Thromboxane B2 Formation Research Articles

NP‐313, 2‐acetylamino‐3‐chloro‐1,4‐naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A2 synthesis and calcium entry

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. We measured platelet aggregation, Ca(2+) 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. 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 B(2) formation and [Ca(2+) ](i) elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A(2) synthase, and protein kinase Cα, whereas it did not affect phospholipase A(2) or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca(2+)](i) increase through its inhibitory effects on Ca(2+) influx, rather than blocking Ca(2+) release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca(2+) influx through store-operated calcium channel but had no effect on Ca(2+) 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. Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A(2) synthesis and Ca(2+) influx through SOCC.

CSH72—A Specific Antagonist of Platelet‐activating Factor in Rabbit Platelets

CSH72 (N-methyl-1-benzyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline HCl) inhibited the aggregation and ATP-release reaction induced by platelet-activating factor (PAF) in a concentration-dependent manner in rabbit washed platelets. The IC50 values of CSH72, kadsurenone and BN52021 on PAF (3·6 nM)-induced platelet aggregation were 61·5 ± 7·2, 18·0 ± 2·8 and 10·6 ± 2·3 μM, respectively. CSH72 also concentration-dependently inhibited [3H]PAF (3·6 nM) binding to washed platelets with an IC50 value of 46·1 ± 1·9 μM. The concentration-response curve of PAF-induced platelet aggregation was shifted to the right by CSH72 with pA2 and pA10 values of 5·47 and 4·68, respectively. CSH72 (300 μM) significantly inhibited PAF (3·6 nM)-induced thromboxane B2 formation but exhibited no effects on those induced by arachidonic acid (100 μM) and thrombin (0·1 units mL−1). CSH72 (300 μM) also completely abolished [3H]inositol monophosphate formation induced by PAF but not that caused by collagen or thrombin in the presence of indomethacin (20 μM). It is suggested that CSH72 is a specific PAF-receptor antagonist as an antiplatelet aggregatory agent.

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.

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.

Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis

Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50 μM), collagen (500 μg/ml), arachidonic acid (AA) (1.0 mM) and calcium ionophore A-23187 (20 μM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6 μM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists—collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation.

Nutrition classics. Proceedings of the National Academy of Sciences of the United States of America, August 1975, Volume 72: Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. By Mats Hamberg, Jan Svensson, and Bengt Samuelsson.

An unstable [t1/2 at 37 degrees = 32 +/- 2 (SD) sec] intermediate, thromboxane A2, was detected in the conversion of prostaglandin G2 into 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (thromboxane B2) in platelets. The intermediate was trapped by addition of methanol, ethanol, or sodium azide to suspensions of washed human platelets incubated for 30 sec with arachidonic acid or prostaglandin G2. The structures of the resulting derivatives demonstrated that the intermediate possessed an oxane ring as in thromboxane B2 but lacked its hemiacetal hydroxyl group. Additional experiments using 18O2 or [2H8]arachidonic acid in the formation of thromboxane B2 and CH3O2H for the trapping of thromboxane A2, together with information on the t1/2 of the intermediate, indicated the presence of an oxetane structure in thromboxane A2. Incubation of arachidonic acid or prostaglandin G2 with washed platelets led to formation of an unstable factor that induced irreversible platelet aggregation and caused release of [14C]serotonin from platelets that had been incubated with [14C]serotonin. The properties and the mode of formation of this factor indicated that it was identical with thromboxane A2. Furthermore, evidence is presented that the more unstable and major component of rabbit aorta contracting substance (RCS) formed in platelets and guinea pig lung is also thromboxane A2.

Splitomicin suppresses human platelet aggregation via inhibition of cyclic AMP phosphodiesterase and intracellular Ca ++ release

Splitomicin is derived from β-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 μg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 μM) or ADP (10 μM). Splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 μM) was combined with PGE1 (0.5 μM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition, splitomicin (300 μM) attenuated intracellular Ca ++ mobilization, and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin, collagen, AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca ++ mobilization, TXB2 formation and ATP release.

Platelet aggregation and thromboxane B2 formation after ethanol abuse: is there a relationship to stroke?

