PAF antagonism in vitro and in vivo by aglafoline from Aglaia elliptifolia Merr.

Aglafoline, isolated from Aglaia elliptifolia Merr, inhibited in a selective and concentration-dependent manner the aggregation and ATP release reaction induced in washed rabbit platelets by PAF (platelet-activating factor). The IC50 values of aglafoline, BN52021 and kadsurenone on PAF (3.6 nM)-induced platelet aggregation were about 50, 12 and 18 microM, respectively. Aglafoline also inhibited [3H]PAF (3.6 nM) binding to washed rabbit platelets with an IC50 value of 17.8 +/- 2.6 microM. The concentration-response curve of PAF-induced platelet aggregation was shifted to the right by aglafoline with pA2 and pA10 values of 5.97 and 5.04, respectively. Although thromboxane B2 formation caused by collagen and thrombin was partially suppressed by aglafoline, thromboxane B2 formation caused by ionophore A23187 and arachidonic acid was not affected. Aglafoline inhibited the [3H]inositol monophosphate formation caused by PAF but not that caused by collagen or thrombin in the presence of indomethacin (20 microM). The cAMP content of washed rabbit platelets was not affected by aglafoline. Rat femoral intravenous administration of aglafoline (10 mg/kg) did not affect blood pressure. However, aglafoline (10 mg/kg) both prophylactically and therapeutically antagonized PAF (2.5 micrograms/kg)-induced hypotensive shock in rats. Intravenous PAF (30 ng/kg) caused severe bronchoconstriction in guinea pigs. This effect was completely blocked by aglafoline. This implies aglafoline is an effective PAF antagonist not only in vitro, but also in vivo.     

Effect of anethole dithiolthione on human platelet aggregation.

Anethole dithiolthione (ADT) (10 mumol/l) inhibited platelet aggregation and the formation of thromboxane (Tx)B2 in plasma in response to adenosine diphosphate (ADP), epinephrine and arachidonic acid (AA). ADT partially inhibited platelet aggregation and TxB2 formation in plasma induced by thrombin, phorbol myristate acetate and calcium ionophore A23187 and increased the lag time of collagen-induced aggregation at concentrations in the range 10-40 mumol/l. ADT (100 mumol/l) completely inhibited the aggregation of washed platelets challenged with thrombin. ADT had no additive effect on the inhibition of thrombin-induced platelet aggregation by acetylsalicylic acid. ADT was a more effective inhibitor of AA-induced platelet aggregation than butylated hydroxytoluene. ADT inhibited the release of 3H-AA from platelet phospholipids in response to ADP and collagen. It is suggested that ADT inhibits platelet aggregation by inhibiting thromboxane synthesis and preventing AA release.     

Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic AMP level of rabbit platelets.

Dicentrine is an antiplatelet agent isolated from the Chinese herb Lindera megaphylla. We examined the in vitro effects of dicentrine on various aspects of platelet reactivity. Dicentrine inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA), collagen, ADP, platelet-activating factor (PAF), thrombin and U46619. Dicentrine also inhibited the thromboxane B2 formation caused by AA, collagen and thrombin in washed intact platelets or that induced by AA in lysed platelet homogenate, while prostaglandin D2 formation caused by AA was not increased. The generation of inositol monophosphates (in the presence of indomethacin) caused by thrombin, collagen and PAF was not suppressed significantly, nor did dicentrine suppress fibrinogen-induced aggregation of elastase-treated platelets. Dicentrine inhibited the intracellular Ca2+ increase in quin-2/AM-loaded platelets caused by thrombin, PAF, collagen and AA. The cyclic AMP level was elevated by dicentrine in a concentration-dependent manner. These data indicate that the inhibitory effect of dicentrine on platelet aggregation and ATP release was due to the inhibition of thromboxane formation and the elevation of the level of cyclic AMP.     

Inhibition of Platelet Thromboxane Formation and Phosphoinositides Breakdown by Diisoeugenol

Abstract— Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B₂ formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.     

Comparison of the actions of some platelet-activating factor antagonists on platelets and aortic smooth muscles.

