Effect of calcium concentration and inhibitors on the responses of platelets stimulated with collagen: contrast between human and rabbit platelets.

1. Variations in the concentration of Ca2+ [Ca2+] in the suspending medium have different effects on the responses of human and rabbit platelets to collagen. 2. When rabbit platelets are stimulated with a low concentration of collagen (0.5 micrograms/ml), aggregation, release of granule contents, and formation of thromboxane are maximal when the suspending medium contains [Ca2+] in the physiological range (0.5-2.0 mM), and very slight in a medium with no added Ca2+. 3. In contrast, human platelets respond most strongly when the suspending medium contains no added Ca2+ [( Ca2+] approx. 20 microM); this is attributable to the enhanced formation of thromboxane A2 (TXA2) upon close platelet-to-platelet contact in this medium. 4. When TXA2 formation is blocked by inhibition of cyclo-oxygenase with aspirin or indomethacin, rabbit platelet aggregation and release in response to 1.25-10 micrograms/ml collagen is also maximal at [Ca2+] of 0.5-2.0 mM and least at 20 microM; human platelets do not aggregate and the extent of release is relatively independent of [Ca2+]. 5. In 1 mM [Ca2+], use of apyrase and/or ketanserin with rabbit platelets in which TXA2 formation is blocked shows that released ADP and serotonin make large contributions to aggregation and release in response to high concentrations of collagen; human platelet aggregation is largely dependent on TXA2. 6. Use of fura-2-loaded platelets shows that the collagen-induced rise in cytosolic [Ca2+] is only slightly inhibited by aspirin or indomethacin in rabbit platelets, but almost completely inhibited in human platelets. 7. Responses of rabbit platelets to collagen are less dependent on TXA2 than those of human platelets. Released ADP and serotonin make major contributions to the responses of rabbit platelets to collagen.     

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.     

Resveratrol reduces oxidative stress induced by platinum compounds in blood platelets

The effects of resveratrol (trans-3,4',5-trihydroxystilbene) on the oxidative stress in blood platelets induced by platinum compounds [cisplatin and selenium-cisplatin conjugate] were studied in vitro. The production of thiobarbituric acid reactive substances (TBARS), the level of conjugate diene, the generation of superoxide anion radicals (O2-*) and other reactive oxygen species (O2-*, H2O2, singlet oxygen and organic radicals) were measured by chemiluminescence in blood platelets treated with platinum compounds. Cisplatin at the concentration of 10 microg/ml, as well as selenium-cisplatin conjugate (10 microg/ml) induced oxidative stress in blood platelets: an increase in TBARS, conjugate diene, chemiluminescence and generation of O2-*. In the presence of resveratrol (a natural compound with antioxidant activity) at the concentrations of 1-25 microg/ml, the chemiluminescence, the levels of O2-*, conjugate diene and TBARS were reduced (p < 0.05). We showed that resveratrol at different concentrations (1-25 microg/ml) had a protective effect against oxidative stress in platelets caused by platinum compounds (10 microg/ml) and it diminished platelet lipid peroxidation and reactive oxygen species generation induced by platinum compounds.     

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.     

Thiopental enhances human platelet aggregation by increasing arachidonic acid release.

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.     

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.     

Disaggregation of Platelet Aggregates Formed by the Action of Thrombin in the Presence of Hydrogen Peroxide

The aggregation and change in the intracellular Ca2+ concentration induced by thrombin (0.005-0.22 U/ml) in the presence of H2O2 (0.05-0.6 mM) was investigated. Under the chosen experimental conditions (incubation time of platelets with H2O2 not more than 15 sec), H2O2 neither accelerated nor inhibited the thrombin-induced platelet aggregation. However, platelet aggregates formed by the action of thrombin in the presence of H2O2 were unstable and disaggregated. Disaggregation was abolished by catalase added after thrombin. The disaggregation effect was dose-dependent; the process of disaggregation was confirmed by electron microscopy. Hydrogen peroxide did not influence thrombin-induced increase in the intracellular Ca2+ concentration, but dose-dependently accelerated Ca2+ extrusion from the platelet cytoplasm.     

Angiotensin II stimulates rapid serine phosphorylation of transcription factor Stat3

Lipid peroxidation (LPO) in rat testis and heart microsomes was compared using the ADP/Fe2+ as initiator with and without ascorbate at different concentrations. The extent of LPO was estimated by the levels of TBARS and PUFA. Without ascorbate, LPO was higher in heart than in testis despite elevated levels of catalase in heart. With increased ascorbate concentrations, a biphasic effect of LPO was observed. For a concentration 0.2 mM, ascorbate acted as pro-oxidant and increased TBARS correlated with decreased PUFA were observed both in testis and heart. Above 0.2 mM, ascorbate acts as antioxidant but differences in the rate of LPO were observed. In heart decreased TBARS correlated with increased PUFA whereas in testis TBARS only decreased, PUFA were not significantly modified. These results suggest different mechanisms in LPO initiation in the two organs. Increasing concentrations of H2O2 produced directly elevated TBARS levels in testis while a lag phase was observed in heart before the increase, suggesting that H2O2 was the essential ROS produced by ascorbate-ADP/Fe2+. The effects of scavengers such as catalase and ethanol showed an inhibitory effect on TBARS production only in testis, suggesting the role of H2O2/OH as an initiator of LPO. In heart, catalase produced a slight increase in TBARS levels whereas no modification was observed with ethanol, suggesting a possible direct activation by ADP/Fe2+ through a metal-oxo intermediate.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.     

