Bakkenolide G,a Natural PAF-receptor Antagonist

Because platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) participates in many physiopathological responses, including inflammatory reaction, endotoxic shock, allergic diseases and platelet aggregation, PAF-receptor antagonists are important in the treatment of these diseases. A biologically active compound, bakkenolide G, extracted from the plant Petasites formosanus selectively and concentration-dependently inhibited PAF-induced platelet aggregation and ATP release. The IC50 of bakkenolide G for PAF (2 ng mL^?1)-induced platelet aggregation was 5.6 ± 0.9 μm . Bakkenolide G also concentration-dependently inhibited PAF-induced intracellular signal transductions, including thromboxane B₂ formation, and increased intra-cellular calcium concentration and phosphoinositide breakdown without affecting those caused by thrombin (01 units mL^?1), collagen (10 μg mL^?1), arachidonic acid (100 μm ) and U46619 (1 μm ). Bakkenolide G shifted the concentration-response curves of PAF-induced platelet aggregation parallel to the right; the Schild plot slope and the pA₂ value were 1.31 ± 0.31 and 6.21 ± 0.75, respectively. Moreover, bakkenolide G concentration-dependently competed with [³H]PAF binding to platelets, with an IC50 value of 2.5 ± 0.4 μm . These data strongly indicate that bakkenolide G is a specific PAF-receptor antagonist as an antiplatelet aggregatory agent.     

Antiplatelet Mechanism of 2‐Chloro‐3‐(4‐hexylphenyl)‐amino‐1,4‐naphthoquinone (NQ304), an Antithrombotic Agent

Abstract: The effects of 2‐chloro‐3‐(4‐hexylphenyl)‐amino‐1,4‐naphthoquinone (NQ304), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein IIb/IIIa and intracellular signals were investigated using human platelets. NQ304 inhibited thrombin‐, arachidonic acid‐ and thapsigargin‐induced aggregation of washed human platelets with the IC₅₀ values of 22.2±0.7, 6.5±0.2, and 7.6±0.1 μM, respectively. NQ304 significantly inhibited fluorescein isothiocyanate‐conjugated fibrinogen binding to human platelet surface glycoprotein IIb/IIIa receptor by 75%, but failed to inhibit the fibrinogen binding to purified glycoprotein IIb/IIIa receptor. This result suggests that NQ304 inhibit platelet aggregation by suppression of an intracellular pathway that involves exposure of the glycoprotein IIb/IIIa receptor, rather than by direct inhibition of fibrinogen‐glycoprotein IIb/IIIa binding. NQ304 significantly inhibited thrombin‐induced increase in intracellular Ca²⁺ mobilization at the dose of 30 μM and ATP secretion in a dose‐dependent manner. It also inhibited thrombin‐ and arachidonic acid‐induced thromboxane A₂ formation in human platelet dose‐dependently. In conclusion, the antiplatelet mechanism of NQ304 may be due to the reduction of the thromboxane A₂ formation, inhibition of adenosine triphosphate release and intracellular calcium mobilization.     

Effects of cryptolepine on collagen-induced aggregation and on the mobilization and metabolism of arachidonic acid by rabbit platelets

1. We examined the effect of cryptolepine on collagen-induced aggregation and on the mobilization, and metabolism of arachidonic acid in rabbit platelets.2. Preincubation of platelets with cryptolepine (50–100 μM) did not affect the primary wave of aggregation but resulted in a dose-dependent, surmountable inhibition of the secondary wave of aggregation induced by collagen (5 μg/ml). The inhibition by cryptolepine was greater when cryptolepine was incubated with the platelets after the peak of the primary wave of aggregation.3. Cryptolepine (50–100 μM) dose-dependently inhibited thrombin (1.5 U/ml) and A23187 (2.5 μM)-induced release of ¹⁴C[AA] from platelet membrane phospholipid pools. The percentage inhibition of A23187-induced ¹⁴C[AA] release was 31.3 ± 4.3% (50 μM) and 79.3 ± 5.4% (100 μM), while thrombin-induced release was inhibited by 39.2 ± 2.4% (50 μM) and 68.2 ± 3.6% (100 μM).4. At near maximal concentration (100 μM) which significantly inhibited secondary aggregatory response and ¹⁴C[AA] release, cryptolepine had no effect on the platelet metabolism of ¹⁴C[AA] to thromboxane B₂, HHT and 12 HETE.5. The present findings suggest that cryptolepine inhibited collagen-induced secondary aggregation through a selective antiphospholipase-like activity. There was not effect on platelet cyclooxygenase and lipoxygenase activities of platelets.     