The pharmacological actions of five platelet-activating factor (PAF) antagonists were compared in rabbit platelets and rat thoracic aorta. In PAF (2 ng/ml)-induced aggregation of washed rabbit platelets, WEB 2086 and WEB 2170 much were more potent inhibitors than BN 52021, kadsurenone and denudatin B, and the IC50 values were calculated to be 0.1, 0.3, 5, 8 and 10 micrograms/ml, respectively. WEB 2086, WEB 2170 and BN 52021 did not affect the platelet aggregation caused by collagen (10 micrograms/ml), ADP (20 microM), arachidonic acid (100 microM) or thrombin (0.1 U/ml). Kadsurenone and denudatin B suppressed ATP release, thromboxane B2 formation and the rise in intracellular calcium of washed rabbit platelets caused by collagen and thrombin, while WEB 2086, WEB 2170 and BN 52021 did not have an effect. Norepinephrine (3 microM) induced a sustained contraction in rat thoracic aorta. Pretreatment with these PAF antagonists (20-100 micrograms/ml) caused inhibition of the aortic contraction in the following order: kadsurenone greater than denudatin B greater than WEB 2086 greater than BN 52021 greater than WEB 2170. In high potassium (60 mM)-induced contraction of rat aorta, kadsurenone and denudatin B caused marked relaxation, while WEB 2086, WEB 2170 and BN 52021 had only a slight effect. It is concluded that WEB 2086, WEB 2170 and BN 52021 are specific PAF antagonists in rabbit platelets, and weak relaxants in rat aorta. Two other PAF antagonists, kadsurenone and denudatin B, may inhibit some aspects of signal transduction, e.g., thromboxane formation or intracellular Ca2+ mobilization in rabbit platelets, and cause vasorelaxation in rat aorta by inhibiting calcium influx.     

Antiplatelet Effect of Gingerol Isolated from Zingiber officinale

The purpose of this investigation was to determine the antiplatelet mechanism of gingerol. Gingerol concentration-dependently (0·5–20 μm ) inhibited the aggregation and release reaction of rabbit washed platelets induced by arachidonic acid and collagen, but not those induced by platelet-activating factor (PAF), U46619 (9,11-dideoxy-9α,11 α-methano-epoxy-PGF_2α) and thrombin. Gingerol also concentration-dependently (0·5–10μ m ) inhibited thromboxane B₂ and prostaglandin D₂ formation caused by arachidonic acid, and completely abolished phosphoinositide breakdown induced by arachidonic acid but had no effect on that of collagen, PAF or thrombin even at concentrations as high as 300 μ m . In human platelet-rich plasma, gingerol and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by adenosine 5′-diphosphate (ADP, 5 μ m ) and adrenaline (5 ä m ) but had no influence on the primary aggregation. The maximal antiplatelet effect was obtained when platelets were incubated with gingerol for 30 min and this inhibition was reversible. It is concluded that the antiplatelet action of gingerol is mainly due to the inhibition of thromboxane formation.     

Antiplatelet effect of marchantinquinone, isolated from Reboulia hemisphaerica, in rabbit washed platelets

Platelet activation is involved in serious pathological situations, including atherosclerosis and restenosis. It is important to find efficient antiplatelet medicines to prevent fatal thrombous formation during the course of these diseases. Marchantinquinone, a natural compound isolated from Reboulia hemisphaerica, inhibited platelet aggregation and ATP release stimulated by thrombin (0.1 units mL(-1)), platelet-activating factor (PAF; 2 ng mL(-1)), collagen (10 microg mL(-1)), arachidonic acid (100 microM), or U46619 (1 microM) in rabbit washed platelets. The IC50 values of marchantinquinone on the inhibition of platelet aggregation induced by these five agonists were 62.0 +/- 9.0, 86.0 +/- 7.8, 13.6 +/- 4.7, 20.9 +/- 3.1 and 13.4 +/- 5.3 microM, respectively. Marchantinquinone inhibited thromboxane B2 (TxB2) formation induced by thrombin, PAF or collagen. However, marchantinquinone did not inhibit TxB2 formation induced by arachidonic acid, indicating that marchantinquinone did not affect the activity of cyclooxygenase and thromboxane synthase. Marchantinquinone did inhibit the rising intracellular Ca2+ concentration stimulated by the five platelet-aggregation inducers. The formation of inositol monophosphate induced by thrombin was inhibited by marchantinquinone. Platelet cAMP and cGMP levels were unchanged by marchantinquinone. The results indicate that marchantinquinone exerts antiplatelet effects by inhibiting phosphoinositide turnover.     

