Antiplatelet activity of [5-(2-methoxy-5-chlorophenyl)furan-2-ylcarbonyl]guanidine (KR-32570), a novel sodium/hydrogen exchanger-1 and its mechanism of action

The antiplatelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg/mL), thrombin (0.05 U/mL), arachidonic acid (100 microM), a thromboxane (TX) A2 mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2, 1 microM) and a Ca2+ ATPase inhibitor thapsigargin (0.5 microM) (IC50 values: 13.8 +/- 1.8, 26.3 +/- 1.2, 8.5 +/- 0.9, 4.3 +/- 1.7 and 49.8 +/- 1.4 microM, respectively). KR-32570 inhibited the collagen-induced liberation of [3H]arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at 50 microM. The TXA2 synthase assay showed that KR-32570 also inhibited the conversion of the substrate PGH2 to TXB2 at all concentrations. Furthermore, KR-32570 significantly inhibited the [Ca2+]i mobilization induced by collagen at 50 microM, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen (10 microg/mL)-induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, TXA2 synthase, the mobilization of cytosolic Ca2+ and NHE-1.     

Antiplatelet activity of carnosic acid, a phenolic diterpene from Rosmarinus officinalis

Carnosic acid is a major phenolic diterpene derived from Rosmarinus officinalis and has been reported to have antioxidant, antibacterial, anticancer, antiobese and photoprotective activities. This study investigated the antiplatelet activity of carnosic acid. carnosic acid significantly inhibited collagen-, arachidonic acid-, U46619- and thrombin-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 39+/-0.3, 34+/-1.8, 29+/-0.8 and 48+/-2.9 microM, respectively, while it failed to inhibit PMA-(a direct PKC activator) and ADP-induced platelet aggregation. In agreement with its antiplatelet activity, carnosic acid blocked collagen-, arachidonic acid-, U46619- and thrombin-mediated cytosolic calcium mobilization. accordingly, serotonin secretion and arachidonic acid liberation were also inhibited in a similar concentration-dependent manner. However, in contrast to the inhibition of arachidonic acid-induced platelet aggregation, carnosic acid had no effect on the formation of arachidonic acid-mediated thromboxane A2 and prostaglandin D2, thus indicating that carnosic acid has no effect on the cyclooxygenase and thromboxane A2 synthase activity. Overall, these results suggest that the antiplatelet activity of carnosic acid is mediated by the inhibition of cytosolic calcium mobilization and that carnosic acid has the potential of being developed as a novel antiplatelet agent.     

Pharmacology of the thromboxane receptor antagonist and thromboxane synthase inhibitor BM-531

BM-531 (N-tert-butyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA2 receptors labeled with [3H]SQ-29548 (IC50 = 0.0078 microM) is higher than sulotroban (IC50 = 0.93 microM) and SQ-29548 (IC50 = 0.021 microM). Moreover, BM-531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet-rich plasma BM-531 prevents platelet aggregation induced by arachidonic acid (600 microM) (ED100 = 0.125 microM), U-46619, a stable TXA2 agonist (1 microM) (ED50 = 0.482 microM) or collagen (1 microgram/mL) (percentage of inhibition: 42.9% at 10 microM) and inhibits the second wave of ADP (2 microM)-induced aggregation. On the other hand, when BM-531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100) is significantly prolonged. In addition, at the concentrations of 10 and 1 microM, BM-531 totally prevents the production of TXB2 by human platelets activated by arachidonic acid. Finally, at 10 microM, BM-531 significantly prevents rat fundus contractions induced by U-46619 but not by prostacyclin. These results suggest that BM-531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.     

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

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

Antiplatelet activity of epigallocatechin gallate is mediated by the inhibition of PLCgamma2 phosphorylation, elevation of PGD2 production, and maintaining calcium-ATPase activity