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 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.     

Inhibition of rat platelet aggregation by a Prudhoe Bay crude oil and its aliphatic,aromatic, and heterocyclic fractions

Wasjed platelets isolated from rats 24 hr after oral treatment with a Prudhoe Bay crude oil (PBCO) showed a substantial inhibition of aggregation induced by ADP, arachidonic acid, or epinephrine. In vitro addition of a dimethyl sulfoxide extract of PBCO or its aliphatic, aromatic, or heterocyclic fractions to washed platelets also resulted in an inhibition of aggregation. ADP release was inhibited in platelets to which an extract of PBCO or its fractions were added in vitro or in platelets isolated from rats treated in vivo with PBCO. Thromboxane B₂ release was increased in platelets isolated from rats intubated with PBCO or in platelets to which a dimethyl sulfoxide extract of the aromatic or heterocyclic fraction was added. However, thromboxane B₂ release was inhibited in platelets to which PBCO or the aliphatic fraction extracts were added. The results indicate that PBCO inhibits platelet aggregation presumably by bringing about alterations in the platelet plasma membrane. Inhibition of ADP release could contribute to the inhibition of aggregation but thromboxane B₂ is believed not to play a significant role.     

Antiplatelet and calcium inhibitory properties of eugenol and sodium eugenol acetate

• 1.1. Eugenol (3-methoxy-4-hydroxy-propenylbenzene) or sodium eugenol acetate (4-0-acetic acid sodium-3-methoxy-l-propenylbenzene) (0.25, 0.5, 1 mM) concentration-dependently inhibited arachidonic acid (AA)., collagen-, epinephrine- and ADP-induced platelet aggregation.
• 2.2. Eugenol or sodium eugenol acetate inhibited collagen-induced aggregation of washed rabbit platelets synergistically with creatine phosphatelcreatine phosphokinase (CP/CPK, 5 mM/10 U/ml) or p-bromophenacyl bromide (p-BPB, 10 μM), and they also potentiated the inhibitory action of imidazole (0.5 mM) on AA-induced aggregation.
• 3.3. Eugenol or sodium eugenol acetate (0.25, 0.5, 1 mM) concentration-dependently inhibited AA-induced thromboxane B₂ and prostaglandin E₂ formation.
• 4.4. The rise of intracellular Ca²⁺ caused by collagen, epinephrine, ADP, and AA were inhibited by eugenol or sodium eugenol acetate (1 mM).

     

Inhibition of collagen-induced platelet activation by arachidonyl trifluoromethyl ketone

Collagen-induced platelet activation is associated with, and markedly potentiated by, the release of arachidonic acid and its subsequent conversion to thromboxane A₂. The precise mechanism of arachidonic acid release is unknown. An inhibitor of isolated cytosolic phospholipase A₂ (cPLA₂), arachidonyl trifluoromethyl ketone (AACOCF₃), was used to examine the role that cPLA₂ plays in this process. AACOCF₃ inhibited platelet aggregation in response to collagen and arachidonic acid but not to thrombin, calcium ionophore, phorbol ester, or a thromboxane mimetic. Thromboxane formation stimulated by thrombin or collagen was inhibited by AACOCF₃. However, AACOCF₃ did not inhibit collagen-induced [¹⁴C]arachidonic acid release. These data are consistent with the inhibitory effects of AACOCF₃ on collagen-induced aggregation involving an action on the conversion of arachidonic acid to thromboxane.     

Inhibition in vitro of platelet aggregation and arachidonic acid metabolism by flavone

The effects of flavone on platelet aggregation and arachidonic acid (AA) metabolism were tested in vitro. When incubated at a concentration of 50 μM, flavone completely suppressed platelet aggregation induced by 150 μM AA in thirty-six out of forty-three subjects tested. A lower concentration (10 μM) was effective in about 50% of the donors. Flavone also inhibited the second wave of aggregation induced by epinephrine and ADP. Platelet thromboxane formation, estimated both by radioimmuno-assay measurements and by studies of ¹⁴C-labeled AA metabolism, was depressed by flavone. Flavone-treated platelets preferentially utilized [¹⁴C]AA for the lipoxygenase pathway while cyclo-oxygenase activity was depressed. Adenosine 3':5'-cyclic monophosphate (cAMP) was measured in flavone-treated and control platelets. While their baseline levels were similar, flavone-treated platelets showed a lower stimulation of cAMP induced by prostacyclin (PGI₂) than did controls. Phosphodiesterase activity was not affected by flavone as judged from the decay rates of PGI₂-stimulated cAMP levels. From these findings we conclude that the antiaggregating activity of flavone is not a consequence of changes in platelet cAMP but is due to inhibition of cyclo-oxygenase.     

