Inhibiting action of anti-arrhythmia agents of the phenothiazine series--etmozin and its diethylamino analog--on platelet aggregation and metabolism of endogenous arachidonic acid

Effect of the cardiotropic drugs of the phenothiazine series ethmozine, and its diethylamine analogue (DAAE), on platelet aggregation and formation of arachidonic acid metabolites has been studied. Both drugs inhibit the ADP-induced aggregation in the platelet-rich plasma. Ethmozine inhibits only the second (irreversible) wave of aggregation, while DAAE inhibits both the first (reversible) and the second one. 50% inhibition (ID50) of the second wave of aggregation is observed at the following concentrations of the two agents: 300-500 micrograms/ml (ethmozine) and 20 micrograms/ml (DAAE). DAAE completely inhibits the irreversible aggregation of platelets washed off plasma, induced by arachidonic acid (ID50 approximately 30 micrograms/ml) and Ca2+-ionophore A23187 (ID approximately 55 micrograms/ml); the aggregation, induced by thrombin is inhibited by 80-90% (ID approximately 130 micrograms/ml). Formation of arachidonic acid metabolites in platelets effected by these inducers was measured by the accumulation of malondialdehyde (MDA). DAAE fails to inhibit MDA formation induced by exogenous arachidonic acid, but completely prevents the synthesis of MDA induced by A23187 and thrombin. These data suggest that DAAE inhibits the release of endogenous arachidonic acid from membrane phospholipids catalysed by phospholipase A2, but does not affect its subsequent metabolic transformations. In all probability, ethmozine and DAAE, just as other phenothiazines, affect platelets via the inhibition of Ca2+-calmodulin-dependent reactions and processes.     

The roles of ATP and calcium in morphological changes and cytotoxicity induced by 1,4-benzoquinone in platelets

To understand the mechanism of 1,4-benzoquinone-induced cytotoxicity in platelets, the roles of ATP and calcium in platelet toxicity and morphological changes were investigated. Using scanning electron microscopy, morphological changes including membrane blebbing were observed in rat platelets 5 min after exposure to 1,4-benzoquinone, which were significantly different from shape changes (pseudopod formation) observed in response to physiological agonists. Benzoquinone-induced membrane blebbing of platelets was associated with rapid depletion of intracellular ATP and was independent of the presence of extracellular calcium. Benzoquinone-induced platelet lysis observed between 20 and 30 min was dependent on extracellular calcium and associated with increased cytosolic calcium. Cytotoxicity induced by 1,4-benzoquinone was inhibited by antagonists of calmodulin, suggesting that calmodulin could play an important role in platelet toxicity. These results suggested that the progression of events for benzoquinone-induced cytotoxicity in platelets was as follows: 1,4-benzoquinone depletes intracellular ATP; membrane blebbing occurs; calcium homeostasis is disrupted, activation of calmodulin-dependent processes results; finally cytotoxicity occurs.     

Synaptotagmin II could confer Ca(2+) sensitivity to phagocytosis in human neutrophils

To understand the mechanism of 1,4-benzoquinone-induced cytotoxicity in platelets, the roles of ATP and calcium in platelet toxicity and morphological changes were investigated. Using scanning electron microscopy, morphological changes including membrane blebbing were observed in rat platelets 5 min after exposure to 1,4-benzoquinone, which were significantly different from shape changes (pseudopod formation) observed in response to physiological agonists. Benzoquinone-induced membrane blebbing of platelets was associated with rapid depletion of intracellular ATP and was independent of the presence of extracellular calcium. Benzoquinone-induced platelet lysis observed between 20 and 30 min was dependent on extracellular calcium and associated with increased cytosolic calcium. Cytotoxicity induced by 1,4-benzoquinone was inhibited by antagonists of calmodulin, suggesting that calmodulin could play an important role in platelet toxicity. These results suggested that the progression of events for benzoquinone-induced cytotoxicity in platelets was as follows: 1,4-benzoquinone depletes intracellular ATP; membrane blebbing occurs; calcium homeostasis is disrupted, activation of calmodulin-dependent processes results; finally cytotoxicity occurs.     

