Xa

Treatment of blood coagulation factor Xa with insolubilized hexyl-agarose derivative of prostaglandin E1 (PGE1) results in the generation of two sulfhydryl groups in the protein molecule. The reduced factor Xa was found to be a potent inhibitor of platelet aggregation and thromboxane A2 synthesis induced by ADP. In contrast to the inhibition of thromboxane formation, the reduced factor Xa had no effect on the formation of PGE2 indicating that thromboxane synthetase might be selectively inhibited by the reduced factor Xa. Incubation with oxidized glutathione reversed the inhibitory activity of factor Xa previously exposed to the insolubilized hormone. Soluble PGE1 also reduces factor Xa, but more slowly than the insolubilized PGE1. PGE1 also exhibits reducing ability as tested with redox dyes. Reduction of factor Xa by dithiothreitol also transformed the coagulation factor into an inhibitor of platelet aggregation and thromboxane A2 formation. These experiments indicate that reduction of factor Xa leads to a reversible alteration of the molecule which inhibits platelet aggregation induced by ADP. This effect of reduced factor Xa is probably mediated through the inhibition of thromboxane A2 synthesis.     

Involvement of phosphoinositide metabolism in potentiation by adrenaline of ADP-induced aggregation of rabbit platelets.

Changes in phosphoinositide metabolism were examined in washed rabbit platelets stimulated with 0.5 microM-ADP, 50 microM-adrenaline, or ADP and adrenaline in combination. Adrenaline does not stimulate platelet aggregation when used alone, but does potentiate aggregation stimulated by ADP. In platelets prelabelled with [32P]Pi and [3H]glycerol, adrenaline was found to potentiate the ADP-induced changes in platelet phospholipids, causing larger increases in the amount and labelling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid than was observed with ADP alone. The combination of ADP and adrenaline did not produce a greater decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) than was produced by ADP alone. In platelets prelabelled with [3H]inositol, adrenaline potentiated the increases in labelling of inositol phosphate and inositol bisphosphate stimulated by ADP; no increase in inositol trisphosphate labelling was detected with ADP alone or with the combination of ADP and adrenaline. Phentolamine, an alpha-adrenergic-receptor antagonist, blocked potentiation by adrenaline of ADP-induced changes in phosphoinositide metabolism. Propranolol and sotalol, beta-adrenergic-receptor antagonists, augmented the potentiation; this is consistent with the concept that the effect of adrenaline is mediated by beta-adrenergic receptors. The effect of adrenaline on phosphoinositide metabolism appears to be to potentiate the mechanisms by which ADP causes turnover of PIP and possibly degradation of PI, rather than the mechanism by which PIP2 is decreased.     

Pomolic acid, triterpenoid isolated from Licania pittieri, as competitive antagonist of ADP-induced aggregation of human platelets

Pomolic acid (PA), triterpenoid isolated from Licania pittieri, has previously shown a potent ability to inhibit adenosine diphosphate (ADP)- and epinephrine-induced human platelet aggregation. To investigate whether PA could be an antagonist of ADP-activated receptors of human platelets (P2Y(1) and P2Y(12)), pharmacological studies were conducted to examining its ability to modulate the platelet shape change induced by a selective P2Y(1) receptor agonist MRS2365 and also the nature of its possible interaction with ADP receptors by analyzing the characteristics of log concentration-response curves of ADP constructed in the absence and in the presence of fixed concentrations of PA, using in vitro platelet aggregation assays. PA did not interfere with the activation of P2Y(1) receptor by MRS2365 to induce platelet shape change and displayed a competitive antagonism of ADP-induced platelet aggregation, which most probably involves competition for a single binding site in platelets. The estimated equilibrium dissociate constant (K(b)) of PA as ADP receptor antagonist was 15.4±0.06nM. Together, these findings give indirect evidence for the idea that PA could be a potent competitive antagonist of P2Y(12) receptor, and open the possibility to consider it as new member of the non-nucleotide generation of antiplatelet drugs.     

