益康唑和克霉唑对血小板聚集及TXB_2和PGE_2生成的影响

益康唑,克霉唑,咪康唑和酮康唑体外抑制AA诱导兔血小板聚集比抑制ADP诱导大鼠血小板聚集的作用强。其中益康唑和克霉唑抑制AA诱聚兔血小板的IC_(50)分别是6.4及11.6μmol/L。放射免疫法测定大鼠血小板产生的TXB_2及PGE_2发现益康唑和克霉唑在5～50 nmol/L浓度下,能抑制TXB_2产生,同时增加PGE_2生成量,并呈良好的剂量效应相关。浓度为0.1～100μmol/L时,TXB_2的生成仍被抑制,而PGE_2的生成量反而逐渐降低。两药对TXB_2生成的抑制作用比Daz强。     

Sex difference in the inhibitory effect of aspirin on prostacyclin production of rat aortae

The effect of aspirin on the prostacyclin (PGI2) production of rat aorta was investigated, and the influence of sex hormones on the effect of aspirin was studied by the treatments of hormone administration, ovariectomy and castration. There was no significant sex difference in the arterial production of PGI2 between male and female rats. However, the PGI2 production was decreased with aspirin treatment, and the effect of aspirin was more efficient in male rats. The inhibitory effect of aspirin was reduced in the rats treated with estradiol and the castrated male rats, but it was potentiated in the rats treated with testosterone and the ovariectomized female rats. These results suggest that sex hormones may regulate the effect of aspirin on the PGI2 production in the aorta.     

Influence of nonsteroidal anti-inflammatory drugs on the antiplatelet effects of aspirin in rats

Low-dose aspirin acts by irreversibly acetylating internal cyclooxygenase-1 (COX-1) on platelets, thereby suppressing platelet aggregation. Because nonsteroidal anti-inflammatory drugs (NSAIDs) also inhibit COX-1, the antiplatelet effects of aspirin may be suppressed when it is co-administered with NSAIDs. In this study, the influences of ibuprofen, loxoprofen sodium and etodolac on the antiplatelet effects of aspirin were investigated in male Sprague-Dawley (SD) rats. Aspirin and/or NSAIDs were administered orally at single or multiple daily doses. Platelet aggregation (ADP and collagen were added as stimuli) and serum thromboxane B(2) (TXB(2)) concentrations were measured. The maximum inhibitions of aggregation in the aspirin before ibuprofen group were 41.0±7.8% for ADP and 38.7±5.4% for collagen at 6 h after administration; similar values were seen in the aspirin group; however, percent inhibitions in the aspirin before ibuprofen multiple administration group were lower than those in the aspirin group. Thus, the inhibitory effects of daily low-dose aspirin on platelets are competitively inhibited by the prolonged use of multiple daily doses of ibuprofen. In contrast, serum TXB(2) concentrations in all groups were lower than those in the control group (drug-free). This suggests that the relationship between the inhibition of platelet COX-1 and the suppression of platelet aggregation is nonlinear. When aspirin was administered with loxoprofen sodium, similar effects were observed; however, with etodolac, the antiplatelet effects in all groups were equal to those in the aspirin group. Accordingly, if co-administration with NSAIDs is necessary with low-dose aspirin, a selective COX-2 inhibitor, such as etodolac, should be used.     

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.     

Gender differences in the effect of aspirin on retinal ischemia, prostanoid synthesis and nitric oxide production in experimental type 1-like diabetes

BACKGROUND: The protective effect of acetylsalicylic acid (aspirin) against cardiovascular events is known to be weaker in women than in men. The present study was designed to test whether this effect of aspirin differed between sexes in an experimental model of diabetes with retinal ischemia. METHODS: We compared nondiabetic rats and rats after 1, 2 and 3 months of diabetes that were given 2 mg/kg/day p.o. of aspirin from the first day of diabetes. The variables recorded were platelet aggregation, production of thromboxane B(2) (TxB(2)), 6-keto-prostaglandin F(1alpha) and aortic nitric oxide, and the percentage of the retinal surface occupied by horseradish peroxidase (HRP)-permeable vessels. RESULTS: In female rats made diabetic, TxB(2) synthesis was more markedly reduced, and the percentage of HRP-permeable retinal vessels was less markedly reduced, than in their male counterparts. The response to aspirin treatment was weaker in female than in male diabetic rats in terms of inhibition of TxB(2) synthesis, increased nitric oxide production, and prevention of the increase in the percentage of retinal surface covered by HRP-permeable vessels. CONCLUSION: Aspirin was less effective in preventing retinal ischemia in experimental diabetes in female than in male rats.     