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.     

Anti-platelet activity of diacetylated obovatol through regulating cyclooxygenase and lipoxygenase activities

Obovatol has been reported biological activities such as muscle relaxative, anti-gastric ulcer, anti-allergic and anti-bacterial activities. The present study was undertaken to investigate the effect of diacetylated obovatol, an obovatol derivative, on rabbit platelet aggregation, and their possible molecular mechanisms. Effects of diacetylated obovatol on platelet activation including aggregation and serotonin secretion were examined. In addition, we investigated the effect of diacetylated obovatol on archidonic acid and metabolites liberation and intracellular calcium mobilization. Diacetylated obovatol concentration-dependently inhibited the washed rabbit platelet aggregation induced by collagen and arachidonic acid, suggesting that diacetylated obovatol may selectively inhibits collagen- and arachidonic acid-mediated signal transduction. In accordance with these results, diacetylated obovatol showed a concentrationdependent decrease in cytosolic Ca²⁺ mobilization and serotonin secretion. However, diacetylated obovatol did not inhibit arachidonic acid liberation; on the other hand, diacetylated obovatol inhibited the formation of arachidonic acid metabolites such as thromboxane A₂, prostaglandin D₂ and 12-HETE through interfering with cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. The results demonstrated that diacetylated obovatol has antiplatelet activities through inhibition of COX-1 and LOX activities.     

Novel inhibitory actions on platelet thromboxane and inositolphosphate formation by xanthones and their glycosides

Xanthones and their glycosides were tested for their antiplatelet activities in washed rabbit platelets. Tripteroside acetate and norathyriol acetate were the most potent inhibitors. Tripteroside acetate inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, platelet-activating factor (PAF), collagen, ionophore A23187 and thrombin. The IC50 values of tripteroside acetate toward arachidonic acid- (100 microM) and collagen- (10 micrograms/ml) induced platelet aggregation were 10 and 30 micrograms/ml respectively. It inhibited thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, thrombin and ionophore A23187 and also that caused by the incubation of lysed platelet homogenate with arachidonic acid. Tripteroside acetate decreased the formation of inositolphosphate caused by thrombin, collagen and PAF, whereas it had no direct effect on fibrinogen-platelet interaction. It is concluded that xanthone derivatives inhibited platelet aggregation and release reaction by diminishing thromboxane formation and phosphoinositide breakdown.     

“In vitro” and “ex vivo” effects of picotamide,a combined thromboxane A2-synthase inhibitor and -receptor antagonist,on human platelets

Summary Picotamide (G 137), a new non prostanoid inhibitor of in vitro arachidonic acid induced platelet aggregation, has been further characterized in in vitro and ex vivo studies.When whole blood was activated with collagen in the presence of picotamide 5×10^–4 M, thromboxane B₂ production was decreased, and 6-keto-PGF₁ generation was significantly increased, suggesting a reorientation of platelet endoperoxide metabolism following blockade of thromboxane synthetase. Picotamide also inhibited platelet aggregation and clot retraction induced by the endoperoxide analogue U46619 in human platelets, indicating thromboxane A₂-receptor antagonism, possibly of competitive nature.A single oral dose of picotamide 1 g in 24 healthy volunteers produced a significant inhibition of collagen, arachidonic acid and U46619-induced platelet aggregation. Serum levels of thromboxane B₂ were also reduced.Chronic administration of picotamide 1.2 g/d to patients with vascular disease resulted in a prompt and persistent fall in their increased plasma levels of -thromboglobulin.The results indicate that picotamide is a combined thromboxane B₂-synthetase inhibitor and thromboxane A₂-receptor antagonist in human platelets, and that it may prove useful as an antithrombotic agent.     