Effects of monovalent cations and anions on ADP-induced aggregation of bovine platelets, and mechanism thereof

The effects of monovalent cations - inorganic alakali metal cations and organic quanternary ammonium cations - and monovalent inorganic anions on ADP-induced aggregation of bovine platelets were investigated. In the presence of K⁺, Rb⁺, Cs⁺, choline or tetramethylammonium, aggeregation proceeded. However, aggregation was markedly restricted in media containing Li⁺, Na⁺, tetrabutylammonium or dimethyldibenzylammonium. With anions, aggregation proceeded in the order Cl⁻ > Br⁻ > I⁻ > Clo₄⁻ > SCN⁻. The effects of cations significantly depended on Ca²⁺ concentration, whereas those of the anions depended little of Ca²⁺. Anions such as SCN⁻ and ClO₄⁻ markedly decreased the fluorescence of the surface charge probe 2-p-tuluidinylnaphthalene-6-sulfonate, whereas cations had less pronouced effects. The relative effects of the anions on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that inhibition of platelet aggregation by the anions is due to a change in the surface change of the platelet plasma membrane. On the other hand, kinetic analysis suggests that the effects of monovalent cations on platelet aggregation are due to their competition with Ca²⁺ during the process of aggregation.     

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quin2. In the presence of 10 microM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+]i evoked by Ca2+ ionophore; aggregation was suppressed but shape-change persisted.     

Effects of alcohols on ADP-induced aggregation and membrane fluidity of gel-filtered bovine blood platelets

Summary The effects of four alcohols—n-propyl,n-butyl,n-amyl andn-hexyl alcohol—on the ADP-induced aggregation of gel-filtered bovine platelets were examined. All four alcohols inhibited the aggregation, the order of their effects beingn-propyln-amyl<n-hexyl. Comparison of the inhibitory effects of the alcohols with their physico-chemical properties showed that their degrees of inhibition depended on their hydrophobicities. Moreover, it was suggested that their interaction with the lipid layer of the membrane was important for the inhibition. Studies on the effects of alcohols on the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene-labeled platelets showed that the membrane fluidity of the platelets increased in the same concentration range in which aggregation inhibition was observed. Since the alcohols inhibited aggregation without affecting Ca²⁺ mobilization in the platelets, as revealed in this study, it was concluded that inhibition of platelet aggregation was due to perturbation of membrane lipids by the alcohols. This hypothesis is supported by several recent studies on the effects of cholesterol and cations, which suggest that a relatively rigid membrane favors platelet aggregation.     

The effect of PGE1 on ADP-induced aggregation in refractory platelets

ADP-induced aggregation of refractory platelets can be enhanced by PGE₁ in concentrations which suppress (by up to 65%) the aggregation of normal platelets. The same degree of inhibition is obtained in normal and refractory platelets in the presence of cyclic AMP. The results suggest that PGE₁ does not stimulate the synthesis of cyclic AMP in refractory platelets.     

Inhibition of aggregation of human platelets by 8,15-dihydroperoxides of 5,9,11,13-eicosatetraenoic and 9,11,13-eicosatrienoic acids

The dihydroperoxy derivative of arachidonic acid, 8,15-dihydroperoxy-5,9,11,13-eicosatetraenoic acid, at , is an effective inhibitor of the aggregation of human blood platelets, induced by ADP, epinephrine and collagen. The nature of the effect is time dependent. If ADP is added 60 sec after the inhibitor, the first phase of aggregation occurs but the second phase is suppressed. When the time is prolonged to 240 sec, the first phase of aggregation is almost completely eliminated. The corresponding dihydroxy derivative is without effect on aggregation. The corresponding dihydroperoxy derivative obtained from 8,11,14-eicosatrienoic acid is also an effective inhibitor.     