Effect of the purified phospholipases A2 from snake and bee venoms on rabbit platelet function

Effects of seven purified phospholipases A2 from the venoms of snakes (Naja naja atra, Trimeresurus mucrosquamatus and T. gramineus) and honey bee (Apis mellifera) on rabbit washed platelet suspension in the absence of bovine serum albumin have been studied. Only phospholipases A2 from N. n. atra, T. mucrosquamatus and A. mellifera venoms induced platelet aggregation with small amounts of 14C-serotonin release. They showed tachyphylaxis and also cross-tachyphylaxis in inducing platelet aggregation. The former two phospholipases A2 exhibited biphasic responses in which irreversible aggregations appeared at concentrations of 1-10 micrograms/ml. At higher concentrations, they elicited the reversible aggregation. Exogenous Ca2+ was essential to their activity. Indomethacin and EDTA completely abolished both phospholipase A2 induced platelet shape change and aggregation, while mepacrine, prostaglandin E1, verapamil and nitroprusside inhibited only the aggregation response. p-Bromophenacyl bromide-modified phospholipases A2, which almost completely lost enzymatic activity, failed to induce platelet aggregation. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol inhibited the phospholipase A2-induced platelet aggregation. These phospholipases A2 induced thromboxane B2 formation which was inhibited by EDTA and indomethacin, but not by prostaglandin E1. Pre-treatment of platelet suspension with phospholipase A2 from N. n. atra or A. mellifera venom (50 micrograms/ml) inhibited platelet aggregation induced by sodium arachidonate or collagen, but not that induced by thrombin or ionophore A-23187. Exogenous sodium arachidonate or lysophosphatidylcholine also showed unaltered inhibitory spectrum on platelet aggregation. It is concluded that phospholipases A2 induce platelet aggregation by virtue of their enzymatic activity, cleaving the membrane phospholipids resulting in arachidonic acid release and formation of thromboxane A2. On the other hand, the cleaved products, lysophosphatidylcholine, arachidonic acid or arachidonate metabolites (via lipoxygenase pathway) may be responsible for anti-platelet activity.     

Mechanism of Action of p-Chlorobiphenyl on the Inhibition of Platelet Aggregation

p-Chlorobiphenyl (1–50 μm ) concentration-dependently inhibited the aggregation and release reaction of rabbit washed platelets induced by arachidonic acid and collagen, but not those induced by platelet-activating factor (PAF), U46619 and thrombin. The IC50 values of p-chlorobiphenyl on the arachidonic acid and collagen-induced platelet aggregation were 2.9 ± 0.5 and 12.8 ± 2.3 μm , respectively. The formation of both platelet thromboxane B₂ and prostaglandin D₂ caused by arachidonic acid was inhibited by p-chlorobiphenyl concentration-dependently. In myo-[³H]inositol-labeled and fura-2-loaded platelets, [³H]inositol monophosphate generation and the rise in intracellular Ca²⁺ stimulated by arachidonic acid were inhibited by p-chlorobiphenyl. In human platelet-rich plasma, p-chlorobiphenyl and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by adenosine 5′-diphosphate and adrenaline without affecting the primary aggregation. It is concluded that p-chlorobiphenyl may be a cyclo-oxygenase inhibitor and its antiplatelet action is mainly due to the inhibition of thromboxane formation.     

