Inhibition of platelet thromboxane formation and phosphoinositides breakdown by osthole from Angelica pubescens

Osthole, isolated from Chinese herb Angelica pubescens, inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin in washed rabbit platelets. It showed a weak activity in platelet-rich plasma. Osthole inhibited the thromboxane B2 formation caused by arachidonic acid, collagen, ionophore A23187 and thrombin in washed platelets, and also the thromboxane B2 formation caused by the incubation of lysed platelet homogenate with arachidonic acid. The generation of inositol phosphates in washed platelets caused by collagen, PAF and thrombin was suppressed by osthole. These data indicate that the inhibitory effect of osthole on platelet aggregation and release reaction was due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Suppression by beta-lactam antibiotics of thromboxane A2 generation and arachidonic acid release in rabbit platelets in vitro

High concentrations of latamoxef and some other beta-lactam antibiotics suppressed thromboxane A2 generation as determined from the thromboxane B2 level in the in vitro aggregation of rabbit platelets in agonist-induced stimulations. In aggregation induced with a low concentration of collagen, the suppression of thromboxane B2 generation correlated well with the suppressions of aggregation and serotonin (5-HT) release; collagen produced thromboxane A2-dependent aggregation and 5-HT release as judged from the inhibitory action of indomethacin. Latamoxef also suppressed thromboxane B2 generation when platelet stimulation was induced by thrombin or platelet activating factor at concentrations at which it did not affect either aggregation or 5-HT release. However, latamoxef did not affect platelet responses including thromboxane B2 generation induced by exogenous arachidonic acid or calcium ionophore, A23187. Beta-lactam antibiotics also interfered with arachidonic acid release from the membrane phospholipids of platelets which had been prelabelled with [3H]arachidonic acid and aggregated with collagen. These results suggest that in the in vitro aggregation of platelets, beta-lactam antibiotics interfere with some of the receptor-stimulated processes which lead to arachidonic acid release from the membranes and this, in turn, suppresses thromboxane B2 generation.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

Antiplatelet effects of protopine isolated from Corydalis tubers

Protopine inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, PAF, collagen and ionophore A23187. Although the platelet aggregation caused by thrombin was not inhibited by protopine (100 micrograms/ml), the release reaction was partially suppressed. In rabbit platelet-rich plasma, protopine also inhibited the platelet aggregation caused by ADP, arachidonic acid, PAF and collagen. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was suppressed by protopine. Protopine inhibited the intracellular calcium increase caused by arachidonic acid in quin-2/AM loaded rabbit platelets. In the presence of indomethacin, the intracellular calcium increase caused by collagen and PAF was completely suppressed by protopine, and the intracellular calcium increase caused by thrombin was partially inhibited. The phosphoinositides breakdown caused by collagen and PAF was inhibited by protopine, but that by thrombin was not affected significantly. Protopine did not cause the elevation of cyclic AMP level of platelets. It is concluded that the antiplatelet effects of protopine is due to inhibition on thromboxane formation and phosphoinositides breakdown and then lead to the decrease of intracellular calcium concentration.     

Platelet aggregation and endogenous 5-ht secretion in presence of ca, sr and ba. effects of calcium antagonists

Washed rat platelets aggregation and endogenous serotonin release were studied after thrombin stimulation in the presence of different concentrations of Ca²⁺, Sr²⁺ or Ba²⁺. The extent of platelet aggregation and release was found to depend upon the external concentration of these cations. For all of them, an optimum concentration could be defined. Higher concentrations were shown to inhibit both aggregation and release. Efficiency to support thrombin-induced aggregation was in the order Ca²⁺>Sr²⁺>Ba²⁺. Complete inhibition of aggregation and release induced by thrombin was obtained after a 30 second preincubation with 38 uM nitrendipine, 1 nM Cd^2- or 1 mM Mn²⁺. Inhibition was obtained in the presence of Ca²⁺, Sr²⁺ or Ba²⁺. These results are consistent with the hypothesis that Sr²⁺ and Ba²⁺ are able to support platelet activation acting as Ca²⁺ substitutes. Following thrombin stimulation, they could penetrate the platelets and mimick a rise in cytoplasmic Ca²⁺.     

