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.     

Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion.

It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.     

内毒素对人血小板聚集,释放的体外作用

大肠杆菌 O55B5内毒素在体外可抑制人血小板自发性聚集,并使血小板内 cAMP、钙调素明显增高,但对肾上腺素诱发的血小板聚集无作用,也不能引起血小板致密颗粒和α-颗粒的释放。此外还观察到内毒素组无血小板上清液中5-HT 含量减少。     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable malonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E1 completely blocked both aggregoserpentin-induced aggregation and release reaction. Furthermore, platelet aggregoserpentin lowered basal and prostaglandin E1-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A2, resulting in the formation of platelet activating factor, but not of prostaglandins.     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.     

How does fluoroaluminate activate human platelets?

Platelet activation induced by NaF or fluoroaluminate (AlF4-) was studied. The latter has been described to substitute for the gamma-phosphate group of the GTP molecule. With 10 mM-NaF, a concentration unable to induce any measurable Ca2+ mobilization (as measured with Indo 1), addition of AlCl3 potentiated platelet aggregation, thromboxane synthesis, diacylglycerol formation and p43 phosphorylation, without any increase in intracellular Ca2+. Neither phosphoinositide hydrolysis nor phosphatidic acid formation could be detected. AlF4- induced the release through a granule centralization within a microtubule bundle, although no myosin light-chain phosphorylation could be detected. Addition of flurbiprofen (10 microM) resulted in only partial inhibition of diacylglycerol formation, with no effect on the release reaction or on p43 phosphorylation. The present results suggest that AlF4- does not stimulate a G-protein governing the phosphoinositide-specific phospholipase C. The AlF4(-)-induced diacylglycerol formation is discussed. Moreover, these results bring evidence that there is no correlation between granule centralization and myosin light-chain phosphorylation.     

CV-3988 - a specific antagonist of platelet activating factor (PAF)

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.     

BF066, a novel dual target antiplatelet agent without significant bleeding

In this study, we report BF066, a novel adenine derivative, inhibits platelet activation and thrombosis via the adenosine receptor (A(2A)) activation and phosphodiesterase (PDE) inhibition. BF066 inhibits platelet aggregation and ATP releasing induced by multiple platelet agonists in a dose-dependent manner. The inhibition of BF066 on ADP-induced aggregation is potentiated by adenosine and can be dramatically antagonized by the A(2A) antagonist SCH58261. BF066 also inhibits the PDE activity and platelet spreading on fibrinogen. In FeCl(3)-injured mouse mesenteric arterial thrombosis model, BF066 prevents thrombus formation effectively, similar to clopidogrel. Intriguingly, at dose achieving similar antithrombotic effect compared to clopidogrel, BF066 does not increase bleeding significantly. Taken together, these results suggest that BF066 may be an effective and safe antiplatelet agent targeting both PDE and A(2A). Considering the successful use of combined antiplatelet therapy, BF066 may be further developed as a novel dual target antiplatelet agent.     

Cyclic AMP inhibits synthesis of prostaglandin endoperoxide (PGG2) in human platelets.

Dibutyryl-cAMP but not dibutyryl-cGMP inhibited platelet aggregation and release of ¹⁴C-serotonin and ADP when induced by collagen and arachidonate but not when induced by the endoperoxide PGG₂^* (TXB₂) induced by addition of collagen to platelet rich plasma (PRP) was decreased by dibutyryl-cAMP and agents known to increase the concentration of cAMP (PGE₁, PGD₂, theophylline and acetyl choline).PGE₂ in concentrations known to decrease cAMP levels increased the formation of TXB₂ whereas concentrations of PGE₂ known to increase cAMP levels decreased the amount of TXB₂ formed. That this was due to an effect on the cyclooxygenase was indicated by inhibition of the transformation of arachidonic acid by DB-cAMP and by high concentrations of PGE₂. Additional support for regulation of the cyclo-oxygenase by cAMP and its relevance to platelet aggregation was obtained by demonstrating stimulation of PGG₂ induced aggregation by low concentrations of PGE₂ and the absence of this effect in the presence of a cyclo-oxygenase inhibitor.     

Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sepharyl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release action in rabbit platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable molonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E₁ completely blocked both aggregoserpentine-induced aggregation and release reaction. Furthermore, platelet aggregoserpentine lowered basal and prostaglandin E₁-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A₂, resulting in the formation of platelet activating factor, but not of prostaglandins.     

The role of thrombocyte prostanoids and products secreted by dense granules in the platelet attachment, spreading and aggregation on collagen substrates

The role of platelet prostanoids and substances released from dense bodies (ADP and serotonin) in the initial attachment, spreading and aggregation of platelets on surfaces coated with I, III, IV and V genetic types of collagen was investigated. A positive linear correlation was found to exist between thrombi-like aggregate formation on collagen substrates and platelet prostanoid synthesis. No correlation was established between platelet aggregate formation and 14C-serotonin release. The cyclooxygenase inhibitor indomethacin and the antagonists of PG endoperoxides and TXA2 (13-APA and BM 13.177) completely block thrombi-like aggregate formation. Neither 13-APA nor BM 13.177 affect platelet spreading, while indomethacin inhibits this process by 25%. The ADP-scavenger CP/CPK inhibits platelet aggregation and spreading by 25-30%. The inhibitors of cyclooxygenase and CP/CPK do not influence the initial attachment of platelets. The data obtained suggest that thrombi-like aggregate formation on collagen substrates is mediated by the synthesis of PG endoperoxides and TXA2; however, in platelet spreading this synthesis plays a limited role. Spreading and aggregation of platelets on collagen substrates is only partly mediated by ADP and serotonin. Initial attachment of platelets does not depend on ADP and serotonin release and PG endoperoxide/TXA2 synthesis.     

SQ-27986 inhibition of platelet aggregation is mediated through activation of platelet prostaglandin D2 receptors

SQ-27986, a oxabicycloheptane derivative, potently inhibits ADP-, collagen- and arachidonic acid-induced platelet aggregation in human platelet-rich plasma. Human platelet aggregation induced by ADP is inhibited by SQ-27986 (EC50 = 22nM), and the inhibitory action of SQ-27986 can be prevented with N-0164, a PGD2 antagonist. By comparison, ADP-induced rat platelet aggregation is unaffected by SQ-27986 (IC50 greater than 80 microM). Washed human platelets treated with SQ-27986 exhibit elevated cAMP levels and activated cAMP-dependent protein kinase. Elevation of platelet cAMP levels (greater than 4 fold basal) and activation of the cAMP-dependent protein kinase (greater than 4 fold) are observed with SQ-27986 concentrations above 100 nM. The SQ-27986-induced elevation of cAMP can be prevented by N-0164. Lysed platelets treated with SQ-27986 showed stimulated adenylate cyclase activity. SQ-27986 competes with [3H]prostaglandin D2 binding to isolated platelet membranes (EC50 for SQ-27986 is 20 nM, which was more potent than cold PGD2 itself). Radiolabeled Iloprost binding is virtually unaffected by SQ-27986 (EC50 greater than 100 microM), indicating that SQ-27986 does not interact with platelet prostacyclin receptors. These studies indicate that SQ-27986 inhibits platelet aggregation by activating platelet adenylate cyclase via stimulation of platelet PGD2 receptors.     

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.     