The effect of the antiarrhythmic, prajmalium bitartrate, on human thrombocyte function

Studies of the in vitro effects of the antiarrhythmic drug prajmalium bitartrate (PBT, Neo-Gilurytmal) showed inhibitory effects on platelet aggregation and thromboxane production. PBT inhibited the primary and secondary phases of aggregation induced by adrenaline (epinephrine) or adenosine diphosphate (ADP). Platelet aggregation stimulated with collagen, platelet activating factor (PAF), and the thromboxane mimetic 9,11-azo-prostaglandin H2 (U 44064) was inhibited. The secondary phase of aggregation induced by ristocetin and aggregation caused by arachidonic acid (AA) were inhibited in samples from some donors (responders) but not in others (nonresponders). Platelet aggregation by the ionophore calcimycine (A 23187) was not inhibited, but small doses of calcimycine abolished the PBT-induced inhibition of aggregation caused by ADP. Thromboxane production of platelets with collagen of ADP was inhibited by higher concentrations of PBT, whereas AA-induced thromboxane synthesis remained unchanged. The observed antiplatelet activities of PBT are thought to result from calcium and sodium channel blocking properties of the drug.     

Inhibitory effect of curcumin, a food spice from turmeric, on platelet-activating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca2+ signaling.

Curcumin, a dietary spice from turmeric, is known to be anti-inflammatory, anticarcinogenic, and antithrombotic. Here, we studied the mechanism of the antiplatelet action of curcumin. We show that curcumin inhibited platelet aggregation mediated by the platelet agonists epinephrine (200 microM), ADP (4 microM), platelet-activating factor (PAF; 800 nM), collagen (20 microg/mL), and arachidonic acid (AA: 0.75 mM). Curcumin preferentially inhibited PAF- and AA-induced aggregation (IC50; 25-20 microM), whereas much higher concentrations of curcumin were required to inhibit aggregation induced by other platelet agonists. Pretreatment of platelets with curcumin resulted in inhibition of platelet aggregation induced by calcium ionophore A-23187 (IC50; 100 microM), but curcumin up to 250 microM had no inhibitory effect on aggregation induced by the protein kinase C (PKC) activator phorbol myrsitate acetate (1 microM). Curcumin (100 microM) inhibited the A-23187-induced mobilization of intracellular Ca2+ as determined by using fura-2 acetoxymethyl ester. Curcumin also inhibited the formation of thromboxane A2 (TXA2) by platelets (IC50; 70 microM). These results suggest that the curcumin-mediated preferential inhibition of PAF- and AA-induced platelet aggregation involves inhibitory effects on TXA2 synthesis and Ca2+ signaling, but without the involvement of PKC.     

The dopamine D(1) receptor agonist SKF-82958 serves as a discriminative stimulus in the rat

The antiplatelet effect of the pyridazinone analogue, 4, 5-dihydro-6-[4-[2-hydroxy-3-(3,4 dimethoxybenzylamino)propoxy]naphth-1-yl]-3(2H)-pyridazinone (HCL-31D), was investigated in vitro with rabbit platelets. HCL-31D dose-dependently inhibited the platelet aggregation and ATP release induced by collagen (10 microg/ml), arachidonic acid (100 microM) or thrombin (0.1 U/ml) with an IC(50) of about 0.95-5.41 microM. HCL-31D (0.5-5 microM) increased the platelet cyclic AMP level in a dose-dependent manner. Furthermore, HCL-31D potentiated cyclic AMP formation caused by prostaglandin E(1) but not that caused by 3-isobutyl-1-methylxanthine (IBMX). HCL-31D also attenuated phosphoinositide breakdown and intracellular Ca(2+) elevation induced by collagen, arachidonic acid or thrombin. HCL-31D inhibited the formation of thromboxane B(2) induced by collagen or thrombin but not by arachidonic acid. In addition, HCL-31D did not affect platelet cylooxygenase and thromboxane synthase activity. These data indicate that HCL-31D is an inhibitor of phosphodiesterase and that its antiplatelet effect is mainly mediated by elevation of cyclic AMP levels.     