Sex difference in the effect of aspirin on rat platelet aggregation and arachidonic acid metabolism

The rat platelet aggregation induced by collagen was stronger in males than in females. The platelet malondialdehyde (MDA) production was more in males than in females, and the platelet cyclooxygenase activity was higher in males than in females. Aspirin at a dose of 10 mg/kg inhibited the collagen-induced aggregation in males, but not in females. Aspirin at a dose of 5 mg/kg blocked the MDA production only in males, but aspirin at a dose of 10 mg/kg inhibited the MDA production in both sexes. The effect of aspirin on the cyclooxygenase activity was only in males, but aspirin at a dose of 10 mg/kg inhibited the MDA production in both sexes. The effect of aspirin on the cyclooxygenase activity was similar to that on the MDA production. In gonadectomized rats, the MDA production and the cyclooxygenase activity were decreased by castration, and they were increased by ovariectomy. Aspirin at a dose of 5 mg/kg failed to inhibit them in castrated rats. Besides, in in vitro experiments, aspirin also inhibited the MDA production and the aggregation. Nevertheless, there was no sex difference in the content of arachidonic acid, a substrate of platelet cyclooxygenase. It is suggested that there is a sex difference in rat platelet cyclooxygenase activity, and it is closely related to the sex difference in the antiplatelet effect of aspirin.     

Vasoactive eicosanoids in the rat heart: Clues to a contributory role of cardiac throm☐ane to post-ischaemic hyperaemia

To assess the potential role of vasoactive cardiac eicosanoids in the modulation of coronary flow, we measure thromboxane(TX)B2, 6-keto-prostaglandin(PG)F1 alpha, PGE2 and sulphido-peptide leukotrienes (LTC4, D4, E4) in the coronary effluent of isolated perfused rat heart in both baseline and post-ischaemic conditions. Leukotrienes were undetectable. The order of production rate for the other eicosanoids was 6-keto-PGF1 alpha > TXB2 > PGE2. Production of such substances was increased about seven-fold over control after 5 min. global ischaemia; experiments with hypoxia showed that this was due to an actual increase in synthesis and not to a washout effect. A platelet source for TXB2 was excluded by 111In platelet labelling experiments. We assessed relative sensitivity to inhibition of cardiac TX synthesis relative to production of 6-keto-PGF1 alpha to inhibition by aspirin, ibuprofen, diclofenac and the specific thromboxane synthase inhibitor OKY-046. Aspirin, ibuprofen and diclofenac decreased 6-keto-PGF1 alpha production at a concentration always greater than required for a similar extent of TX inhibition. On the other hand a selective inhibition (> 90%) of TX was observed in the presence of OKY-046. Regression analysis of various 6-keto-PGF1 alpha/TXB2 ratios, as obtained in these different conditions, vs coronary flow, showed no correlation in baseline conditions, but a significant positive correlation (r = 0.59, P < 0.01) for post-ischaemic values. These data suggest a role for cardiac eicosanoids, including a non-platelet, cardiac-derived TX, in modulating the hyperaemic response in the isolated rat heart.     

Platelet thromboxane A2/prostaglandin H2 receptors in human volunteers on low doses of aspirin

Administration of aspirin (81 mg/day for 2-3 weeks) in nine healthy volunteers (out of an initial ten subjects, only nine qualified) resulted in a greater than 95% decrease of thromboxane B2 production by thrombin-stimulated platelets. At the same time, ligand binding studies with a thromboxane A2 antagonist, 125I-PTA-OH, measurements of shape change, and aggregation of platelets stimulated with U46619, a prostaglandin H2 analogue, indicated that administration of aspirin to normal human subjects does not result in the up-regulation of platelet thromboxane A2/prostaglandin H2 receptors.     