Reduced functions of intracellular Ca in aggregation, secretion and protein phosphorylation of permeabilized platelets from stroke-prone spontaneously hypertensive rats

Aggregation, secretion and 47kDa protein (P47) phosphorylation by various agonists such as thrombin, ADP and ionophore A23187 were markedly reduced in platelets from stroke-prone spontaneously hypertensive rats (SHRSP) compared with those of age-matched Wistar Kyoto rat (WKY) platelets, suggesting defective functions of intracellular Ca²⁺ in SHRSP platelets (Tomita et al. Hypertension 1989: 14: 304–315). To clarify the mechanism of the platelet hypofunctions, saponin permeabilized platelets were prepared to compare the responses of platelets from both rats in varying concentrations of extracellular Ca²⁺. The leakage of lactate dehydrogenase from saponin (15 μg/ml)-treated platelets was approx. 5 % of total activity; the degree of the leakage in both platelets did not differ. In saponin-treated platelets, extracellular Ca²⁺ alone did not induce either aggregation or secretion in both strains. However, in the presence of 1-oleoyl-2-acetylglycerol (10 μg/ml), Ca²⁺ dose dependently stimulated both aggregation and secretion. Under this condition, Ca²⁺ sensitivity of aggregation, secretion and P47 phosphorylation in SHRSP platelets were significantly reduced compared with those in WKY platelets. These results strongly suggest that intracellular Ca²⁺ functions are impaired in SHRSP platelets.     

Effect of cobra venom phospholipase A2 on platelet aggregation in comparison with those produced by arachidonic acid and lysophophatidylcholine

Cobra venom phospholipase A₂ induced a biphasic effect on washed rabbit platelets. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The aggregation and thromboxane B₂ formation were inhibited by indomethacin, mepacrine, tetracaine and imipramine, while PGE₁ and sodium nitroprusside inhibited only the aggregation, but not the thromboxane B₂ formation. The second phase was an inhibitory effect on platelet aggregation induced by arachidonic acid, PAF, ADP or collagen but not that by thrombin or ionophore A23187. The longer the incubation time of cobra venom phospholipase A₂ with platelets, the more the inhibitory effect. The aggregating and anti-aggregating effects could be overcome by bovine serum albumin. Lysophosphatidylcholine (Lyso-PC) and arachidonic acid showed synergistic inhibition in platelet aggregation. Lyso-PC decreased thromboxane B₂ formation in platelets formed by collagen. The inhibitory effect of Lyso-PC on platelet aggregation was more marked at lower calcium concentrations. It is concluded that the aggregating effect of exogenous addition of venom phospholipase A₂ is due to thromboxane formation and the antiplatelet effect is similar to those produced by arachidonic acid and lysophosphatidylcholine.     

Inhibition of rabbit platelet aggregation by 1,4-naphthoquinones

The effects of four 1,4-naphthoquinone derivatives on the aggregation of rabbit platelets were examined. All the four 1,4-naphthoquinone derivatives inhibited the platelet aggregation of washed rabbit platelets induced by thrombin (0.1 U/ml) and the IC50 is: 2-chloro-3-methyl-1,4-naphthoquinone (CMN), 5 micrograms/ml; 3-methyl-5,8-dihydroxy-1,4-naphthoquinone, 13 micrograms/ml; 5,8-dihydroxy-1,4-naphthoquinone, 18 micrograms/ml; 3-methyl-1,4-naphthoquinone (vitamin K3), 53 micrograms/ml. CMN was the most potent in inhibiting the aggregation and release reaction induced by ADP, arachidonic acid, PAF, ionophore A23187, collagen and thrombin in a dose-dependent manner in washed platelets, platelet-rich-plasma and whole blood. The thromboxane B2 formation caused by collagen and ionophore A23187 was inhibited by CMN. However, the thromboxane B2 formation by arachidonic acid was markedly increased. The platelet inhibitory effect of CMN could not be antagonized either by raising the concentrations of extracellular Ca++ or by wash out. The phosphoinositides breakdown induced by thrombin was inhibited by CMN. Phospholipids (PE, PC, PI) could slightly antagonize the antiplatelet effect of CMN. It is concluded that the inhibitory effect of CMN on rabbit platelet aggregation may be due to the inhibition of phosphoinositides breakdown caused by the inducers.     