Regulation of human platelet activation--analysis of cyclooxygenase and cyclic AMP-dependent pathways

We have studied the regulation of human platelet activation by cyclic AMP (cAMP), and the cyclooxygenase products by examining the effect of prostacyclin (PGI2) and indomethacin on platelet aggregation, release reaction and thromboxane B2 (TxB2) generation induced by the full dose range of common platelet agonists in both platelet-rich plasma and washed platelets. Platelet aggregation, [14C]-5HT and TxB2 release induced by "threshold" and "supramaximal" concentrations of ADP, adrenaline, platelet-activating factor (PAF) and U46619 were totally abolished by low concentrations of PGI2 (3-6 nM). In contrast, platelet activation induced by submaximal concentrations of collagen, thrombin and the calcium ionophore A23187 was only partially inhibited by PGI2 (3-3000 nM). PAF-induced release reaction like that induced by ADP and adrenaline was totally dependent on the cyclooxygenase products and aggregation, while U46619-induced release reaction was only partially dependent on aggregation and the cyclooxygenase products. While both PGI2 (18-3000 nM) and indomethacin (10 microM) abolished collagen-induced aggregation and the aggregation-mediated release reaction, neither inhibitor significantly inhibited platelet adhesion or the adhesion-mediated release reaction. Maximal thrombin-induced aggregation and release reaction was also not significantly inhibited by PGI2 (300 nM) or indomethacin (10 microM). Thromboxane (TxB2) generation induced by sub-maximal to maximal concentrations of collagen, thrombin and A23187 was, although significantly inhibited, not abolished by PGI2. These results demonstrate that PAF is a "weak" agonist similar to ADP and adrenaline, U46619 is an agonist intermediate between weak and strong which induces a release reaction that is only partially dependent on aggregation, but unlike the strong agonists, is totally susceptible to inhibition by PGI2, PGI2 is an indirect inhibitor of phospholipase activation, which does not significantly inhibit non-aggregation-mediated arachidonate mobilization, induced by the strong agonists, and the so-called third pathway in the collagen and thrombin-induced release reaction, which is insensitive to indomethacin, is also insensitive to elevators of cAMP such as PGI2.     

Antiplatelet Effects of Some Aporphine and Phenanthrene Alkaloids in Rabbits and Man

Two aporphines (boldine and laurolitsine) and five phenanthrene alkaloids (litebamine, secoboldine, N-cyanosecoboldine, N-methylsecoglaucine and N-methylsecopredicentrine) were evaluated in-vitro for their ability to inhibit platelet aggregation. All seven alkaloids inhibited aggregation of rabbit platelets and inhibited the release of ATP induced by arachidonic acid and collagen in rabbit platelets. Those aggregations induced by platelet-activating factor (PAF), thrombin, U46619 and ADP were inhibited by the three N-substituted secoboldine derivatives only. Thromboxane B₂ formation caused by arachidonic acid was also suppressed by these compounds. They did not affect the generation of [³H]inositol monophosphate caused by collagen, PAF and thrombin in the presence of indomethacin. Platelet cyclic AMP level was unaffected by litebamine, but was increased by N-methyl-secoglaucine. Litebamine suppressed the secondary aggregation, but not the primary aggregation, induced by ADP and adrenaline in platelet-rich plasma from man, whereas N-methylsecoglaucine inhibited both primary and secondary aggregation. It is concluded that the antiplatelet effect of these seven aporphine and phenanthrene alkaloids is mainly a result of inhibition of thromboxane A₂ formation; N-methylsecoglaucine has additional antiplatelet activity as a result of increasing the levels of platelet cyclic AMP.     