Formation of thromboxane B2 (TXB2), a metabolite of the potent platelet-aggregating and vasoconstrictor agent thromboxane A2 (TXA2), during ADP-induced platelet aggregation was studied in 10 healthy men and in 10 male alcoholics during the 2-week period of detoxification. None of the alcoholics had anemia or thrombo-embolic disease. The platelets of the alcoholics were more sensitive for ADP and synthesized as much as triple the amount of TXB2 compared to those of the nonalcoholic donors. The effect was most striking during the rebound thrombocytosis and suggests that it could possible contribute to the increased incidence of various thrombotic diseases in the alcoholic.

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 Ca 2+ 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 Ca 2+ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.

FcγRII mediates platelet aggregation caused by disintegrins and GPIIb/IIIa monoclonal antibody, AP2

Disintegrins, snake venom-derived Arg-Gly-Asp (RGD)-containing polypeptides, and GPIIb/IIIa antagonist (AP2) block fibrinogen binding to GPIIb/IIIa of activated platelets, however, the combination of these two agents caused platelet aggregation. We hypothesize that disintegrin initially binds to specific epitope of GPIIb/IIIa, causing conformational change, and the recruitment of FcgammaRII, which can be bound by AP2, and finally triggering platelet aggregation. We prepared human platelet suspensions and measured platelet aggregation, Ca2+ mobilization, thromboxane B2 formation, and signal transduction. Disintegrin (e.g., accutin) and AP2 (a monoclonal antibody [mAb]-raised against GPIIb/IIIa) individually inhibited human platelet aggregation caused by collagen. However, as both accutin and AP2 were sequentially added into platelet suspension, platelet aggregation occurred. Accutin/AP2 caused shape change, cytosolic Ca2+ mobilization, P-selectin expression, and thromboxane A2 formation. Tirofiban, FcgammaRII mAb, or indomethacin completely inhibited platelet aggregation caused by accutin/AP2. Accutin/AP2 also caused tyrosine phosphorylation of signal molecules. Disintegrins enhanced AP2 binding to platelets, and AP2 also promoted disintegrin binding to platelets. FcgammaRII mAb inhibited the enhanced fluorescein isothiocyanate-disintegrin binding to platelet caused by AP2. Immunoprecipitation of the lysates of disintegrin/AP2-treated platelets using FcgammaRII Ab showed complex formation of GPIIb/IIIa and FcgammaRII. FcgammaRII mediates platelet aggregation caused by disintegrin and AP2, triggering a phospholipase C, phospholipase A2, Src-, Syk kinases, and Ca2+-dependent activation process. AP2 triggers platelet aggregation via binding to accessible FcgammaRII and the conformation-altered GPIIb/IIIa caused by disintegrin.

Impact of naproxen sodium at over-the-counter doses on cyclooxygenase isoforms in human volunteers

Over the last few years, there has been concern regarding the cardiovascular safety of selective cyclooxygenase (COX)-2 inhibitors and high-dose regimens of nonsteroidal anti-inflammatory drugs (NSAIDs). On the other hand, those compounds which elicit an almost complete (> 95%) and continuous suppression of platelet COX-1 may represent an exception. Apart from aspirin, which irreversibly inactivates COX-1, high-dose naproxen causes sustained COX-1 inhibition throughout the dose interval in some individuals. The present study examines whether naproxen sodium after a single dose administration and at steady state using "over-the-counter (OTC) doses" produces sufficient COX-1 inhibition. COX-2 inhibition was assessed concomitantly. Ex vivo inhibition of COX enzymes and the pharmacokinetics of naproxen were assessed in four volunteers receiving 220 mg naproxen sodium b.i.d. for 7 days. Blood samples were obtained pre-dose, at specified time points after the first dose on Day 1, and 12 hours after the previous evening dose on Days 2, 3, 4, 5 and 8. Recovery was assessed up to 36 hours after the last dose. Coagulation-induced thromboxane B2 formation and lipopolysaccharide-induced prostaglandin E2 synthesis were measured ex vivo in human whole blood as indices of COX-1 and COX-2 activity. Maximal inhibition after a single dose and at steady state were as follows: 94% and 93% (COX-1), and 79% and 85% (COX-2). A greater than 95% COX-1 inhibition was observed transiently in 2 of 4 volunteers at the time of maximal plasma concentration after a single-dose administration and in 1 of 4 volunteers throughout the 12-hour dose interval at steady state. For both isoenzymes, COX inhibition correlated with naproxen plasma levels (ex vivo IC50 values of 35.48 micromol/l (COX-1) and 64.62 micromol/l (COX-2)). Administration of naproxen sodium at OTC doses was associated with a profound inhibition of both COX enzymes. Although low-dose naproxen may elicit a virtually complete COX-1 inhibition in some individuals, it does not mimic the reliable, sustained and complete COX-1 blockade produced by aspirin. In conclusion, prolonged treatment with 220 mg naproxen sodium b.i.d. is not expected to provide sufficient cardioprotection in all patients, but may influence platelet function in some.