Platelet-mediated vascular contractions. Inhibition by flunarizine, a calcium-entry blocker

Flunarizine, a calcium (Ca2+)-entry blocker, selective for vascular tissues, inhibits in a concn-dependent way the contraction of isolated rat caudal artery preparations induced by mediators derived from thrombin-stimulated rat platelets. This inhibition is slow in onset and is of prolonged duration. Specific measurements and pharmacological analysis show 5-hydroxytryptamine (5HT) and thromboxane A2 (TXA2) to be the main mediators involved. Comparison with exogenously added agonists shows amplification between 5HT and TXA2 at the level of the vascular smooth muscle cells. Combined treatment with ketanserin, a selective 5HT2 receptor antagonist, and suprofen, a fatty acid cyclo-oxygenase inhibitor, shows that flunarizine inhibits the 5HT-induced as well as the prostaglandin-induced components of the contraction. The compound does not affect the release of 5HT from the platelet and does not interfere with the biosynthesis of TXA2 from endogenous platelet arachidonic acid; it reduces the amounts of TXB2 and HHT and increases the production of HETE from exogenous [14C]arachidonic acid by washed rat platelets.     

Effect of SAS 650 (Furofenac) on malondialydehyde production by platelets

The activity of SAS 650, a new anti-inflammatory drug, on ex vivo and in vitro MDA production by platelets was compared to that of aspirin. The drug induced dose-dependent inhibition of in vitro MDA production by rat and guinea-pig platelets and also had good activity after 30 second of incubation in rat platelets, quicker than aspirin. SAS 650 preincubation reduced the in vitro inhibitory effect of ASA, as shown also by ex vivo experiments. The results of the present study support the involvement of SAS 650 in the platelet cyclooxygenase pathway.     

低密度脂蛋白对人血小板磷脂酰肌醇代谢产物及钙摄取的影响

观察了低密度脂蛋白(LDL)或高密度脂蛋白(HDL)加LDL对人血小板磷脂酰肌醇循环中的重要物质磷脂酰肌醇4,5-二磷酸(PIP_2),磷脂酸(PA),以及~(45)Ca~(2+)摄取的影响,同时还观察了LDL,HDL对猪肺微粒体酶催化前列腺素内过氧化物生成血栓素B_2(TXB_2)的影响.结果表明,LDL引起血小板PIP_2短暂下降,继之增加,同时伴有PA的增加,未观察到HDL拮抗LDL的作用,此外,LDL能刺激Ca~(2+)内流入血小板.HDL明显抑制TXB_2生成。     

trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets.

1. The phytoestrogenic compound trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. 2. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets. 3. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 microM. trans-Resveratrol at 0.1, 1.0 and 10.0 microM produced 20+/-6, 37+/-6 and 57+/-4% inhibition respectively of the effect of thrombin (0.01 u ml(-1)) to increase [Ca2+]i. 4. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 microM trans-resveratrol producing 10+/-5, 30+/-5 and 50+/-7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. 5. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. 6. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol. 7. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to inhibit platelet aggregation induced by thrombin.     

Mechanisms of Forssman-induced bronchospasm and their inhibition.

1 The bronchospasm induced in the guinea-pig by the injection of Forssman antiserum was biphasic in nature in both the sublethal and the lethal reaction. 2 The development of both phases of the bronchospasm in the sublethal reaction was dependent upon the presence of the intact complement system and circulating platelets. In the lethal reaction the phase II bronchospasm did not appear to depend on these factors. 3 The compounds used in this study inhibited phase I bronchospasm of the sublethal reaction in the order, methysergide greater than indomethacin greater than aspirin = sulphinpyrazone and phase II in the order, indomethacin greater than sulphinpyrazone greater than aspirin. Methysergide was inactive. 4 Aspirin, indomethacin and sodium salicylate all prevented the inhibitory action of sulphinpyrazone in reducing the phase II bronchospasm of the sublethal reaction in the order, indomethacin greater than sodium salicylate greater than aspirin, when the drugs were administered prior to sulphinpyrazone. 5 The inhibitory action of aspirin on the sulphinpyrazone effect could be prevented by administering sulphinpyrazone before aspirin. All drug-induced inhibitions of sulphinpyrazone by aspirin, indomethacin and sodium salicylate were dose-dependent.     