Platelet membrane potential as a modulator of aggregating mechanisms

The membrane potential of platelets suspended in physiological medium and membrane potential changes induced by high potassium concentrations, ouabain and cooling have been measured using a cyanine fluorescent dye (3,3'-dipropylthiodicarbocyanine) [corrected]. The membrane potential of platelets suspended in physiological medium was -63.8 mV. High potassium concentrations, ouabain and cooling induced depolarization of platelet membrane. Depolarization using the above procedures enhanced platelet aggregation induced by ADP, adrenaline and collagen. These results suggest that the membrane potential could modulate platelet activity.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

The interaction of beta-adrenoceptor blocking drugs with platelet aggregation, calcium displacement and fluidization of the membrane

beta-Adrenoceptor blocking drugs interfere with adenosine diphosphate-stimulated platelet aggregation. Alprenolol, exaprolol, K? 1124 and propranolol inhibited the aggregation, metipranolol decreased the extent and rate of aggregation significantly. Atenolol potentiated the aggregation measured by amplitude significantly. The interaction of beta-adrenoceptor blocking drugs with aggregation correlated with the displacement of calcium ions from binding sites in isolated platelets and the fluidization of the whole platelets and isolated platelet membrane as measured with electron spin resonance of the spin probe. The most potent were highly liposoluble drugs alprenolol, exaprolol, metipranolol and propranolol which increased the calcium displacement and membrane fluidity, the least active was atenolol decreasing these phenomena. The inhibition by beta-adrenoceptor blocking drugs of stimulated platelet aggregation is rather a result of unspecific than specific receptor interaction.     

Alterations of wheat-germ agglutinin binding pattern on cell surface of blood platelets after thrombin stimulation

An attempt was made to demonstrate wheat-germ agglutinin (WGA) binding sites on platelet surfaces after thrombin stimulation, by means of a post-embedding cytochemical technique using colloidal gold as marker at an ultrastructural level. In unstimulated platelets washed with EDTA, an intense uniform labeling of WGA-gold complexes was found on the surface membrane. When washed platelets were stimulated by thrombin in the absence of Ca2+, only a release reaction was induced. WGA labeling on the surface membranes of these platelets decreased dramatically. However, the labeling intensity of WGA-gold complexes on the surface membrane of aggregated platelets induced by thrombin in the presence of Ca2+ increased significantly compared to that of thrombin-stimulated platelets in the absence of Ca2+. In contrast to the uniform labeling on the surface membranes of unstimulated platelets, clusters of gold label were often found on the surface membrane of the aggregated platelets, although there was no significant quantitative difference in the labeling intensity between these two groups. Thus, we present direct morphological evidence demonstrating qualitative and quantitative alterations of WGA labeling on the surface membrane of platelets after thrombin stimulation. The possibility is considered that WGA-binding glycoproteins in the surface membrane are involved in the aggregation response after thrombin stimulation.     

The effect of halofenate and clofibrate on aggregation and release of serotonin by human platelets.

The hypolipidemic drugs halofenate and clofibrate inhibit the aggregation of human platelets induced by ADP, collagen and epinephrine. The effect of the drugs is primarily on the “second wave” of aggregation which is associated with the platelet release reaction. The drugs also inhibit the release of serotonin from platelets suggesting that the effect on aggregation is attributable to an inhibition of the release reaction. Halofenate is much more potent than clofibrate in inhibiting both the release of serotonin and the second wave of aggregation.     

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A2, thromboxane A2-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholipase A2. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A2-like acitivity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A2. The results indicate that formation of thromboxane A2-like activity enhances aggregation in rat platelets, but that aggregation is not induced.     