Significant effects of tipranavir on platelet aggregation and thromboxane B2 formation in vitro and in vivo

In the past, bleeding events have been described for patients with haemophilia taking HIV-1 protease inhibitors. Recently, the FDA published a warning concerning intracranial haemorrhage in patients taking the HIV-1 protease inhibitor tipranavir co-administered with ritonavir. We investigated (i) platelet aggregation in vivo in HIV-1-infected adult patients (n = 5) immediately before and 2 and 4 h after dosing of tipranavir/ritonavir 500/200 mg. To further characterize the effects, we then evaluated (ii) platelet aggregation and (iii) thromboxane B2 (TxB2) formation (ELISA) with increasing tipranavir concentrations (TPV(conc)) in vitro of up to 100,000 ng/mL. Platelet aggregation was stimulated either with 2 microM ADP (ADP) or 10 mg/L collagen (COL). TPV(conc) were measured with validated EPI-LC-MS/MS. Intraindividual comparisons of values at time points and TPV(conc), respectively, were carried out with repeated samples ANOVA. Platelet aggregation (mean, maximal light transmission A(max)) was significantly decreased in patients 4 h post-dose in collagen- (from 79.8% to 57.1%; P < 0.001) and in ADP-stimulated (from 58.5% to 54.0%; not significant) samples at a median (range) TPV(conc) of 62 500 ng/mL (22,990-67,500). These results could be reproduced in vitro at TPV(conc) 50,000 ng/mL (A(max)ADP/A(max)COL = 20.7/36.9%; P = 0.003/<0.001) and 100 000 ng/mL (A(max)ADP/A(max)COL = 14.5/17.1%; P < 0.001/<0.001). Median (range) TxB2 concentrations were reduced (P = 0.07) from 327 ng/mL (187-500) at baseline to 265 ng/mL (152-428) at 5000 ng/mL and were significantly reduced (P < 0.001) to 187 ng/mL (81-362) at a TPV(conc) of 50,000 ng/mL, respectively. Five HIV-1-infected patients on tipranavir-containing highly active antiretroviral therapy presented marked decreases in platelet aggregation. In vitro these effects were reproduced and decreased TxB2 formation was also demonstrated. Inhibition of platelet aggregation while receiving tipranavir treatment might contribute to increased risk of bleeding.

Antiplatelet and antithrombotic activity of indole‐3‐carbinol in vitro and in vivo

Indole-3-carbinol, a natural compound found in cruciferous vegetables, is known to have anticancer activity. In the present study, the antiplatelet and antithrombotic activities of indole-3-carbinol were investigated in vitro and in vivo. Indole-3-carbinol significantly inhibited collagen-induced platelet aggregation in human platelet rich plasma (PRP) in a concentration-dependent manner. Indole-3-carbinol significantly inhibited fibrinogen binding to the platelet surface glycoprotein IIb/IIIa (GP IIb/IIIa) receptor by flow cytometric analysis. In addition, the levels of thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) in collagen stimulated PRP were significantly inhibited in a concentration-dependent manner by indole-3-carbinol. Furthermore, indole-3-carbinol dose-dependently suppressed the death of mice with pulmonary thrombosis induced by intravenous injection of collagen and epinephrine. These results suggest that indole-3-carbinol can be a potent antithrombotic agent with antiplatelet activity through the inhibition of GP IIb/IIIa receptor and thromboxane B2 formation.