The mechanism of the inhibitory effect of protease inhibitors on platelet aggregation and cellular synthesis of prostaglandins. I. The effect on the release of arachidonic acid from phospholipids

The inhibitory effect of proteinase inhibitors on platelet aggregation was investigated. The proteinase inhibitors tested were SBTI, leupeptin and FOY (a synthetic proteinase inhibitor). Also, synthetic substrates for serine proteinases (TLME, ATEE) were tested. They completely inhibited the secondary aggregation of platelets induced by ADP or epinephrine. They also completely inhibited the platelet aggregation induced by collagen or thrombin. The aggregation induced by arachidonic acid was completely inhibited by all the proteinase inhibitors and synthetic substrates. The aggregation induced by Ca ionophore A 23187 was completely inhibited by leupeptin, FOY, TLME or ATEE but not by SBTI. It is generally accepted that platelet prostaglandin metabolism plays an important role in platelet aggregation. As the first step to elucidate the possible mechanism of the inhibitory effect of the proteinase inhibitors, their effect on the release of arachidonic acid from platelet phospholipids was investigated. The release of arachidonic acid from ¹⁴C-arachidonate incorporated gel filtered platelets (¹⁴C-AA-GFP) by thrombin or A 23187 was directly measured in the presence or absence of a proteinase inhibitor or synthetic substrate, utilizing thin layer chromatography (TLC) and a scintillation counting. The release was almost completely blocked when the aggregation of ¹⁴C-AA-GFP by thrombin or A 23187 was completely inhibited by a proteinase inhibitor or a synthetic substrate.     

Chloroquine: a multipotent inhibitor of human platelets in vitro

Chloroquine inhibited human platelet aggregation in vitro both at receptor- and nonreceptor-operated stimuli. The inhibition was dose-dependent, recorded on isolated platelets as well as in platelet-rich plasma, and followed the rank order of stimuli: adrenaline (second phase)>phorbol 12-myristate 13 acetate>adenosine diphosphate>adrenaline (first phase)>thrombin>calcium ionophore A23187. In thrombin-activated platelets, chloroquine decreased in a dose-dependent manner phospholipase A(2)-induced arachidonic acid liberation from membrane phospholipids, malondialdehyde formation (a marker of membrane phospholipid peroxidation), and thromboxane generation, considered the most potent autoaggregatory agent. Chloroquine only slightly altered the arachidonic acid cascade of platelets stimulated with A23187 and phorbol 12-myristate 13 acetate. Histamine formation and liberation induced with thrombin and A23187 were not affected by chloroquine. On the other hand, thrombin-stimulated serotonin secretion was significantly decreased with chloroquine in the concentration of 10 micromol/L. This indicated that chloroquine might interfere with stimulated secretion from platelets. The results suggest that chloroquine inhibited activated platelets: first, intracellularly; second, in a close relationship to the intraplatelet Ca(2+) mobile pool; and third, most probably at the site of platelet phospholipase A(2) activation.     

Effects of Cathepsin G Pretreatment of Platelets on their Subsequent Responses to Aggregating Agents