Alterations of wheat-germ agglutinin binding pattern on cell surface of blood platelets after thrombin stimulation

Summary An attempt was made to demonstrate wheatgerm agglutinin (WGA) binding sites on platelet surfaces after thrombin stimulation, by means of a post-embedding cytochemical technique using colloidal gold as marker at an ultrastructural level. In unstimulated platelets washed with EDTA, an intense uniform labeling of WGA-gold complexes was found on the surface membrane. When washed platelets were stimulated by thrombin in the absence of Ca²⁺, only a release reaction was induced. WGA labeling on the surface membranes of these platelets decreased dramatically. However, the labeling intensity of WGA-gold complexes on the surface membrane of aggregated platelets induced by thrombin in the presence of Ca²⁺ increased significantly compared to that of thrombin-stimulated platelets in the absence of Ca²⁺. In contrast to the uniform labeling on the surface membranes of unstimulated platelets, clusters of gold label were often found on the surface membrane of the aggregated platelets, although there was no significant quantitative difference in the labeling intensity between these two groups. Thus, we present direct morphological evidence demonstrating qualitative and quantitative alterations of WGA labeling on the surface membrane of platelets after thrombin stimulation. The possibility is considered that WGA-binding glycoproteins in the surface membrane are involved in the aggregation response after thrombin stimulation.     

Aggregation-induced alterations in human platelet membranes: A spin label study

Washed human platelets were labeled with stearate or methyl stearate spin probe. The order parameter of stearate spin probe was decreased markedly when the platelets were aggregated by thrombin, ADP or epinephrine. Central peak width of methyl stearate spin probe was also reduced in the aggregation induced by thrombin and ADP. However, isolated plasma membrane did not show any decrease of the order parameter by the addition of thrombin. Aspirin inhibited both the platelet aggregation and the decrease of the order parameter. The results suggest that ESR spectral changes might be caused by reorganization of membrane constituents rather than their quantitative or qualitative alteration.     

Interaction of terbium with human platelets

The effect of terbium on platelets has been studied by aggregation experiments and by fluorescence measurements. TbCl3 does not substitute for CaCl2 in the aggregation of platelets induced by ADP, but it may even inhibit, probably by a competition mechanism. It was impossible to observe a sensitized emission of Tb3+ in the presence of platelets. Instead the lanthanide, like Ca2+, significantly increases the aggregation of platelets induced by A23187. The fluorescence yield of this compound is greater in the presence of platelets than in buffer alone. Energy transfer appears to take place from the aromatic amino acids of the platelet membrane to the bound ionophore.     

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A₂, thromboxane A₂-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholinase A₂. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A₂-like activity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A₂.The results indicate that formation of thromboxane A₂-like activity enhances aggregation in rat platelets, but that aggregation is not induced.     

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E2 formed by the incubation of guinea-pig lung homogenate with arachidonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

The behavior of platelets at foreign surfaces

Many conditions affect the interaction of platelets with foreign surfaces, including the type of surface, modifications of the surface, conditions of blood flow, the adsorbed layer of plasma proteins, changes in this protein layer with time, and the animal species in which experiments are done. Platelets probably never adhere directly to a foreign surface in vivo, because upon exposure of the surface to blood, plasma proteins, principally fibrinogen, are adsorbed almost immediately. When platelets adhere to such a surface and spread on it, they are activated in much the same way as when they are exposed to a strong aggregating and release-inducing agent, but in contrast to aggregation caused by some agonists, adhesion is not dependent on the formation of TXA2 or the release of ADP. It does appear to depend on external Ca2+. Much less is known about the initial adhesion reaction than about platelet aggregation (thrombus formation) on the adherent platelets, although the morphological changes resulting from adhesion have been described. It is surmised that the metabolic and cytoskeletal changes upon adhesion are similar to those that are involved in the response of platelets to other activating agents. The consequences of adhesion include the formation of thrombi and thromboemboli, thrombocytopenia, reduced platelet survival, reduced platelet function in response to hemostatic stimuli, and the appearance in the circulation of products released or formed by activated platelets. Many efforts are being made to develop surfaces and to set up conditions that will minimize platelet adhesion, but it has not yet been possible to find a foreign surface that has and can maintain the nonthrombogenic characteristics of the normal endothelium.     