The effect of calcium channel blockers on blood platelet function, especially calcium uptake

The effects of organic and inorganic calcium antagonists on washed platelets from rat and human have been studied. Platelet aggregation was assessed by turbidimetry. Endogenous serotonin release was measured on the same sample by means of electrochemically treated carbon fiber electrodes. The organic calcium antagonist, nitrendipine, and the inorganic calcium channel blockers (Co2+, Mn2+, Cd2+, La3+) drastically inhibited rat and human platelet aggregation induced by thrombin, ADP or adrenaline in the presence of 0.32 mM Ca2+. In our conditions, the thrombin-induced release of endogenous serotonin was found to be external Ca2+-dependent and completely inhibited by 20 microM nitrendipine or 1 mM Cd2+. In addition, Ba2+ or Sr2+ ions can be substituted for Ca2+ to bring about platelet aggregation as well as endogenous serotonin secretion. In Ba2+ or Sr2+-containing media, rat platelet aggregation and/or serotonin secretion can be inhibited by either nitrendipine or Cd2+. Finally, we have also studied the thrombin- and external Ca2+-dependence of radiolabeled calcium uptake by rat platelets. We found that the thrombin-induced 45Ca uptake was inhibited by either 18 microM nitrendipine or 1 mM Cd2+. These results provide strong evidence for the existence of an influx of divalent cations (Ca2+, Sr2+, Ba2+) triggering platelet function. They also suggest, although they do not prove, that the translocation of these cations occurs through an agonist-operated channel as proposed by Hallam and Rink (FEBS Lett. 186 (1986) 175-179).     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation.

The characteristics of hydroperoxide activation of 5-lipoxygenase were examined in the high speed supernatant fraction prepared from rat polymorphonuclear leukocytes. Stimulation of 5-lipoxygenase activity by the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) reaction product was strongly dependent on the presence of thiol compounds. Various reducing agents such as mercaptoethanol and glutathione (0.5-2 mM) inhibited the reaction and increased the concentrations of 5-HPETE (1-10 microM) necessary to achieve maximal arachidonic acid oxidation. The requirement for 5-HPETE was not specific and could be replaced by H2O2 (10 microM) but not by the 5-hydroxyeicosatetraenoic acid (5-HETE) analogue. Furthermore, gel filtration chromatography of the soluble extract from leukocytes resolved different fractions which can increase the hydroperoxide dependence or fully replace the stimulation by 5-HPETE. Maximal activity of the 5-HPETE-stimulated reaction required Ca2+ ions (0.2-1 mM) and ATP with the elimination of the HPETE requirement at high ATP concentrations (2-4 mM). In addition, NADPH (1-2 mM), FAD (1 mM), Fe2+ ions (20-100 microM) and chelated Fe3+ (0.1 mM-EDTA/0.1 mM-FeCl3) all markedly increased product formation by 5-lipoxygenase whereas NADH (1 mM) was inhibitory and Fe3+ (20-100 microM) alone had no effect on the reaction. The stimulation by Fe2+ ions and NADPH was also observed under various conditions which increase the hydroperoxide dependence such as pretreatment of the enzyme preparation with glutathione peroxidase or chemical reduction with 0.015% NaBH4. These results provide evidence for an hydroperoxide activation of 5-lipoxygenase which is not product-specific and is modulated by thiol levels and several soluble components of the leukocytes. They also indicate that stimulation of 5-lipoxygenase activity can contribute to increase lipid peroxidation in iron and nucleotide-promoted reactions.     

Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue

Free radicals and reactive oxygen species (ROS) participate in physiological and pathological processes in the thyroid gland. Bivalent iron cation (ferrous, Fe(2+)), which initiates the Fenton reaction (Fe(2+) + H2O2 --> Fe(3+) + *OH + OH(-)) is frequently used to experimentally induce oxidative damage, including that caused by lipid peroxidation. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of carcinogenesis. In turn, melatonin is a well-known antioxidant and free radical scavenger. The aim of the study was to estimate the effect of melatonin on basal and iron-induced lipid peroxidation in homogenates of the porcine thyroid gland. In order to determine the effect of melatonin on the auto-oxidation of lipids, thyroid homogenates were incubated in the presence of that indoleamine in concentrations of 0.0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, or 5.0 mM. To study melatonin effects on iron-induced lipid peroxidation, the homogenates were incubated in the presence of FeSO(4) (40 microM) plus H2O2 (0.5 mM), and, additionally, in the presence of melatonin in the same concentrations as above. The degree of lipid peroxidation was expressed as the concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. Melatonin, in a concentration-dependent manner, decreased lipid peroxidation induced by Fenton reaction, without affecting the basal MDA + 4-HDA levels. In conclusion, melatonin protects against iron + H2O2-induced peroxidation of lipids in the porcine thyroid. Thus, the indoleamine would be expected to prevent pathological processes related to oxidative damage in the thyroid, cancer initiation included.     

