The evaluation and structure-activity relationships of 2-benzoylaminobenzoic esters and their analogues as anti-inflammatory and anti-platelet aggregation agents

Forty-seven 2-benzoylaminobenzoic esters were synthesized and evaluated in anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Most 2-benzoylamino-4-chlorobenzoic acid derivatives showed selective inhibitory effects on arachidonic acid (AA)-induced platelet aggregation. Among them, compounds 6b and 7b exhibited more potent inhibitory effects (ca. 200-fold) than aspirin. Additionally, compounds 1a and 5a showed strong inhibitory effects on neutrophil superoxide generation with IC(50) values of 0.65 and 0.17 microM, respectively. However, compounds 6d and 6e exhibited dual inhibitory effects on platelet aggregation and neutrophil elastase (NE) release; therefore, these two compounds may be new leads for development as anti-inflammatory and anti-platelet aggregatory agents.     

Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets

The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.     

Pharmacologic antagonism of thromboxane A2 receptors by trimetoquinol analogs in vitro and in vivo.

Although (-)-(S)-trimetoquinol [1-(3,4,5-trimethoxy-benzyl)- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ] is recognized as a potent bronchodilator, (+)-(R)-TMQ is a selective antagonist of human platelet aggregation and serotonin secretion induced by thromboxane A2 (TXA2) agonists. To confirm the pharmacological actions of TMQ analogs, the interaction of the drugs with TXA2 receptors was examined in human platelets and in a mouse sudden death model. The inhibitory potencies of TMQ analogs (pIC50 values) for displacement of [3H]SQ 29,548 binding to platelets showed excellent correlation with the respective pIC50 (-log IC50) values for U46619-induced aggregation (r = 0.99, P less than 0.01) and serotonin secretion (r = 0.99, P less than 0.01) in human platelet-rich plasma and for whole blood aggregation (r = 0.99, P less than 0.01). In each system, the rank order of inhibitory potencies was rac-iodoTMQ greater than or equal to (+)-(R)-TMQ greater than rac-TMQ much greater than (-)-(S)-TMQ. Antithrombotic effects of TMQ analogs were evaluated in a mouse sudden death model. In vivo antithrombotic potencies of these compounds were consistent with the in vitro potencies as TXA2 receptor antagonists in platelet systems. Administration of rac-iodoTMQ, (+)-(R)-TMQ and rac-TMQ 15 min before the injection of U46619 (800 micrograms/kg, iv) protected mice against U46619-induced sudden death. On the other hand, (-)-(S)-TMQ did not protect animals against death. Protection of U46619-induced cardiopulmonary thrombosis by TMQ analogs was seen at doses of 3-100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)     

The PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban synergistically inhibit TXA2-dependent platelet activation

The stable PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban (BM 13177) were investigated for possible synergistic effects on platelet aggregation in human platelet rich plasma in vitro. Iloprost and sulotroban synergistically inhibited U 46619, collagen, and the second wave of ADP-induced platelet aggregation. Iloprost and sulotroban at concentrations showing little or no inhibition alone resulted, in combination, in marked or complete inhibition of U 46619 or collagen induced aggregation. Combination of iloprost 10(-10) M, which had no effect on the concentration-response curve (CRC) to U 46619, with sulotroban 5 x 10(-6) M, which shifted the CRC to U 46619 by a factor of 3 to the right, resulted in a rightward shift of the U 46619 CRC by a factor of 4.5. To attain a 4.5-fold shift with either compound alone, a concentration of 5 x 10(-10) M iloprost or 10(-5) M sulotroban was required. A similar mutual enhancement of inhibitory effects was seen for combinations of the PGI2-analogue cicaprost (ZK 96.480) with sulotroban or the TXA2-receptor antagonist SQ 29548 with iloprost. When the TXA2-dependent part of collagen-induced aggregation was fully inhibited by sulotroban, the concentrations of iloprost necessary for 90% inhibition were reduced by a factor of 2.5 - 3. In the presence of acetylsalicylic acid, the synergistic action of sulotroban and iloprost was reduced and merely additive effects against U 46619-induced platelet aggregation were found, suggesting that the release of endogenous TXA2 plays an important role for the synergistic effect of the two compounds. The combination of a PGI2-analogue and a TXA2-antagonist may lead to a safer and more effective control of platelet activation than with either compound alone.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.     