Dipyrone elicits substantial inhibition of peripheral cyclooxygenases in humans: new insights into the pharmacology of an old analgesic

Dipyrone (INN, metamizol) is a common analgesic used worldwide. Its widespread prescription or over-the-counter use in many countries (e.g., Brazil, Israel, Mexico, Russia, Spain) requires insight into its mode of action. This study therefore addressed the impact of its metabolites 4-methyl-amino-antipyrine (MAA) and 4-amino-antipyrine (AA) on peripheral cyclooxygenases (COX). Pharmacokinetics of metabolites and ex vivo COX inhibition were assessed in five volunteers receiving dipyrone at single oral doses of 500 or 1000 mg. Coagulation-induced thromboxane B2 formation and lipopolysaccharide-induced prostaglandin E2 synthesis were measured in vitro and ex vivo in human whole blood as indices of COX-1 and COX-2 activity. In vitro, metabolites elicited no substantial COX-1/COX-2 selectivity with MAA (IC50=2.55 micromol/L for COX-1; IC50=4.65 micromol/L for COX-2), being approximately 8.2- or 9-fold more potent than AA. After administration of dipyrone, MAA plasma concentrations remained above the IC50 values for each isoform for at least 8 h (500 mg) and 12 h (1000 mg) postdose. COX inhibition correlated with MAA plasma levels (ex vivo IC50 values of 1.03 micromol/L [COX-1] and 0.87 micromol/L [COX-2]). By contrast, plasma peak concentrations of AA after the 1000 mg dose were 2.8- and 6.5-fold below its IC50 values for COX-1 and COX-2, respectively. Maximal inhibitions of COX-1 and COX-2 were 94% and 87% (500 mg), 97% and 94% (1000 mg). Taken together, dipyrone elicits a substantial and virtually equipotent inhibition of COX isoforms via MAA. Given the profound COX-2 suppression by dipyrone, which was considerably above COX-2 inhibition by single analgesic doses of celecoxib and rofecoxib, a significant portion of its analgesic action may be ascribed to peripheral mechanisms. In view of the observed COX-1 suppression, physicochemical factors (lack of acidity) rather than differential COX-1 inhibition may be responsible for dipyrone's favorable gastrointestinal tolerability compared with acidic COX inhibitors.

Determination of Aspirin Responsiveness by Use of Whole Blood Platelet Aggregometry

Insufficient platelet inhibition is associated with an increased cardiovascular risk in up to 30% of patients taking regular doses of aspirin. We describe an assay to study aspirin responsiveness. We performed impedance aggregometry on diluted whole blood with 1 mg/L collagen and 0.5 mmol/L arachidonic acid (AA). We measured thromboxane B(2) (TXB(2)) by RIA. We examined 66 healthy control individuals, 144 aspirin users with stable coronary artery disease (CAD), and 245 CAD patients treated with aspirin and clopidogrel. Nonresponsive samples were incubated with excess DL-lysinmonoacetylsalicylic acid. Assay imprecision (CV) was 9.8% and 8.2% at mean (SD) 6-min impedance of 13.7 (2.8) Omega and 13.6 (2.3) Omega for collagen and AA, respectively. Collagen induced stronger aggregation (P = 0.0199) in women [n = 28, 14.6 (2.4) Omega] than in men [n = 38, 13.1 (2.9) Omega], even after sample incubation with 0.1 mmol/L acetylsalicylic acid (ASA) or 1 micromol/L terbogrel, a combined inhibitor of thromboxane synthase and receptors. The sex association persisted in aspirin users, but not if clopidogrel was also taken. A 6-min impedance >8 Omega with collagen (mean - 2 SD of the controls) was taken as evidence of nonresponsiveness, particularly if incubation with ASA did not inhibit aggregation further (>2 Omega). Compared with AA, collagen identified more nonresponsive samples among aspirin users (15%) and CAD patients who also received clopidogrel (10%). Incubation with ASA improved inhibition of aggregation in 70% of samples and consistently reduced TXB(2) formation during aggregation. Impedance aggregometry may prove useful to study aspirin responsiveness, and incubation with ASA may help to identify nonresponders and classify resistance.