Inhibitory effect of 8-bromo cyclic GMP on an extracellular Ca2+-dependent arachidonic acid liberation in collagen-stimulated rabbit platelets

The inhibitory effect of cyclic GMP on collagen-induced platelet activation was studied using 8-bromo cyclic GMP (8brcGMP) in washed rabbit platelets. Addition of collagen (1 micrograms/ml) to platelet suspension caused shape change and aggregation associated with thromboxane (TX) A2 formation. 8brcGMP (10-1000 microM) inhibited collagen-induced platelet aggregation and TXA2 formation in a concentration-dependent manner. 8brcGMP did not affect platelet cyclooxygenase pathways, but markedly inhibited collagen-induced arachidonic acid (AA) liberation from membrane phospholipids in [3H]AA-prelabeled platelets, indicating that the inhibitory effect of 8brcGMP on collagen-induced aggregation is due to an inhibition of AA liberation. In [32P]orthophosphate-labeled platelets, collagen stimulated phosphorylation of a 20,000 dalton (20-kD) and 40-kD proteins. 8BrcGMP stimulated phosphorylation of a specific protein having molecular weight of 46-kD and inhibited collagen-induced both 20- and 40-kD protein phosphorylation. Collagen could stimulate the AA liberation without activation of phospholipase C or Na+-H+ exchange, but could not in the absence of extracellular Ca2+. These findings suggest that cyclic GMP inhibits collagen-induced AA liberation which is mediated by an extracellular Ca2+-dependent phospholipase A2. However, cyclic GMP seems to inhibit the Ca2+-activated phospholipase A2 indirectly, since 8brcGMP had no effect on Ca2+ ionophore A23187-induced platelet aggregation or AA liberation. It is therefore suggested that cyclic GMP may regulate collagen-induced increase in an availability of extracellular Ca2+ which is responsible for phospholipase A2 activation in rabbit platelets.     

Effect of a potent cyclooxygenase inhibitor, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), on human platelets

Because the metabolites of arachidonic acid participate in many physiopathological responses, including inflammation and platelet aggregation, cyclooxygenase inhibitors are important in the treatment of associated diseases. A biologically active compound, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), selectively and concentration dependently inhibited aggregation of platelets from man and ATP release caused by arachidonic acid (200 microM) and collagen (10 microg mL(-1)) without affecting the aggregation caused by thrombin (0.1 U mL(-1)) and U46619 (2 microM). The IC50 value (drug concentration inhibiting maximum response by 50%) of KTC-5 for aggregation induced by arachidonic acid and collagen was 0.11+/-0.04 microM and 0.20+/-0.03 microM, respectively. This inhibitory effect of KTC-5 was reversible and time dependent. KTC-5 specifically inhibited intracellular calcium mobilization initiated by arachidonic acid or collagen without affecting that caused by thrombin or U46619 in human platelets. Furthermore, KTC-5 inhibited thromboxane B2 and prostaglandin D2 formation provoked by arachidonic acid. The IC50 value of KTC-5 for arachidonic-acid-induced thromboxane B2 formation was 0.07+/-0.02 microM. Based on these observations, the data indicated that KTC-5 potently inhibited human platelet aggregation and ATP release mainly via the inhibition of the cyclooxygenase-1 activity. Moreover, KTC-5 inhibited lipopolysaccharide-induced prostaglandin E2 formation in RAW264.7 cells in the presence of external arachidonic acid with an IC50 value of 0.17+/-0.06 microM. Immunoblot analysis showed that KTC-5 did not affect the cyclooxygenase-2 expression in the presence of lipopolysaccharide on RAW264.7 cells. This result indicated that KTC-5 affects the activity of cyclooxygenase-2. According to these data, we concluded that KTC-5 is a cyclooxygenase inhibitor for both subtypes.     

Effect of cefaclor on guinea pig platelet aggregation in vitro

Effects of cefaclor (3-chloro-7-D-(2-phenyl-glycinamido)-3-cephem-4-carboxylic acid) on PAF, ADP, collagen, endotoxin, and thrombin-induced platelet aggregation were examined in vitro with the use of guinea pig platelet-rich plasma and washed platelets. PAF, even at concentrations lower than its minimum effective concentration, enhanced ADP- or endotoxin-induced platelet aggregation and prolonged the time to attain the maximum aggregation. PAF also enhanced collagen-induced platelet aggregation and shortened the lag time. Cefaclor (CCL) inhibited the PAF, ADP or thrombin induced platelet aggregation and shortened their maximum aggregation times at higher concentrations such as 300 micrograms/ml or more. CCL also inhibited the collagen-induced platelet aggregation and prolonged the lag time, but showed no effect on endotoxin-induced platelet aggregation. The effect of CCL was almost the same as that of latamoxef (LMOX). CCL and LMOX, however, showed no effect on cellular Ca2+ increase produced by PAF, ADP, or thrombin, suggesting that the inhibitory effect of CCL and LMOX on platelet aggregation is caused by the inhibition of fibrinogen binding to the glycoprotein IIb/IIIa complex.     