An antithrombotic agent, NQ301, inhibits thromboxane A2 receptor and synthase activity in rabbit platelets

In previous studies we have reported that NQ301, a synthetic 1,4-naphthoquinone derivative, displays a potent antithrombotic activity, and that this might be due to antiplatelet effect, which was mediated by the inhibition of cytosolic Ca(2+) mobilization in activated platelets. In the present study, the effect of NQ301 on arachidonic acid cascade in activated platelets has been examined. NQ301 concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen (10 microg/ml), arachidonic acid (100 microM) and U46619 (1 microM), a thromboxane A2 receptor agonist, with IC50 values of 0.60+/-0.02, 0.78+/-0.04 and 0.58+/-0.04 microM, respectively. NQ301 also produced a shift to the right of the concentration-effect curve of U46619, indicating a competitive type of antagonism on thromboxane A2/prostaglandin H2 receptor. NQ301 slightly inhibited collagen-induced arachidonic acid liberation. In addition, NQ301 potently suppressed thromboxane B2 formation by platelets that were exposed to arachidonic acid in a concentration-dependent manner, but had no effect on the production of prostaglandin D2, indicating an inhibitory effect on thromboxane A2 synthase. This was supported by thromboxane A2 synthase activity assay that NQ301 concentration-dependently inhibited thromboxane B2 formation converted from prostaglandin H2. Moreover, NQ301 concentration-dependently inhibited 12-hydroxy-5,8,10,14-eicosatetraenoic acid formation by platelets that were exposed to arachidonic acid. Taken together, these results suggest that NQ301 has a potential to inhibit thromboxane A2 synthase activity with thromboxane A2/prostaglandin H2 receptor blockade, and modulate arachidonic acid liberation as well as 12-hydroxy-5,8,10,14-eicosatetraenoic acid formation in platelets. This may also be a convincing mechanism for the antithrombotic action of NQ301.     

NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A(2) synthesis and calcium entry

BACKGROUND AND PURPOSE

1,4-Naphthoquinones exhibit antiplatelet activity both in vivo and in vitro. In the present study, we investigated the antiplatelet effect of a novel naphthoquinone derivative NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone and its mechanism of action.

EXPERIMENTAL APPROACH

We measured platelet aggregation, Ca²⁺ mobilization, thromboxane B2 formation and P-selectin expression and examined several enzymatic activities. Furthermore, we used the irradiated mesenteric venules in fluorescein sodium–treated mice to monitor the antithrombotic effect of NP-313 in vivo.

KEY RESULTS

NP-313 concentration-dependently inhibited human platelet aggregation induced by collagen, arachidonic acid, thapsigargin, thrombin and A23187. NP-313 also inhibited P-selectin expression, thromboxane B₂ formation and [Ca²⁺]_i elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A₂ synthase, and protein kinase Cα, whereas it did not affect phospholipase A₂ or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca²⁺]_i increase through its inhibitory effects on Ca²⁺ influx, rather than blocking Ca²⁺ release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca²⁺ influx through store-operated calcium channel but had no effect on Ca²⁺ influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. Nevertheless, it had little effect on cyclic AMP and cyclic GMP levels. Also, intravenously administered NP-313 dose-dependently inhibited the thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A₂ synthesis and Ca²⁺ influx through SOCC.     

Antiplatelet effects of piplartine,an alkamide isolated from Piper tuberculatum: possible involvement of cyclooxygenase blockade and antioxidant activity

Objectives Piplartine (piperlongumine; 5,6‐dihydro‐1‐[1‐oxo‐3‐(3,4,5‐trimethoxyphenyl]‐2(1H) pyridinone) is an alkaloid amide isolated from Piper species (Piperaceae). It has been reported to show multiple pharmacological activities in vitro and in vivo. Methods We evaluated the in‐vitro antiplatelet effect of piplartine isolated from the roots of P. tuberculatum, on human platelet aggregation induced in platelet‐rich plasma by the agonists collagen, adenosine 5′‐diphosphate (ADP), arachidonic acid (AA) and thrombin. Key findings Piplartine (100μg/ml) caused a 30% inhibition in platelet aggregation when collagen was the agonist. At 200 μg/ml, piplartine significantly inhibited the aggregation induced by arachidonic acid (100%), collagen (59%) or ADP (52%) but not that induced by thrombin. The highest concentration of piplartine (300 μg/ml) inhibited thrombin‐ (37%), ADP‐ (71%) and collagen‐ (98%) induced aggregation. The inhibitory effect of piplartine on ADP‐induced platelet aggregation was not modified by pretreatment with pentoxifylline (a phosphodiesterase inhibitor), l ‐arginine (a substrate for nitric oxide synthase) or ticlopidine (a P2Y₁₂ purinoceptor antagonist). However, aspirin, a well‐known inhibitor of cyclooxygenase, greatly increased the inhibitory effect of piplartine on arachidonic‐acid‐induced platelet aggregation. Conclusions The mechanism underlying the piplartine antiplatelet action is not totally clarified. It could be related to the inhibition of cyclooxgenase activity and a decrease in thromboxane A₂ formation, similar to that occurring with aspirin. This and other possible mechanisms require further study.     