We have previously reported that green tea catechins displayed a potent antithrombotic effect by inhibition of platelet aggregation. In the present study, the antiplatelet and antithrombotic activities of epigallocatechin gallate (EGCG), the major catechin derived from green tea, were extensively investigated. EGCG inhibited arterial thrombus formation and U46619-, collagen-, and arachidonic acid (AA)-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 61 +/- 3, 85 +/- 4, and 99 +/- 4 microM, respectively. In line with the inhibition of collagen-induced platelet aggregation, EGCG revealed blocking of the collagen-mediated phospholipase (PL) Cgamma2 and protein tyrosine phosphorylation, and it caused concentration-dependent decreases of cytosolic calcium mobilization, AA liberation, and serotonin secretion. In addition, the platelet aggregation, intracellular Ca2+ mobilization, and protein tyrosine phosphorylation induced by thapsigargin, a Ca2(+)-ATPase pump inhibitor, were completely blocked by EGCG. Contrary to the inhibition of AA-induced platelet aggregation, EGCG failed to inhibit cyclooxygenase and thromboxane (TX) A2 synthase activities, but it concentration-dependently elevated AA-mediated PGD2 formation. In contrast, epigallocatechin (EGC), a structural analogue of EGCG lacking a galloyl group in the 3' position, slightly inhibited collagen-stimulated cytosolic calcium mobilization, but failed to affect other signal transductions as did EGCG in activated platelets and arterial thrombus formation. These results suggest that antiplatelet activity of EGCG may be attributable to its modulation of multiple cellular targets, such as inhibitions of PLCgamma2, protein tyrosine phosphorylation and AA liberation, and elevation of cellular PGD2 levels, as well as maintaining Ca2(+)-ATPase activity, which may underlie its beneficial effect on the atherothrombotic diseases.     

Antiplatelet effect of NQ12: a possible mechanism through the arachidonic acid cascade

NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC(50) values of 0.71 +/- 0.2, 0.82 +/- 0.3, and 0.45 +/- 0.1 microM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A(2) receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA(2) formation, but had no effect on the PGD(2) production, indicating an inhibitory effect on TXA(2) synthase, which was supported by a TXA(2) synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA(2) formation converted from PGH(2). On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA(2) synthase activity and TXA(2) receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12.     

Pharmacological characterization of cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 receptor.

1. The pharmacological effects of cinnamophilin, a new lignan, isolated from Cinnamomum philippinense, was determined in vitro in human platelet, rat isolated aorta and guinea-pig isolated trachea and in vivo in mice and guinea-pigs. 2. Cinnamophilin inhibited dose-dependently human platelet-rich plasma (PRP) aggregation induced by arachidonic acid (AA), collagen and U-46619 with IC50 of 5.0 +/- 0.4, 5.6 +/- 0.6 and 3.0 +/- 0.4 microM, respectively. The second wave of ADP- or adrenaline-induced platelet aggregation was inhibited by cinnamophilin, while the first wave was only slightly inhibited by cinnamophilin above 30 microM. 3. Cinnamophilin was found to be a thromboxane A2 (TXA2) receptor blocking agent in human platelet, rat aorta and guinea-pig trachea as revealed by its competitive antagonism of U-46619-induced aggregation of human-PRP, contraction of rat aortic rings and guinea-pig tracheal rings with pA2 values of 7.3 +/- 0.2, 6.3 +/- 0.1 and 5.2 +/- 0.2, respectively. 4. [3H]-inositol monophosphate formation and the rise of intracellular Ca2+ caused by U-46619 in human platelet was suppressed by cinnamophilin (10 microM). 5. Cinnamophilin induced a dose-dependent inhibition of thromboxane B2 (TXB2) formation, while the prostaglandin E2 (PGE2) formation was increased. Cinnamophilin did not affect unstimulated platelet adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. When the platelets were challenged with AA, a dose-dependent rise in cyclic AMP was observed. Dazoxiben (a pure TX synthase inhibitor) and SQ 29548 (a pure TXA2 receptor antagonist) did not affect cyclic AMP levels in AA-treated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design, synthesis and biological evaluation of a sulfonylcyanoguanidine as thromboxane A2 receptor antagonist and thromboxane synthase inhibitor

The synthesis and the structure of N-isopropyl-N'-[2-(3'-methylphenylamino)-5-nitrobenzenesulfonyl] urea (14) was drawn from two thromboxane A2 receptor antagonists structurally related to torasemide. Compound 14 showed an IC50 value of 22 nM for the thromboxane A2 (TXA2) receptor of human washed platelets. Compound 14 prevented platelet aggregation induced by arachidonic acid (0.6 mM) and U-46619 (1 microM) with an IC50 value of 0.45 and 0.15 microM, respectively. Moreover, 14 relaxed the rat isolated aorta and guinea-pig trachea precontracted by U-46619, a TXA2 agonist. Its efficacy (IC50) was 20.4 and 5.47 nM, respectively. Finally, 14 (1 microM) completely inhibited TXA2 synthase of human platelets. The pKa value and the crystallographic data of 14 were determined and used to propose an interaction model between the TXA2 antagonists related to torasemide and their receptor.     