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.     

Effects of N-acetylglucosamine and platelet inhibitors on the synergistic interaction of platelets and aggregating agents in the presence of wheat germ agglutinin

Low concentrations of wheat germ agglutinin (4 micrograms/ml) have been shown to act synergistically to induce platelet aggregation with epinephrine, collagen, arachidonate and ionophore A23187. Aggregation ceased on the addition of the haptenic sugar N-acetylglucosamine at any time following the onset of aggregation with these agonists and a small degree of disaggregation was observed during the reversible first wave with the biphasic aggregating agents epinephrine and ADP. Cyclooxygenase inhibitors such as indomethacin and aspirin blocked the second wave of aggregation with the biphasic aggregating agents epinephrine and ADP but a synergistic response continued to be shown with the first wave in the presence of these inhibitors. Release of [14C]serotonin and the mobilization of [3H]arachidonate by epinephrine and collagen were markedly stimulated in the presence of wheat germ agglutinin but there was no increase of either radiolabel in the case of ADP. Platelet shape change, but not aggregation, occurred with low levels of wheat germ agglutinin and the synergistic response with ADP, collagen or ionophore A23187 occurred without further shape change. Wheat germ agglutinin did not affect the basal or stimulated levels of cyclic AMP. The membrane fluidity of platelets was not affected by the lectin or by thrombin as shown by the lack of change in fluorescence polarization with diphenylhexatriene. It is suggested that the binding of wheat germ agglutinin to the platelet surface induces platelet activation by mechanisms similar to those of other agonists and that it may affect the distribution of membrane-bound Ca2+ by a reversible perturbation of the platelet membrane.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases.

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.     

Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol.

Ethanol has an inhibitory effect on some platelet functions, but the mechanisms by which it exerts this effect are not known. Using suspensions of washed platelets, we observed that ethanol (1-9 mg/ml) did not affect the aggregation of rabbit platelets stimulated with ADP (0.5-10 microM). When platelets were prelabelled with 5-hydroxy[14C]tryptamine, aggregation and secretion of granule contents in response to thrombin (0.01-0.10 unit/ml) were not inhibited by ethanol, but these responses to thrombin at lower concentrations (less than 0.01 unit/ml) were inhibited by ethanol (2-4 mg/ml). Platelets were prelabelled with [3H]inositol so that increases in inositol phosphates upon stimulation could be assessed by measuring the amount of label in these compounds. ADP-induced increases in IP (inositol phosphate) and IP2 (inositol bisphosphate) were not affected by ethanol. IP3 (inositol trisphosphate) was not changed by ADP or ethanol. Although ethanol did not affect the increases in IP, IP2 and IP3 caused by stimulation of platelets with thrombin at concentrations greater than 0.01 unit/ml, ethanol did inhibit the increases observed at 2 and 3 min in these inositol phosphates caused by lower concentrations of thrombin (less than 0.01 unit/ml). Since ADP did not cause formation of IP3 in rabbit platelets, and since no thromboxane B2 was detected in platelets stimulated with the lower concentrations of thrombin, it is unlikely that the inhibitory effect of ethanol in IP3 formation was due to effects on further stimulation of platelets by released ADP or by thromboxane A2. Ethanol may inhibit platelet responses to thrombin by inhibiting the production of the second messenger, IP3.     

Na+/H+ exchange and aggregation of human platelets activated by ADP: the exchange is not required for aggregation

Isolated human blood platelets, loaded with the pH-sensitive fluorescence dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein show cytoplasmic alkalinization upon stimulation with thrombin but acidification with ADP stimulation. In both cases a Na+/H+ exchange is activated. This can be revealed by the sensitivity of the induced pH changes to amiloride and to 5-N-(3-aminophenyl)amiloride (APA), known inhibitors of the Na+/H+ exchanger, and by a dependence on sodium in the external medium. ADP-induced platelet aggregation is not affected by omission of sodium from the external medium. Furthermore, aggregation is barely inhibited (less than 10%) by amiloride or APA at concentrations up to 50 microM while the Ki values in affecting the Na+/H+ exchange are 5.9 and 1.6 microM for amiloride and APA, respectively. Platelet aggregation is inhibited by amiloride or APA at concentrations higher than 50 microM, but this inhibition is apparently due to a secondary effect of the agents. It is concluded that platelet aggregation induced by ADP is not dependent on activation of Na+/H+ exchange.