Inhibition by antioxidants of agonist evoked cytosolic Ca++ increase, ATP secretion and aggregation of aspirinated human platelets

The synthetic antioxidants butylated hydroxytoluene (BHT), nordihydroguaiaretic acid and the one-electron donor 1,1'-dimethylferrocene, inhibit cytosolic Ca++ increase, shape change, aggregation and ATP secretion in aspirinated washed human platelets stimulated by thrombin, vasopressin and platelet-activating factor. The antioxidants also inhibit cytosolic Ca++ increase originating from intracellular stores in the presence of EGTA. The effect of phorbol ester (TPA), which promotes platelet aggregation and secretion without raising the cytosolic Ca++, is also antioxidant-sensitive. Since agonist activation of aspirinated platelets does not involve cyclooxygenase or lipoxygenase metabolites, it is suggested that other yet unknown free radical-dependent pathways are involved in the mechanism of platelet activation, both in the protein kinase C-independent events leading to the cytosolic Ca++ increase, and in those, largely protein kinase C-dependent, leading to aggregation and ATP secretion.     

Antithrombotic effects of peroxynitrite: Inhibition and reversal of aggregation in human platelets

The inhibition of platelet aggregation by peroxynitrite, a reactive oxygen species derived from the interaction of nitric oxide (NO) and superoxide, was examined in platelet-rich plasma. In this report, we have used a preparation of peroxynitrite that was free of H₂0₂ and MnO₂. As such, peroxynitrite dose-dependently (50–200 μA) inhibited aggregation of human platelets stimulated by ADP (5 μM), collagen (0.5 μg), thrombin (0.5 UlmL) and U46619 (1 PM). In addition, peroxynitrite reversed platelet aggregation induced by collagen, ADP, and thrombin. Peroxynitrite, preincubated with platelet-poor plasma or albumin (7%) for 30 min, did not alter the inhibition of platelet aggregation. This suggested that the inhibitory action of peroxynitrite may be due to nitrosylation of proteins, which by themselves possess activity, rather than conversion to NO or NO donors. Furthermore, we show that peroxynitrite increased the cGMP level only at 200 μM concentrations, further suggesting that the action of peroxynitrite was not completely due to its conversion to NO or NO donors.     

Inhibition of platelet aggregation and arachidonate metabolism in platelets by procyanidins

The effects of procyanidins on platelet aggregation and arachidonate metabolism in platelets were studied. Nine procyanidins were used in this investigation. Procyanidins B-2-S, EEC and C-1 significantly induced the inhibition of platelet aggregation, and the potency of inhibition was comparable with aspirin. Procyanidin B-2-S was used as a representative of procyanidins for further studies on the effect on arachidonate metabolism. In arachidonate metabolism by fatty acid cyclooxygenase pathway, B-2-S inhibited TXB2 and HHT formation by intact platelets treated with exogenous arachidonic acid. It also inhibited TXB2 formation measured by a specific radioimmunoassay when the cells were challenged with calcium ionophore A23187. In cell-free system, B-2-S inhibited both TXB2 and 12-HETE bioxynthesis in platelet microsome and cytosol, respectively. The inhibitory effect on thromboxane biosynthesis might explain the inhibitory effect of procyanidins on platelet aggregation.     

Standardization of ADP-induced thrombocyte aggregation]

It is the purpose of this study to standardize platelet aggregation according to the method of Born. It was found that aggregation is influenced by the time of storage, the pH and temperature of plasma. However, there is no significant correlation between platelet number versus aggregation in healthy subjects. To get reproducible results, the plasma samples should be investigated within 2 hours after venipuncture. During storage temperature of all samples should be constant.     

A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.     

Inhibition of fibrinogen binding to human platelets by S-nitroso-N-acetylcysteine