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

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

Mode of action of 2-(diethylamino)-7-ethoxychromone on human platelets.

The in vitro effect of 2-(diethylamino)-7-ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromosome-derivative exerts a dose-dependent inhibitory effect on aggregation produced by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in a dose-dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident.     

Synthesis, antiproliferative, and antiplatelet activities of oxime- and amide-containing quinolin-2(1H)-one derivatives

Certain oxime- and amide-containing quinolin-2(1H)-one derivatives were synthesized and evaluated for their antiproliferative and antiplatelet activities. These compounds were synthesized via alkylation of hydroxyl precursors followed by the reaction with NH(2)OH or NaN(3) (Schmidt reaction). The preliminary assays indicated that amide derivatives are either weakly active or inactive while the oxime counterparts exhibited potent inhibitory activities against platelet aggregation induced by collagen, AA (arachidonic acid), and U46619 (the stable thromboxan A(2) receptor agonist). Among them, (Z)-6-[2-(4-methoxyphenyl)-2-hydroxyiminoethoxy]quinolin-2(1H)-one (7c) was the most active against AA induced platelet aggregation with an IC(50) of 0.58microM and was inactive against cell proliferation. For the inhibition of U46619 induced aggregation, 7a and 8a-c exhibited very potent activities with IC(50) values in a range between 0.54 and 0.74microM. For the antiproliferative evaluation, N-(biphenyl-4-yl)-2-(2-oxo-1,2-dihydroquinolin-7-yloxy)acetamide (11d) was the most potent with GI(50) values of <10, 10.8, and <10microM against the growth of MT-2, NCI-H661, and NPC-Tw01, respectively, and possessed only a weak antiplatelet activity. Further evaluation of 11d as a potential anticancer agent is on-going.     

Comparison of verapamil and nifedipine in thrombosis models

Calcium blockers and calmodulin antagonists have been reported to inhibit the aggregation of blood platelets in vitro. In the present study, the effects of two calcium blockers, verapamil and nifedipine, were compared in several rodent thrombosis models. In rat and mouse platelet-rich plasma, preincubation with either verapamil or nifedipine had a dose-dependent inhibitory effect on collagen-induced aggregation (P less than 0.01). The concentration required for 50% inhibition of rat platelet aggregation was 0.91 X 10(-4) M for verapamil and 1.77 X 10(-4) M for nifedipine. In in vivo thrombosis models in mice, acute pretreatment with nifedipine had a significant, dose-dependent protective effect (P less than 0.05). At a dose of 500 micrograms/kg, nifedipine inhibited thrombotic sudden death provoked by arachidonic acid, a thromboxane agonist (U46619), or a combination of collagen and epinephrine. In vivo platelet depletion induced by U46619 was also inhibited by this calcium blocker. Thus, nifedipine is protective against a variety of thrombotic stimuli, and its antiplatelet aggregatory effect apparently extends to the in vivo situation. In contrast, no in vivo antithrombotic activity was observed for verapamil. Two additional calcium blockers, perhexilene and diltiazem, and three calmodulin antagonists, W-7, chlorpromazine, and trifluoperazine, were also tested in the U46619-induced thrombotic sudden death model. Of these, only diltiazem (5 and 10 mg/kg) had an acute protective effect.     

Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.     

Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A(2) mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca(2+) increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE(1)-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP-mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin alpha(2)beta(1)- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.     

Inhibitory activity of shiitake flavor against platelet aggregation

Sulfuric-flavored compounds were extracted from shiitake (Lentinus edodes) and their inhibitory activity against platelet aggregation was investigated. Platelet aggregation induced by arachidonic acid and U-46619, the analog of thromboxane A(2), was inhibited by the essential oil from shiitake that contained lenthionine as a major sulfuric compound. This result indicates that the inhibitory site of the shiitake flavor compounds would be different from that of garlic-flavor compounds because the latter inhibits the passage between arachidonic acid and thromboxane A(2). The effect of the synthesized lenthionine was almost equivalent to that of the essential oil, which indicates that the inhibitory activity of the essential oil from shiitake would be mainly attributed to lenthionine.     

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.     

How can we inhibit 5-HT-induced platelet aggregation and why should we bother?