Cathepsin G, a proteolytic enzyme from activated leukocytes, can interact with platelets during inflammation and thrombosis. Platelets that have been exposed to cathepsin G in thrombi may recirculate if they are freed during fibrinolysis. To determine whether some of the subsequent functions of such platelets would be impaired, we investigated the responses of cathepsin G-pretreated platelets to agonists that they would encounter in the circulation. Suspensions of washed human platelets were labeled with [¹⁴C]serotonin and resuspended in Tyrode-albumin solution (with 2 mM Ca²⁺ and apyrase). After 15 minute incubation with 400 nM cathepsin G at 37°C, 52±3% of [¹⁴C]serotonin had been released, and glycoprotein Ib was degraded. The platelets were washed and resuspended in fresh medium to remove cathepsin G and released materials. Ristocetin-induced agglutination was abolished, indicating that the binding site for von Willebrand Factor on glycoprotein Ib had been removed. Aggregation and release of residual [¹⁴C]serotonin in response to 0.1–1.0 U/mL thrombin was blocked or greatly reduced by the cathepsin G pretreatment. This inhibition is probably largely due to cleavage by cathepsin G of some of the protease-activated receptors at the C-terminal side of Ser⁴² so that the tethered ligand is lost. Pretreatment with cathepsin G did not affect responses to ADP or a low concentration of platelet-activating factor in the presence of fibrinogen, indicating that receptors for these agonists were unaffected and that the function of the fibrinogen receptor, GPIIb/IIIa was unchanged. Responses to cathepsin G, the thrombin receptor-activating peptide SFLLRN, collagen, or the thromboxane A₂ mimetic U46619 were partially inhibited, even in the presence of added fibrinogen. Platelet adhesion to a collagen-coated surface was 51±7% inhibited, which may indicate cleavage of a collagen receptor or receptors; this may partly account for strong inhibition of collagen-induced aggregation and release of granule contents; additionally, as shown by inhibition of responses to U46619, the function of the thromboxane A₂ receptor may be compromised. Thus, although cathepsin G activates platelets, if they recirculate after interaction with it, their subsequent adhesion to damaged vessel walls, aggregation, and release of granule contents induced by thrombin and collagen will be diminished.     

Effects of etamsylate on platelet functions and arachidonic acid metabolism

The effects of etamsylate on human platelet aggregation and ATP release as well as on the arachidonate metabolism by the platelet have been studied. Etamsylate enhanced these platelet functions induced by arachidonic acid (AA), thromboxane A2, collagen and calcium ionophore A23187 but not those induced by ADP and epinephrine. In experiments with cyclooxygenase-inhibited platelets, AA-induced platelet aggregation was completely inhibited and it was not enhanced by etamsylate, while A23187-induced aggregation was partially inhibited and this aggregation was enhanced by etamsylate. Platelet AA metabolism including thrombin-induced AA liberation from phospholipids as well as the lipoxygenase and cyclo-oxygenase pathways was not significantly affected by etamsylate. These results suggested that etamsylate enhanced platelet response to thromboxane A2 and calcium ionophore and that this could be included as a mechanism for its potentiating effect on platelet functions.     

Investigation on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.     

Effect of op 1206, a prostaglandin E1 derivative, on guinea-pig platelet functions

OP 1206, 17(S)-methyl-ω-homo-trans-Δ²-prostaglandin E₁, inhibited guinea-pig platelet aggregation induced by ADP, collagen, A 23187, arachidonic acid, labile aggregation stimulating substances (LASS) and thromboxane A₂ (TXA₂)-like substance, and platelet adhesiveness to a glass bead column. The potency of inhibition was 10–16 times stronger than that of prostaglandin E₁ (PGE₁). ADP-induced platelet aggregation was notably disaggregated by the addition of OP 1206 after induction of aggregation. The release of ATP and ADP from guinea-pig platelets induced by collagen was suppressed by OP 1206, of which potency was 9–10 times stronger than that of PGE₁. OP 1206 and PGE₁ increased guinea-pig platelet cyclic AMP levels, and the increased levels were augmented by the pretreatment with theophylline. OP 1206 and PGE₁ inhibited synthesis of guinea-pig platelet malondialdehyde (MDA) induced by thrombin but not by arachidonic acid. This inhibition was released by exogenous calcium. OP 1206 and PGE₁ showed no influence on synthesis of radioactive TXA₂ (measured as a stable form, TXB₂) from [¹⁴C] arachidonic acid. From these results, increased levels of platelet cyclic AMP by OP 1206 as well as PGE₁ may exert their action on platelet functions.     

XC 386: A new antiplatelet agent

XC386 was a new antiplatelet compound, which inhibited the aggregation and release reaction of rat platelet-rich plasma induced by collagen. This inhibition was dose-dependent and the IC₅₀was calculated to be 1 mM on collagen-induced aggregation. In washed platelets, the aggregations induced by ADP and collagen were much more markedly inhibited by XC386 than those induced by thrombin, A23187 and arachidonate. High calcium (4 to 8 mM) could not antagonize the inhibition. XC386 did not alter the malondialdehyde (MDA) and thromboxane B (TXB₂) levels of resting platelets. But MDA formation induce by collagen, thrombin and A23187, and TXB₂ formation induced by collagen and thrombin were significantly inhibited, while both formations induced by arachidonate were not changed. Combination of indomethacin or CP/CPK and XC386 enhanced markedly the inhibitory effect of XC386 on collagen- or A23187-induced aggregation. It was concluded that XC386 might inhibit platelet aggregation before the step of arachidonic acid release by phospholipase A₂.     