Because of the central role of fibrinogen binding in platelet aggregation and recent evidence implicating S-nitrosothiol compounds in the platelet inhibitory effects of endogenous and exogenous organic nitrate compounds, we examined the effect of the S-nitrosothiol S-nitroso-N-acetylcysteine (SNOAC) on fibrinogen binding to gel-filtered human platelets. We found that SNOAC markedly inhibited the binding of fibrinogen to normal human platelets in a dose-dependent fashion and that this inhibitory effect was the result of both an increase in the apparent Kd of the platelet receptor for the fibrinogen molecule (from 6.8 x 10(-7) to 1.8 x 10(-6) M, a 2.7-fold increase) and a decrease in the total number of fibrinogen molecules bound to the platelet (from 76,200 to 38,250, a 50% decrease). In addition, we noted a rapid, dose-dependent rise in platelet cyclic GMP levels following exposure of platelets to SNOAC which was significantly inversely correlated with fibrinogen binding and was accompanied by inhibition of intracellular calcium flux in response to a variety of platelet agonists. Similar dose-dependent inhibition of fibrinogen binding was found in the presence of cyclic GMP analogues and was significantly enhanced by inhibition of platelet cyclic GMP phosphodiesterase. These results describe the inhibition of platelet fibrinogen binding by an S-nitrosothiol compound, help define the biochemical mechanism by which S-nitrosothiols inhibit platelet aggregation, and lend support to the view that cyclic GMP is an important inhibitory intracellular mediator in human platelets.     

Inhibition of adenosine diphosphate-induced intravascular aggregation of rat platelets by 6-oxo-prostaglandin E1

The antiaggregatory effects of 6-oxo-PGE1 were evaluated in the rat using a minimally invasive technique involving 111-Indium labelling of platelets. The antiaggragatory effects on adenosine diphosphate-induced aggregation were compared with those of prostacyclin (PGI2) and prostalandin E1 (PGE1) following slow infusions and bolus injections. The rank order of antiaggregatory potency was PGI2 > 6-oxo-PGE1 > PGE1 while the rank order of duration of antiaggregatory effects was PGE1 > 6-oxo-PGE1 > PGI2. The kinetics of the antiaggregatory effects of these prostaglandins suggests that such action are not mediated by direct effects on platelets, but through a secondary mechanism.     

Mechanisms of the protease-induced aggregation of human platelets

The role of phospholipase C (an enzyme involved in the metabolism of inositol-containing phospholipids), cyclooxygenase and lipoxygenase (the enzymes of arachidonic acid metabolism), and adenylate cyclase and phosphodiesterase (the enzymes of cyclic adenosine 3,5-monophosphate (cAMP) metabolism) in the mechanisms of the aggregation of human platelets induced by the serine protease in low concentrations (thrombin 0.5 mkg per ml, trypsin 1 mkg per ml, and alpha-chymotrypsin 10 mkg per ml) have been investigated with the use of the inhibitor analysis. The effect of neomycin sulfate (phospholipase C inhibitor), indometacin (cyclooxygenase inhibitor), and nordihydrogvayaretic acid (lipoxygenase inhibitor) on protease-induced increase in the content of calcium cations in platelet plasma has been studied. The results of the inhibitor analysis indicated that the enzymes of metabolism of inositol-containing phospholipids, arachidonic acid, and cAMP are involved in the mechanisms of the protease-induced platelet aggregation. The increase in the content of calcium ions, associated with the protease-induced activation of phospholipase C, in cytoplasm may play an important role in the mechanisms of platelet aggregation.     

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.     

Effects of four types of reagents on ADP-induced aggregation and Ca2+ mobilization of bovine blood platelets

The effects of four types of reagents--a stimulant analog (ATP), reagents increasing cAMP (theophylline and (-)-isoproterenol), Ca2+ blockers (chlorpromazine, procaine, dibucaine and tetracaine) and nonspecific membrane-reactive reagents (n-butanol, n-hexanol and linoleate) - on ADP-induced Ca2+ mobilization and aggregation of platelets were investigated. All the reagents tested inhibited the aggregation. Of these reagents, those increasing cAMP and the stimulant analog inhibited the aggregation at least partly by inhibiting Ca2+ mobilization, whereas Ca2+ blockers and nonspecific membrane-reactive reagents must have inhibited the aggregation by different mechanisms, because they had: (1) no effect on ADP-induced Ca2+ mobilization, (2) accelerated it, or (3) themselves stimulated Ca2+ mobilization. The results showed that the inhibitory effects of Ca2+ blockers were at least partly due to competition with Ca2+ for binding sites on the outside of the membrane, whereas the effects of the nonspecific membrane-reactive reagents tested were due to membrane perturbation.