Comparison of the inhibitory effects of some compounds present in crude oils on rat platelet aggregation: role of intra- and extra- cellular calcium

In vitro addition of some representative aliphatic, aromatic or heterocyclic compounds present in petroleum crude oils to washed rat platelets resulted in a concentration-dependent inhibition of aggregation induced by ADP or thrombin. Increasing concentration of extracellular Ca2+ did not alter the pattern of inhibition. ADP-induced intracellular Ca2+ mobilization was unaffected by most of the compounds tested. However, Ca2+ uptake was significantly inhibited when platelets were preincubated with these agents. This suggests that some components of crude oil may inhibit platelet aggregation by bringing about alterations in the platelet plasma membrane.     

Inhibitory effect of disodium quercetin-7,4'-disulfate on aggregation of pig platelets induced by thrombin and its mechanism

AIM: To study the inhibitory effect of semi-synthesized quercetin derivatives--disodium quercetin-7,4'-disulfate (DQD) on the platelet aggregation induced by thrombin and its mechanism. METHODS: Platelet aggregation was analysed by turbidimetry. Cytosolic free calcium concentration ([Ca2+]i) was determined by Fura-2 fluorescence technique. Activity of Ca2+/PL dependent protein kinase C (PKC) was assayed by incubating PKC with histone III S and [gamma-32P]ATP. The cytoskeletal proteins were precipitated by Triton and separated by SDS-PAGE. RESULTS: DQD inhibited the platelet aggregation induced by thrombin (500 U/L), when DQD concentrations were 100, 200, and 400 mumol/L, the inhibition rates were 77%, 86%, and 82% respectively. DQD inhibited Ca2+ influx in platelets induced by thrombin (500 U/L) in the presence of extracellular Ca2+ 1 mmol/L in a concentration-dependent manner (10-80 mumol/L); DQD also had inhibitory effect on intracellular Ca2+ mobilization in the absence of extracellular Ca2+. DQD (10-160 mumol/L) inhibited the cytosolic Ca2+/PL dependent PKC from platelets in a concentration-dependent manner, but had no effect on membrane PKC. DQD (20-200 mumol/L) inhibited the actin polymerization induced by thrombin (500 U/L) in platelets in a concentration-dependent manner. CONCLUSION: DQD inhibited pig platelet aggregation induced by thrombin and its molecular mechanism was due to its inhibition of Ca2+ influx, intracellular Ca2+ mobilization, Ca2+/PL dependent PKC activity, and actin polymerization.     

Gender-Specific Inhibition of Ca2+ Entry Mechanisms of Arterial Vasoconstriction by Sex Hormones

1. The clinical observation that hypertension is more common in males and postmenopausal women than in premenopausal women suggests vascular protective effects of female sex hormones, including hormone-mediated inhibition of vascular tone. The purpose of the present study was to investigate whether the Ca2+ mobilization mechanisms of vascular smooth muscle contraction are modified by gender and sex hormones. 2. Active stress and [45Ca2+] influx were measured in de-endothelialized aortic strips isolated from intact and gonadectomized male and female Sprague-Dawley rats. In normal Krebs' (2.5 mmol/L Ca2+), both phenylephrine (Phe; 10(-5) mol/L) and membrane depolarization by 96 mmol/L KCl increased active stress to 15.5 +/- 1.3 x 10(3) and 14.8 +/- 1.2 x 10(3) N/m2, respectively, and Ca2+ influx to 28.4 +/- 1.4 and 32.3 +/- 1.5 mumol/kg per min, respectively, in intact males. The Phe- and KCl-induced stress and Ca2+ influx were significantly reduced in intact females. Gonadectomy was associated with no significant changes in the Phe- and KCl-induced stress and Ca2+ influx in males, but was associated with significant enhancement in females. In Ca(2+)-free (2 mmol/L EGTA) Krebs', stimulation of intracellular Ca2+ release by Phe or caffeine (25 mmol/L) caused a transient contraction that was not significantly different in all groups of rats. 3. Exogenous application of 17 beta-oestradiol, progesterone or testosterone to aortic strips caused concentration-dependent inhibition of Phe- and KCl-stimulated contractions and Ca2+ influx. 17 beta-Oestradiol was the most effective hormone and its relative potency was intact males, castrated males and ovariectomized females > intact females. 4. Thus, vascular reactivity and Ca2+ entry in aortic smooth muscle are reduced in the presence and enhanced in the absence of female gonads. Both male and female sex hormones cause vascular relaxation, mainly by inhibiting Ca2+ entry, with oestrogen being the most effective, particularly in the absence of female gonads. The results suggest that a cellular mechanism of oestrogen-induced vascular relaxation involving inhibition of Ca2+ entry into vascular smooth muscle is gender dependent.     