Mechanisms involved in the antiplatelet effect of C-phycocyanin

C-phycocyanin (cpc), a biliprotein isolated from Spirulina platensis, has been reported to exert many therapeutic and nutritional values. In the present study, we examined whether cpc has an antiplatelet activity in vitro and further investigated the possible anti-aggregatory mechanisms involved. Our results showed that preincubation of cpc (1-50 microg/ml) with rabbit washed platelets dose-dependently inhibited the platelet aggregation induced by collagen (10 microg/ml) or arachidonic acid (100 microm), with an IC50 of about 10 microg/ml. Furthermore, the thromboxane B2 formation caused by collagen or arachidonic acid was significantly inhibited by cpc due to suppression of cyclooxygenase and thromboxane synthase activity. Similarly, the rise of platelet intracellular calcium level stimulated by arachidonic acid and collagen-induced platelet membrane surface glycoprotein IIb/IIIa expression were also attenuated by cpc. In addition, cpc itself significantly increased the platelet membrane fluidity and the cyclic AMP level through inhibiting cyclic AMP phosphodiesterase activity. These findings strongly demonstrate that cpc is an inhibitor of platelet aggregation, which may be associated with mechanisms including inhibition of thromboxane A2 formation, intracellular calcium mobilization and platelet surface glycoprotein IIb/IIIa expression accompanied by increasing cyclic AMP formation and platelet membrane fluidity.

Phospholipase A2 activity in platelets of patients with primary thrombocythemia

Patients with primary thrombocythemia (PT) have both, bleeding and thrombotic events. Although platelet aggregation tests are usually abnormal, synthesis of thromboxane B2 (TxB2) by platelets is increased. This feature could be the consequence of an increased phospholipase activity or a facilitated metabolism of arachidonate by prostaglandin synthetase pathway. We studied the activity of phospholipase A2 as well the arachidonate metabolism in platelets of patients suffering from PT. Eleven patients and 11 controls were included. Platelets were labelled with [14C]arachidonic acid ([14C]AA). Lost of radioactivity from phospholipids and new radioactive prostanoids were evaluated in calcium ionophore A23187 activated platelets, to explore phospholipase A2 activity. This assay was also carried out in aspirin-incubated platelets. We also studied the formation of prostanoids in platelets activated by radioactive free arachidonic acid. Platelet aggregation studies of patients were abnormal. [14C]AA incorporation in platelet phospholipids was normal. Ionophore activated platelets from patients and controls lost 26.1 ± 8.3% and 24.1 ± 10.5% of radioactivity, respectively, mainly from phosphatidylcholine. The main arachidonate metabolite was 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), which comprised 14.1 ± 5.1% of the radioactivity released from phospholipids in patients, and a similar amount in the controls (14.4 ± 7.5%). Formation of TxB2 was also similar in patients (5.5 ± 1.2%) and controls (4.9 ± 2.9%). Formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was also normal. Ionophore A23187 activation of aspirinized platelets of patients released 19.5 ± 7.4% of radioactivity from phospholipids, which was completely metabolized to HETE. Formation of prostanoids HETE, HHT and TxB2 by arachidonic acid activated platelets of patients was normal. Phospholipase A2 activity as well both cyclooxygenase and lipoxygenase activities in platelets of patients with PT were found to be normal.

Activation of p38 MAPKinase/cPLA2 pathway in homocysteine-treated platelets.

Hyperhomocysteinemia is considered a risk factor in arterial and venous thrombosis. The mechanism by which homocysteine (HCy) supports atherothrombosis is still unknown and may be multifactorial. Earlier in vitro studies demonstrated that HCy induced arachidonic acid release and increased thromboxane B2 (TXB2) formation. In this work, we found that HCy stimulated the rapid and sustained phosphorylation of platelet p38 mitogen-activated protein kinase (p38 MAPK). The effect was time- and dose-dependent. The HCy effect on p38 MAPK phosphorylation was prevented by N-acetyl-L-cysteine and iloprost and was partially inhibited by nordihydroguaiaretic acid. Moreover, the incubation of platelets with HCy led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). In addition HCy promoted cPLA2 activation, assessed as arachidonic acid release. The cPLA2 phosphorylation and activation were both impaired by the inhibition of p38 MAPK through SB203580. This effect was not complete, reaching at the most the 50% of the total. In FURA 2-loaded platelets, HCy induced a dose-dependent intracellular calcium rise suggesting that the calcium elevation promoted by HCy could participate in the cPLA2 activation, leading to arachidonic acid release and TXB2 formation. In conclusion, our data provide insight into the mechanisms of platelet activation induced by HCy, suggesting that the p38 MAPK/cPLA2 pathway could play a relevant role in platelet hyperactivity described in hyperhomocysteinemia.