Antiplatelet effect of 2-chloro-3-(4-acetophenyl)-amino-1,4-naphthoquinone (NQ301): a possible mechanism through inhibition of intracellular Ca2+ mobilization.

The effects of 2-chloro-3-(4-acetophenyl)-amino-1,4-naphthoquinone (NQ301), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein (GP)IIb/IIIa complex and intracellular signals were investigated using human platelets. NQ301 significantly inhibited the collagen-, thrombin-, arachidonic acid-, thapsigargin- and calcium ionophore A23187-induced aggregation of washed human platelets with IC50 values of 13.0+/-0.1, 11.2+/-0.5, 21.0+/-0.9, 3.8+/-0.1 and 46.2+/-0.8 microM, respectively. NQ301 also significantly inhibited FITC-conjugated fibrinogen binding to human platelet surface GPIIb/IIIa complex, but failed to inhibit the fibrinogen binding to purified GPIIb/IIIa complex. These data demonstrate that NQ301 inhibits platelet aggregation by suppression of the intracellular pathway, rather than by direct inhibition of fibrinogen-GPIIb/IIIa complex binding. NQ301 significantly inhibited the increase of cytosolic Ca2+ concentration and ATP secretion, and also significantly increased platelet cAMP levels in the activated platelets. These results suggest that the antiplatelet activity of NQ301 may be mediated by inhibition of cytosolic Ca2+ mobilization, enhancement of cAMP production and inhibition of ATP secretion in activated platelets.     

Anti-platelet activity of KR-32560, a novel sodium/hydrogen exchanger-1 inhibitor.

We investigated the anti-platelet effect of a newly synthesized guanidine derivative KR-32560, a sodium/hydrogen exchanger-1 (NHE-1) inhibitor, together with the elucidation of the possible mode of action. KR-32560 concentration dependently inhibited the aggregation of washed rabbit platelets induced by collagen (10 microg mL(-1)) and arachidonic acid (AA; 100 microM), with IC50 values of 25 and 46 microM, respectively. Whereas, KR-32560 showed weaker potency against aggregation induced by thrombin (0.05 UmL(-1)) and U46619 (1 microM), and had no effect on thapsigargin (0.5 microM)- or A23187 (5 microM)-induced platelet aggregation up to 50 microM. KR-32560 inhibited the collagen-induced [3H]AA liberation in a concentration-dependent manner. In addition, KR-32560 significantly suppressed TXB2 formation in AA-exposed platelets, but had no effect on production of PGD2, indicating an inhibitory effect on TXA2 synthase. This finding was supported by a TXA2 synthase assay that KR-32560 inhibited the conversion of PGH2 into TXB2 with a similar magnitude to suppression of TXB2 formation. Furthermore, KR-32560 significantly inhibited the collagen-induced [Ca2+]i mobilization and serotonin secretion. Taken together, these observations suggest that the anti-platelet activity of KR-32560 may be mediated by the inhibition of cytoplasmic Ca2+ mobilization and AA liberation.     

Antiplatelet mechanism of 2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone (NQ304), an antithrombotic agent

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 microM, 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 microM 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.     

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.     

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.     

Differential effects of ganodermic acid S on the thromboxane A2-signaling pathways in human platelets.

Ganodermic acid S (GAS) [lanosta-7,9(11),24-triene-3beta,15alpha-diacetoxy-26-oic acid], isolated from the Chinese medicinal fungus Ganoderma lucidum (Fr.) Karst (Polyporaceae), exerted a concentration-dependent inhibition on the response of human gel-filtered platelets (GFP) to U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha), a thromboxane (TX) A2 mimetic. GAS at 2 microM inhibited 50% of cell aggregation. GAS at 7.5 microM inhibited 80% of Ca2+ mobilization, 40% of phosphorylation of myosin light chain and pleckstrin, 80% of alpha-granule secretion, and over 95% of aggregation. GAS also strongly inhibited U46619-induced diacylglycerol formation, arachidonic acid release, and TXB2 formation. An immunoblotting study of protein-tyrosine phosphorylation showed that GAS inhibited the formation of phosphotyrosine proteins at the steps involving the engagement of integrin alphaIIbbeta3 and aggregation. However, GAS did not inhibit U46619-induced platelet shape change or the inhibitory effect of U46619 on the prostaglandin E1-evoked cyclic AMP level in GFP. It is concluded that GAS inhibits platelet response to TXA2 on the receptor-Gq-phospholipase Cbeta1 pathway, but not on the receptor-G1 pathway.     