Hydrogen peroxide has a role in the aggregation of human platelets

The aggregation of platelets induced by soluble and particulate stimuli is enhanced by the addition of minute amounts of H2O2. Externally added catalase strongly inhibits the aggregation induced by particulate stimuli and by phorbol myristate acetate (PMA). The addition of aminotriazole to stimulated platelets causes a significant inhibition of intracellular catalase. This indicates the formation of H2O2 inside the platelets during activation. No effects were observed when the platelets were stimulated by the ionophore A23187.     

Influence of sodium conductances on platelet activation

The effects of extracellular Na+ and tetrodotoxin on resting membrane potential, cytosolic free Ca2+ levels and aggregation of human platelets have been studied. Neither the decrease in extracellular Na+-concentration (from 140 mmol/l to 0 mmol/l) nor the addition of tetrodotoxin (10(-7) to 10(-5) mol/l) modified the platelet membrane potential. Zero extracellular Na+ concentration or the presence of tetrodotoxin in the medium inhibited platelet aggregation; however, K+-depolarized platelets showed an unchanged aggregation induced by ADP or thrombin in media with zero or low extracellular Na+ concentrations or in the presence of tetrodotoxin. Moreover, zero extracellular Na+ concentration or tetrodotoxin inhibited calcium mobilization in platelets during activation induced by thrombin. Hence, voltage-dependent activation linked to Na+ influx appears to be necessary for ADP- and thrombin-induced platelet aggregation under control conditions. Mechanisms for the role of Na+ conductances in platelet function are discussed.     

Membrane structure of nonactivated and activated human blood platelets as revealed by freeze-fracture: evidence for particle redistribution during platelet contraction

The distribution of intramembrane particles of nonactivated and activated human blood platelets was studied by freeze-fracture under various experimental conditions to see whether morphological evidence for a structural coupling between the platelet actomyosin system and the fibrin network in a retracting clot could be established. Membrane particles were evenly distributed in nonactivated platelets; the total number (E + P faces) was approximately 1,500/micrometers 2 of membrane, and there were two to three times more particles present on the E face than on the P face. Transformation of discoid platelets to "spiny spheres" by cooling did not change the particle distribution. Platelet activation and aggregation by serum or ADP caused no change in membrane particle density or distribution. Particle distribution was not changed in Ca2+-activated platelets fixed immediately before fibrin formation, but after fibrin formation and during clot retraction, particles were sometimes most frequent on the P face and tended to form distinct clusters, and aggregates of E face pits were observed. Blood platelets contain contractile proteins that are distinct as filaments in platelets in retracting clots. We suggest that the redistribution of particles seen in activated platelets during clot retraction reflects the esablishment of mechanical transmembrane links between the platelet actomyosin system and the fibrin net. The P-face particle clusters may represent sites of force transmission between actin filaments bonded to the inside of the membrane and the fibrin network at the outside. Thus, whereas membrane particles may not be directly involved in the attachment of actin filaments to membranes, the transmission of the force of the contractile system to an exterior substrate apparently involves the intramembrane particles.     

Effects of Welsh onion extracts on human platelet function in vitro

Welsh onion has been consumed for prevention of cardiovascular disorders. However, its underlying mechanisms are still unclear. This study investigated whether Welsh onion extracts can alter human platelet function (ie, platelet adhesion, aggregation, and thromboxane release). To clarify the underlying mechanisms, we also measured the intracellular calcium ([Ca2+]i) and cyclic nucleotide levels in platelets. Our results showed that 1) boiled extracts directly induced platelet aggregation in a dose-dependent manner; 2) raw extracts inhibited platelet adhesion and ADP-evoked platelet aggregation, while boiled extracts enhanced them; 3) raw green extract suppressed ADP-stimulated platelet [Ca2+]i elevation and thromboxane production, whereas boiled green extract enhanced them; 4) raw green extract elevated platelet cAMP level, whereas boiled green extract had no effect on cAMP level. Furthermore, the boiled green extract, but not the raw extract, induced pronounced platelet morphological changes. In conclusion, raw extracts of Welsh onion inhibit platelet function in vitro while boiled extracts activate platelets.