Effect of 4-hydroxy-2,3-trans-nonenal on platelet function

4-Hydroxy-2,3-trans-nonenal (HNE), an aldehyde end-product of lipid peroxidation, potentiated aggregation and increased thromboxane A2 formation in platelets challenged with ADP, thrombin or the ionophore A23187. These effects were observed at HNE concentrations in the range 10-100 microM. Platelet responses to collagen, epinephrine and arachidonic acid were not affected by HNE. Concentrations of HNE in excess of 100 microM inhibited platelet activation. HNE increased the release of 3H-arachidonic acid from prelabelled platelet phospholipids in response to thrombin or ADP. It is proposed that HNE may play an important role in controlling platelet function by regulating the activity of phospholipase A2.     

The role of PAR4 in thrombin-induced thromboxane production in human platelets

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [(3) H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca(2+)](i) and activation of phospholipase A(2). These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.     

Inhibition of platelet aggregation by some flavonoids.

The inhibitory effects of five flavonoids on the aggregation and secretion of platelets were studied. These flavonoids inhibited markedly platelet aggregation and ATP release of rabbit platelets induced by arachidonic acid or collagen, and slightly those by platelet-activating factor. ADP-induced platelet aggregation was also suppressed by myricetin, fisetin and quercetin. The IC50 on arachidonic acid-induced platelet aggregation was: fisetin, 22 microM; kaempferol, 20 microM; quercetin, 13 microM; morin, 150 microM less than IC50 less than 300 microM. The thromboxane B2 formations were also inhibited by flavonoids in platelets challenged with arachidonic acid. Fisetin, kaempferol, morin and quercetin antagonized the aggregation of washed platelets induced by U46619, a thromboxane A2/prostaglandin endoperoxides mimetic receptor agonist. In human platelet-rich plasma, quercetin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine or ADP. These results demonstrate that the major antiplatelet effect of flavonoids tested may be due to both the inhibition of thromboxane formation and thromboxane receptor antagonism.     

Comparative study of antiplatelet drugs in vitro: distinct effects of cAMP-elevating drugs and GPIIb/IIIa antagonists on thrombin-induced platelet responses.

Among various categories of antiplatelet drugs, cAMP-elevating agents and GP IIb/IIIa antagonists have been reported to inhibit platelet aggregation stimulated by a wide variety of platelet agonists. To clarify the qualitative difference between these two agents, their effects on various platelet responses in washed platelets evoked by thrombin (0.05 U/mL) were compared in vitro. Two types of cAMP-elevating drugs, cilostazol (a phosphodiesterase III inhibitor) and prostaglandin E1 (an adenylate cyclase activator), both inhibited platelet aggregation, thromboxane A2 formation, and platelet factor 4 release in a concentration-dependent manner. In addition, both agents suppressed intracellular Ca++ elevation induced by thrombin. However, two classes of GP IIb/IIIa antagonists, abciximab (Fab fragment of antibody) and tirofiban (a synthetic compound), showed no inhibitory effects against thromboxane A2 formation and platelet factor 4 release, although these drugs inhibited platelet aggregation. Essentially the same results were obtained in platelet-rich plasma stimulated with high concentration (100 microM) of thrombin receptor activating peptide. In contrast to these different profiles on thromboxane A2 formation and release reaction, both cAMP-elevating agents and GP IIb/IIIa antagonists potently suppressed procoagulant activity in thrombin-stimulated platelets. These results suggest that the development of platelet procoagulant activity induced by thrombin is exclusively dependent on platelet aggregation or aggregation-dependent processes. These observations also indicate that cAMP-elevating agents possess wider inhibitory effects on platelet responses evoked by strong agonists than GP IIb/IIIa antagonists.     

Effects of ethanol on pathways of platelet aggregation in vitro

Ethanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.