Effects of dilazep on cytoplasmic calcium ion concentrations and arachidonic acid metabolism in activated human platelets

The effects of dilazep (tetrahydro-1H-1,4-diazepine-1,4(5H)-dipropanol bis(3,4,5-trimethoxybenzoate)-di-hydrochloride monohydrate, Comelian), a coronary and cerebral vasodilator and an antiplatelet drug, on the cytoplasmic Ca2+ concentration ([Ca2+]i) and arachidonic acid (AA) metabolism in activated human platelets were investigated. [Ca2+]i (free calcium ion concentration) of aequorin-loaded platelets was estimated by using the platelet ionized calcium aggregometer. AA metabolism was studied by the determination of AA metabolites, hydroxyheptadecatrienoic acid and 12-hydroxyeicosatetraenoic acid, using reversed-phase high performance liquid chromatography. When platelets were preincubated with dilazep (0-0.5 mmol/l), the drug inhibited both platelet aggregation and [Ca2+]i elevation induced by thrombin, AA and collagen in a concentration dependent manner, while only aggregation was inhibited after stimulation with the Ca ionophore A23187 (calcimycin). Both influx and release of Ca2+ into platelet cytoplasm induced by thrombin or AA were inhibited by dilazep, while neither of them was affected when induced by A23187. Oral ingestion of dilazep as a 100-mg capsule significantly depressed the [Ca2+]i elevation induced by thrombin, AA and collagen after 3 h. Dilazep inhibited endogenous AA metabolism by platelets induced by thrombin, although it enhanced exogenous one. Thus, dilazep inhibited platelet aggregation induced by any agonists including A23187, while [Ca2+]i elevation was inhibited by the drug only when the receptor-mediated agonist was used. Furthermore, it is suggested that dilazep inhibited AA liberation from platelet membrane phospholipids, leading to reduced production of all endogenous AA metabolites after platelet activation although metabolites of exogenous AA could be increased.     

Inhibition of rabbit platelet aggregation by nucleoside 5'-alkylphosphates: correlation with inhibition of agonist-induced calcium influx

We investigated the effects of uridine 5'-alkylphosphates on agonist-induced aggregation, increased intracellular calcium concentration [Ca(2+)](i), and Ca(2+) (Mn(2+)) influx in washed rabbit platelets. Uridine 5'-hexadecylphosphate (UMPC16) and uridine 5'-eicosylphosphate (UMPC20) at a concentration of 1 x 10(-5) M inhibited platelet aggregation induced by platelet-activating factor (PAF), thrombin, arachidonic acid, and ADP. UMPC16 did not cause significant interference in the binding of [(3)H-acetyl]PAF to platelets. The inhibition of PAF-induced platelet aggregation by UMPC16 was dependent upon the addition time; UMPC16 was ineffective at 60 sec when the extracellular calcium uptake reached the maximum level in PAF-stimulated platelets. Furthermore, UMPC16 inhibited guanosine 5'-O-(3-thiotriphosphate)-induced platelet aggregation but did not affect ionophore A23187- and calcium-independent agonist phorbol 12-myristate 13-acetate-induced platelet aggregation. UMPC16 markedly inhibited the Ca(2+) (Mn(2+)) influx induced by PAF and ADP, and partly inhibited the [Ca(2+)](i) increase induced by the receptor-mediated stimulation. On the other hand, UMPC16 did not affect the [Ca(2+)](i) increase and Ca(2+) (Mn(2+)) influx induced by ionomycin. These experiments suggest that inhibition of calcium influx associated with receptor-mediated platelet activation may be involved in the action of UMPC16.     

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca²⁺ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.