Different 5-HT receptor antagonists inhibit 5-HT-induced platelet aggregation with different potencies. The inhibitory effects of seven relatively potent antagonists could not be surmounted by increasing the concentration of 5-HT, but the inhibitory effects of seven less potent antagonists could be surmounted by 5-HT. Verapamil has in insurmountable inhibitory effect on 5-HT-induced aggregation at relatively low concentrations. Amlodipine is a very weak inhibitory of 5-HT-induced aggregation. Verapamil is more effective as an inhibitory of 5-HT-induced aggregation than it is of aggregation induced by PAF, adrenaline or ADP. The platelet aggregation obtained in whole blood in response to 5-HT, PAF, U46619 or ADP is not different in patients with peripheral vascular disease and age-sex matched controls.     

The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [1r-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'- biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.     

Platelet activation by tetraprenol via stimulation of phospholipase A2 action

Only tetraprenol (n = 4), among the (n)-polyprenols studied, induced activation of rabbit platelets. Tetraprenol-induced responses, including platelet aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release, were greatly inhibited by a thromboxane A2 (TXA2) receptor antagonist and a cyclooxygenase inhibitor, indicating an essential role for endogenously produced TXA2. The TXA2-mimetic agonist U46619 induced platelet aggregation, Ca2+ mobilization and phospholipase C action but did not induce arachidonic acid release. These results suggest that arachidonic acid is not released via phospholipase C but by phospholipase A2, and this is also supported by the finding that phospholipase C action was inhibited by depletion of extracellular Ca2+, while arachidonic acid release was not. Full arachidonic acid release was found to be induced by the synergistic action of U46619 and tetraprenol. Therefore, the initial, most essential response induced by tetraprenol is a small arachidonic acid release by phospholipase A2, which results in initial TXA2 formation. Further action of phospholipase C as well as Ca2+ mobilization and aggregation were induced by the initially formed TXA2 while further activation of phospholipase A2 required the synergistic action of tetraprenol and TXA2.     

Enrichment of platelet phospholipids with eicosapentaenoic acid and docosahexaenoic acid inhibits thromboxane A2/prostaglandin H2 receptor binding and function

Human platelet lipids were enriched in vitro with different amounts of either docosahexaenoic acid (22:6n-3), eicosapentaenoic acid (20:5n-3) or linoleic acid (18:2n-6). Of the total fatty acid incorporated, between 82 and 95% was associated with the phospholipid (PL) fraction, with the remainder as either neutral lipid or hydroxy fatty acid. Within the PL fraction, the majority (64% of total) of each fatty acid was incorporated into phosphatidylcholine. It was found that platelet aggregation induced by the thromboxane A2/prostaglandin H2 mimetic (15S)-hydroxy-11,9-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) was inhibited after PL enrichment with 22:6n-3 or 20:5n-3, but not after 18:2n-6 enrichment. The specificity of 22:6n-3 and 20:5n-3 for U46619 activation was demonstrated by the finding that neither fatty acid significantly inhibited thromboxane A2/prostaglandin H2-independent aggregation induced by A23187 or thrombin. Furthermore, enrichment with 22:6n-3 or 20:5n-3 resulted in inhibition of [3H]U46619 specific binding, while enrichment with 18:2n-6 did not affect binding. Scatchard analysis revealed that thromboxane A2/prostaglandin H2 receptor affinity for [3H]U46619 decreased 4.8-fold following 22:6n-3 incorporation. These results demonstrate that platelet phospholipid enrichment with 22:6n-3 or 20:5n-3 results in a selective inhibition of thromboxane A2/prostaglandin H2 receptor function.     

Wnt5a Potentiates U46619-Induced platelet aggregation <Emphasis Type="Italic">via</Emphasis> the PI3K/Akt pathway

Platelet aggregation plays crucial roles in the formation of hemostatic plugs and thrombosis. Although it was recently shown that canonical Wnt signaling negatively regulates platelet aggregation, the role of non-canonical Wnt signaling remains unknown. Here, we observed that Wnt5a, one of the non-canonical Wnts, positively regulated platelet aggregation. Platelet aggregation was potentiated by the addition of Wnt5a to collagen-or U46619-induced rat platelet rich plasma (PRP). Treatment with Wnt5a to U46619-stimulated PRP resulted in an increase in the level of phosphorylated Akt, whereas phosphorylation of PKCδ and JNK1 was unaffected. In addition, inhibition of PI3K blocked the potentiating effect of Wnt5a. Taken together, these results suggest that Wnt5a potentiates U46619-induced platelet aggregation via the PI3K/Akt pathway.