Effects of diuretics on calcium excretion and Ca2+-activated ATPase in rat kidney.

Effects of three diuretics on the urinary Ca2+ excretion and on the microsomal Ca2+-activated ATPase were examined in the rat kidney. Furosemide and bumetanide increased Na+, K+, and Ca2+ excretion in the rats. Acetazolamide increased Na+ and K+ excretion but not Ca2+. Urinary inorganic phosphate excretion was not affected during the administration of acetazolamide. It is suggested that acetazolamide inhibits Na+ transport without affecting Ca2+ reabsorption in the rat nephron. Microsomal ATPase of the rat kidney cortex was stimulated by Ca2+ or Mg2+ alone and an additive effect of the two cations was not observed. Microsomal ATPase activated by Ca2+ or Mg2+ was not inhibited by furosemide, bumetanide, and acetazolamide. These data suggest that the inhibitory effect of furosemide and bumetanide on the Ca2+ reabsorption is not related to the inhibition of Ca2+-activated ATPase.     

Irreversible inhibition of thromboxane (TX) A2 synthesis by Y-20811, a selective TX synthetase inhibitor.

As Y-20811, sodium (+-)-4-[alpha-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylb+ ++ enzoic acid, has been reported to inhibit serum thromboxane (TX) A2 production with a long duration of action, its mechanism of action was investigated. When [3H]Y-20811 (3 mg/kg) was administered orally to rats, the peak platelet concentration of Y-20811 was obtained 1 hr after the administration, and the T1/2 was 43 hr. The peak plasma concentration of Y-20811 was also obtained 1 hr after administration, but the elimination of Y-20811 from plasma was faster (T1/2 alpha = 1.5 hr, T1/2 beta = 15 hr) than that observed in platelets. Serum TXA2 (estimated as TXB2) production was inhibited significantly from 1 to 72 hr after the oral administration of unlabeled Y-20811 (3 mg/kg), which temporally resembled the change of the platelet Y-20811 concentration. In platelet-rich plasma, [3H]Y-20811 completely inhibited TXA2 production at about 1500 pg/10(9) platelets, and the IC50 level was about 600 pg/10(9) platelets, which was similar to values obtained in ex vivo studies. In addition, inhibition of TXA2 production by Y-20811 still remained after washing the drug-pretreated microsomes, whereas that of dazoxiben completely disappeared. A similar irreversible inhibition of TXA2 production was observed with aspirin. These results suggest that Y-20811 may firmly combine with platelet TX synthetase and may irreversibly inhibit TXA2 production.     

山豆根碱对大鼠血小板摄取~（45）Ca~（2＋）的影响

用４５Ｃａ２＋摄入法测定了大鼠血小板摄取细胞外Ｃ２＋，观察山豆根碱对血小板摄取Ｃａ２＋的影响、结果表明，静息血小板可迅速摄取细胞外Ｃａ２＋，呈时间和浓度依赖性；ＡＤＰ促进血小板摄取Ｃａ２＋；山豆根碱以浓度依赖方式抑制静息的和ＡＤＰ诱导的血小板摄取细胞外Ｃａ２＋。这提示山豆根碱抑制血小板聚集的作用机制之一与阻止细胞外Ｃａ２＋内流入血小板，降低血小板胞浆内Ｃａ２＋浓度有关。