Characterisation of the Ral GTPase inhibitor RBC8 in human and mouse platelets

The Ral GTPases, RalA and RalB, have been implicated in numerous cellular processes, but are most widely known for having regulatory roles in exocytosis. Recently, we demonstrated that deletion of both Ral genes in a platelet-specific mouse gene knockout caused a substantial defect in surface exposure of P-selectin, with only a relatively weak defect in platelet dense granule secretion that did not alter platelet functional responses such as aggregation or thrombus formation. We sought to investigate the function of Rals in human platelets using the recently described Ral inhibitor, RBC8. Initial studies in human platelets confirmed that RBC8 could effectively inhibit Ral GTPase activation, with an IC₅₀ of 2.2 μM and 2.3 μM for RalA and RalB, respectively. Functional studies using RBC8 revealed significant, dose-dependent inhibition of platelet aggregation, secretion (α- and dense granule), integrin activation and thrombus formation, while α-granule release of platelet factor 4, Ca²⁺ signalling or phosphatidylserine exposure were unaltered. Subsequent studies in RalAB-null mouse platelets pretreated with RBC8 showed dose-dependent decreases in integrin activation and dense granule secretion, with significant inhibition of platelet aggregation and P-selectin exposure at 10 μM RBC8. This study strongly suggests therefore that although RBC8 is useful as a Ral inhibitor in platelets, it is likely also to have off-target effects in the same concentration range as for Ral inhibition. So, whilst clearly useful as a Ral inhibitor, interpretation of data needs to take this into account when assessing roles for Rals using RBC8.     

Inhibitory effect of ginkgolide B on platelet aggregation in a cAMP- and cGMP-dependent manner by activated MMP-9

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 microg/ml)- stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metalloproteinase- 9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular Ca2+ mobilization, and thromboxane A2 (TXA2) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or TXA2 synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular Ca2+ mobilization and TXA2 production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.     

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.     

Acetyl eugenol, a component of oil of cloves (Syzygium aromaticum L.) inhibits aggregation and alters arachidonic acid metabolism in human blood platelets

In continuation of our studies with the oil of cloves--a common kitchen spice and a crude drug for home medicine--we have isolated yet another active component identified as acetyl eugenol (AE); the earlier reported active component being eugenol. The isolated material (IM) was found to be a potent platelet inhibitor; IM abolished arachidonate (AA)-induced aggregation at ca. 12 microM, a concentration needed to abolish the second phase of adrenaline-induced aggregation. Chemically synthesized acetyl eugenol showed similar effects on AA- and adrenaline-induced aggregation. A dose-dependent inhibition of collagen-induced aggregation was also observed. AE did not inhibit either calcium ionophore A23187- or thrombin-induced aggregation. Studies on aggregation and ATP release were done using whole blood (WB). AA-induced aggregation in WB was abolished at 3 micrograms/ml (14.6 microM) which persisted even after doubling the concentration of AA. ATP release was inhibited. Inhibition of aggregation appeared to be mediated by a combination of two effects: reduced formation of thromboxane and increased generation of 12-lipoxygenase product (12-HPETE). These effects were observed by exposing washed platelets to (14C)AA or by stimulating AA-labelled platelets with ionophore A23187. Acetyl eugenol inhibited (14C)TxB2 formation in AA-labelled platelets on stimulation with thrombin. AE showed no effect on the incorporation of AA into platelet phospholipids.     

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.     

Modulation of mast cell activity by a peptide agonist of the thrombin receptor: role of nitric oxide.

The effect of a thrombin receptor agonist peptide (TRAP-6) on the release of nitric oxide (NO) and platelet activating factor (PAF) from resting and calcium-ionophore (A23187)-activated rat peritoneal mast cells (RPMC) was studied using a platelet aggregation bioassay. RPMC spontaneously released NO, which inhibited TRAP-6-, ADP-, and PAF-stimulated platelet aggregation. This effect of NO was abolished by the addition of an NO binding agent, oxyhemoglobin (oxyHb), to the platelet suspension. The RPMC-induced suppression of platelet aggregation was completely inhibited by the NO-synthase inhibitor L-NAME. TRAP-6 and its high affinity analog haTRAP stimulated the rapid release of NO from RPMC. The effect of TRAP-6 was inhibited by pretreatment of the RPMC with L-NAME or with the inhibitor of the constitutive NO-synthase isoform (cNOS) calmidazolium. TRAP-6 inhibited PAF release from A23187-activated RPMC via an NO-dependent mechanism. Platelet aggregation induced by PAF release from activated RPMC was also confirmed in experiments using the PAF receptor antagonist ginkgolide B. Thus, TRAP-6 is a rapidly acting modulator of mast cell reactivity; it stimulates NO release and inhibits PAF secretion.