Influence of genotypes on the pharmacokinetics and pharmacodynamics of piroxicam

Our objective was to evaluate the influence of cytochrome P450 (CYP) 2C9 polymorphisms on the pharmacokinetics and pharmacodynamics of the nonsteroidal anti-inflammatory drug piroxicam. Thirty-five healthy subjects with CYP2C9 genotypes *1/*1 (n=17), *1/*2 (n=9), and *1/*3 (n=9) received a single oral dose of piroxicam (20 mg). Blood samples were collected at various time points up to 240 hours for measurements of the concentrations of piroxicam and thromboxane B2 (TXB2). Piroxicam's area under the plasma concentration-time curve from time 0 to infinity and oral clearance corrected for body weight were 154+/-37 microg.mL-1.h and 2.0+/-0.5 mL.h-1.kg-1, respectively, in CYP2C9*1/*1 individuals, as compared with 256+/-97 mL.h-1 (P=.002) and 1.3+/- 0.4 mL.h-1.kg-1 (P=.002), respectively, in CYP2C9*1/*2 individuals and 259+/- 95 mL.h-1 (P=.002) and 1.3+/- 0.4 mL.h-1.kg-1 (P=.002), respectively, in CYP2C9*1/*3 individuals. There were no significant differences between CYP2C9*1/*2 and CYP2C 9*1/*3 individuals in these pharmacokinetic parameters (P=.95 for area under the plasma concentration-time curve from time 0 to infinity and P=.94 for oral clearance corrected for body weight). The formation of TXB2, reflecting cyclooxygenase type 1 activity, showed significant differences in the area above the effect-time curves (expressed as percent of baseline TXB2.h) between CYP2C9*1/*1 (10,190 +/- 2632) and either CYP2C9*1/*2 (19,255+/-1,291 [P=.00003]) or CYP2C9*1/*3 (18,241+/- 2397 [P=.00003]). The minimum serum TXB2 concentration, however, did not differ among the different genotypes (P=.32, ANOVA). Piroxicam's oral clearance was impaired and its inhibitory effect on cyclooxygenase 1 activity was increased in CYP2C9*1/*2 or CYP2C9*1/*3 individuals, as compared with CYP2C 9*1 homozygous individuals.

Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation

Lycopene is a natural carotenoid antioxidant that is present in tomatoes and tomato products. The pharmacologic function of lycopene in platelets is not yet understood. Therefore, in this study we sought to systematically examine the effects of lycopene in the prevention of platelet aggregation and thrombus formation. We found that lycopene concentration-dependently (2-12 micromol/L) inhibited platelet aggregation in human platelets stimulated by agonists. Lycopene (6 and 12 micromol/L) inhibited phosphoinositide breakdown in platelets labeled with tritiated inositol, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane B2 formation stimulated by collagen. In addition, lycopene (6 and 12 micromol/L) significantly increased the formations of cyclic GMP and nitrate but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of 47,000 Da (P47), a marker of protein kinase C activation, was triggered by PDBu (60 nmol/L). This phosphorylation was markedly inhibited by lycopene (12 micromol/L) in phosphorus-32-labeled platelets. In an in vivo study, thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Lycopene (5, 10, and 20 mg/kg) significantly prolonged the latency period for the induction of platelet-plug formation in mesenteric venules. These results indicate that the antiplatelet activity of lycopene may involve the following pathways: (1) Lycopene may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane B2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization. (2) Lycopene also activated the formations of cyclic GMP/nitrate in human platelets, resulting in the inhibition of platelet aggregation. The results may imply that tomato-based foods are especially beneficial in the prevention of platelet aggregation and thrombosis.