15,16-Dihydrotanshinone I, a major component from Salvia miltiorrhiza Bunge (Dansham), inhibits rabbit platelet aggregation by suppressing intracellular calcium mobilization

Radix Salviae miltiorrhiza (RSM, ‘Dansham’ in Korea, ‘Danshen’ in Chinese), the root of Salviae miltiorrhiza Bunge (Labiate) has been used as Chinese fork medicine for the treatment of cardiovascular diseases such as angina pectoris, coronary heart disease, myocardial infarction, and hypertension. In the present study, we evaluated the inhibitory effects of 15, 16-Dihydrotanshinone I, one of the major ingredients of Salvia miltiorrhiza Bunge, on platelet aggregation, with elucidation of its mechanisms of action. 15,16-Dihydrotanshinone I concentration-dependently inhibited collagen-induced aggregation of rabbit washed platelets with IC₅₀ of 8.7±5.6 μM, the potency being about seven-fold greater than EGCG, an active Green tea catechin component (IC₅₀: 56.6±48.7 μM). 15,16-Dihydrotanshinone I significantly inhibited the intracellular calcium ([Ca²⁺]_i) mobilization in a concentration-dependent manner. 15,16-dihdydrotanshinone I also significantly suppressed collagen (50 μg/mL)-induced liberation of [³H]Arachidonic acid from [³H]Arachidonic acid-incorporated rabbit platelet. In addition, 15,16-Dihydrotanshinone I at 50 μM slightly but significantly inhibited collagen-induced production of thromboxane B₂. These results indicate that 15,16-Dihydrotanshinone I exert potent anti-platelet activity via suppression of [Ca²⁺]_i mobilization and arachidonic acid liberation.     

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a direct thrombin inhibitor with a suppressive effect on thromboxane formation in platelets

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a newly synthetic benzoxazinone derivative, has previously been shown to inhibit rabbit platelet aggregation caused by thrombin and arachidonic acid. In the present study, the mechanism for the antiplatelet effect of HPW-RX2 was further investigated. In human platelets, HPW-RX2 concentration-dependently inhibited platelet aggregation, ATP release, P-selectin expression, and intracellular calcium mobilization caused by thrombin. In contrast, HPW-RX2 had no significant effect on either SFLLRN- or GYPGKF-induced platelet aggregation, indicating that HPW-RX2 did not interfere with platelet thrombin receptors. Moreover, HPW-RX2 inhibited the amidolytic activity of thrombin and prolonged the fibrinogen clotting time. These results suggest that the inhibitory effect of HPW-RX2 on thrombin-induced platelet aggregation is via direct inhibition of thrombin proteolytic activity. Besides the inhibition on thrombin, HPW-RX2 also prevented platelet aggregation, ATP release, and increase in [Ca2+]i caused by arachidonic acid and low concentration collagen. In a parallel manner, both arachidonic acid-induced thromboxane B2 and prostaglandin D2 formations were decreased in platelets treated with HPW-RX2. This indicates that HPW-RX2 is able to inhibit the arachidonic acid cascade at the cyclooxygenase level. This is the first report of a benzoxazinone derivative possessing both thrombin and cyclooxygenase inhibitory properties. The dual effect of HPW-RX2 might provide extra therapeutic benefits for treatment of arterial thrombosis.     

Inhibitory mechanisms of gabapentin, an antiseizure drug, on platelet aggregation

Gabapentin (Neurontin) is an analogue of gamma-aminobutyric acid (GABA) that is effective against partial seizures. Gabapentin has been reported to modulate serotonin release from platelets, but the effects of gabapentin on platelet activation have not been explored. In this study, gabapentin concentration-dependently (60-240 microM) inhibited platelet aggregation in washed platelets stimulated by collagen (1 microg mL(-1)), ADP (20 microM) and arachidonic acid (60 microM). Gabapentin (120 and 240 microM) also concentration-dependently inhibited collagen (1 microg mL(-1))-induced phosphoinositide breakdown, intracellular Ca(2+) mobilization, thromboxane A(2) formation, and p38 MAPK phosphorylation in human platelets. In conclusion, the most important findings of this study suggest that gabapentin inhibits platelet aggregation, at least in part, through the phospholipase C-inositol 1,4,5-trisphosphate-thromboxane A(2)-Ca(2+) pathway. Thus, it is possible that gabapentin treatment, alone or in combination with other antiplatelet drugs, may induce or potentiate inhibition of platelet aggregation, which may affect haemostasis 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.