Studies on the inhibitory effect of etafenone hydrochloride on platelet aggregation

The inhibitory effect of etafenone hydrochloride (etafenone) on platelet aggregation in rabbit platelet rich plasma and the involvement of the arachidonic acid (AA) cascade in the inhibitory mechanism for etafenone on platelet aggregation were studied. 1) Etafenone exhibited a dose-dependent inhibitory effect on collagen (15--20 micrograms/ml)-induced platelet aggregation, and its median inhibitory concentration (IC50) was 1.7 X 10(-5)M. 2) In ADP (20 microM)-induced aggregation, etafenone also exhibited a dose-dependent inhibitory effect, but its IC50 was 2.7 X 10(-4)M and was significantly higher than that in the case of collagen. 3) Etafenone inhibited AA (0.3--0.5mM)-induced platelet aggregation dose-dependently. Its IC50 was 2.8 X 10(-5)M. 4) In thromboxane (TX) A2-induced aggregation, etafenone exhibited a dose-dependent inhibition, and the IC50 was 3.2 X 10(-4)M. 5) Trapidil which was reported to inhibit platelet aggregation via phosphodiesterase (PDE) inhibition had a similar IC50 on ADP- and TXA2-induced platelet aggregation to that of etafenone, but in collagen- and AA-induced aggregation, its IC50 was higher than that of etafenone. 6) Etafenone (3 X 10(-6)--3 X 10(-4)M) dose-dependently inhibited the production of TXB2 in PRP induced by collagen. 7) Etafenone scarcely affected TXA2 synthetase activity in rabbit platelet homogenate. 8) The correlation between the inhibitory effect of etafenone on platelet aggregation and inhibition of AA metabolism activation and PDE inhibition was discussed.     

Biological properties of a specific Galpha q/11 inhibitor, YM-254890, on platelet functions and thrombus formation under high-shear stress

1 The effects of YM-254890, a specific Galpha(q/11) inhibitor, on platelet functions, thrombus formation under high-shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM-254890 concentration dependently inhibited ADP-induced intracellular Ca(2+) elevation, with an IC(50) value of 0.92+/-0.28 microM. 3 P-selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM-254890, with IC(50) values of 0.51+/-0.02 and 0.16+/-0.08 microM, respectively. 4 YM-254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP-stimulated washed platelets. 5 YM-254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM-254890 attenuated ADP-, collagen-, TRAP-, arachidonic acid- and U46619-induced platelet aggregation with IC(50) values of <1 microM, whereas it had no effect on phorbol 12-myristate 13-acetate-, ristocetin-, thapsigargin- or A23187-induced platelet aggregation. 7 High-shear stress-induced platelet aggregation and platelet-rich thrombus formation on a collagen surface under high-shear flow conditions were concentration dependently inhibited by YM-254890. 8 The antithrombotic effect of YM-254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM-254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM-254890 is a useful tool for investigating Galpha(q/11)-coupled receptor signaling and the physiological roles of Galpha(q/11).     

Thromboxane A2-mediated shape change: independent of Gq-phospholipase C--Ca2+ pathway in rabbit platelets.

1. Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in washed rabbit platelets to clarify the mechanisms of induction of shape change and aggregation. 2. The TXA2 agonist, U46619 (1 nM to 10 microM) caused shape change and aggregation in a concentration-dependent manner. A forty-times higher concentration of U46619 was needed for aggregation (EC50 of 0.58 microM) than shape change (EC50 of 0.013 microM). The aggregation occurred only when external 1 mM Ca2+ was present, but the shape change could occur in the absence of Ca2+. 3. SQ29548 at 30 nM and GR32191B at 0.3 microM (TXA2 receptor antagonists) competitively inhibited U46619-induced shape change and aggregation with similar potency, showing that both aggregation and shape change induced by U46619 were TXA2 receptor-mediated events. However, ONO NT-126 at 1 nM, another TXA2 receptor antagonist, inhibited U46619-induced aggregation much more potently than the shape change, suggesting the possible existence of TXA2 receptor subtypes. 4. ONO NT-126 (2 nM to 3 microM) by itself caused a shape change without aggregation in a concentration-dependent manner, independent of external Ca2+. Therefore, ONO NT-126 is a partial agonist at the TXA2 receptor in rabbit platelets. 5. U46619 (10 nM to 10 microM) increased internal Ca2+ concentration ([Ca2+]i) and activated phosphoinositide (PI) hydrolysis in a concentration-dependent manner with a similar concentration-dependency. 6. U46619 (3 nM to 10 microM) also activated GTPase concentration-dependently in the membranes derived from platelets. U46619-induced activation of GTPase was partly inhibited by treatment of membranes with QL, an antibody against Gq/11. 7. The EC50 values of U46619 in Ca2+ mobilization (0.15 microM), PI hydrolysis (0.20 microM) and increase in GTPase activity (0.12 microM) were similar, but different from the EC50 value in shape change (0.013 microM), suggesting that activation of TXA2 receptors might cause shape change via an unknown mechanism. 8. U46619-induced shape change was unaffected by W-7 (30 microM), a calmodulin antagonist or ML-7 (30 microM), a myosin light-chain kinase inhibitor, indicating that an increase in [Ca2+]i might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+]i level, determined by simultaneous recordings. 9. [3H]-SQ29548 and [3H]-U46619 bound to platelets at a single site with a Kd value of 14.88 nM and Bmax of 106.1 fmol/10(8) platelets and a Kd value of 129.8 nM and Bmax of 170.4 fmol/10(8) platelets, respectively. The inhibitory constant Ki value for U46619 as an inhibitor of 3H-ligand binding was similar to the EC50 value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P < 0.001 by Student''s t test) from the effect on shape change. 10. Neither U46619 nor ONO NT-126 affected the adenosine 3'',5''-cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11. The results indicate that TXA2 receptor stimulation causes phospholipase C activation and increase in [Ca2+]i via a G protein of the Gq/11 family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA2 receptor stimulation might occur without involvement of the Gq-phospholipase C-Ca2+ pathway.     

Synthesis and antiplatelet evaluation of novel aryl-sulfonamide derivatives, from natural safrole.

In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antiplatelet prototype compounds, exploring bioisosterism principles for molecular design, we describe in this paper the synthesis of new aryl-sulfonamides derivatives, structurally similar to known thromboxane A2 receptor antagonists. The synthetic route used to access the new compounds described herein starts from safrole, an abundant Brazilian natural product, which occurs in Sassafras oil (Ocotea pretiosa). The results from preliminary evaluation of these novel aryl-sulfonamide compounds by the platelet aggregation inhibitory test, using rabbit PRP, induced by ADP, collagen, arachidonic acid, and U46619, identified the N-[2-(4-carboxymethoxyphenyl)ethyl]-6-methyl-3,4-methylenedioxyphe nyl- sulfonamido derivative as the most active among them, presenting in IC50 value for the U-46619-induced platelet aggregation in rabbit platelet-rich plasma: 329 microM.     

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.     

Cordycepin (3'-deoxyadenosine) inhibits human platelet aggregation induced by U46619, a TXA2 analogue

Cordycepin (3'-deoxyadenosine), which comes from Cordyceps militaris, the Chinese medicinal fungal genus Cordyceps, is known to have anti-tumour activity. In this study, we investigated the novel effect of cordycepin on human platelet aggregation that was induced by U46619, a thromboxane A(2) (TXA(2)) analogue. TXA(2) is an aggregation-inducing autacoidal molecule that is produced in various agonist-activated platelets. Cordycepin completely inhibited U46619-induced platelet aggregation and simultaneously reduced cytosolic free Ca(2+) ([Ca(2+)](i)), which was increased by U46619 (5 microM) up to 66%. Furthermore, the U46619-stimulated phosphorylation of Ca(2+)-dependent proteins (20 kDa of a myosin light chain and 47 kDa of pleckstrin) was strongly inhibited by cordycepin. These results suggest that cordycepin may have a beneficial effect on autacoidal TXA(2)-mediated thrombotic diseases by inhibiting TXA(2)-induced platelet aggregation via suppression of the Ca(2+) level.