Inhibition of cyclooxygenases 1 and 2 by the phospholipase-blocker, arachidonyl trifluoromethyl ketone

Background and purpose:

Arachidonyl trifluoromethyl ketone (ATK) is widely used as an inhibitor of cytosolic group IV phospholipase A₂ (cPLA₂) and calcium-independent group VI phospholipase A₂ (iPLA₂). ATK thus reduces arachidonic acid (AA) substrate for cyclooxygenase (COX; also known as prostaglandin H synthase) and attenuates prostaglandin (PG) synthesis. It has been shown previously, that ATK blocks thromboxane B₂ production induced by exogenous AA in human platelets. It remains, however, unknown whether ATK also directly modulates the activity of cyclooxygenase (COX).

Experimental approach:

Time courses for inhibition of COX by ATK was obtained using osteoblast-like MC3T3-E1 cells, with exogenous AA as substrate and the pure enzymes COX-1 and COX-2. PGE₂ was measured by GC-MS.

Key results:

ATK was a potent inhibitor of COX-1 and COX-2 with IC₅₀ values of 0.5 and 0.1 μM in MC3T3-E1 cells and of 1.7 and 2.6 μM using the pure enzymes. Inhibition was reversible, with slow- and tight-binding characteristics. The arachidonyl carbon chain was essential, as the saturated palmitoyl analogue had no effect.

Conclusions and implications:

Attenuation of PG synthesis by ATK is taken to be the consequence of PLA₂ inhibition and the findings of many studies are interpreted on that basis. If there are, however, alternative routes for AA liberation (such as phospholipase C/diacyl glycerol lipase or phospholipase D), this interpretation can lead to false conclusions. As ATK is a widely used and important pharmacological tool in eicosanoid research, knowledge of its interactions with other major enzymes of the cascade is of considerable importance.     

Inhibition of human platelet activation by polymorphonuclear leukocytes.

1. The effect of unstimulated human polymorphonuclear leukocytes (PMNs) on platelet activation was examined. 2. Human platelet aggregation and adenosine 5'-triphosphate (ATP) release induced by collagen (1-2 micrograms ml-1); thrombin (0.01-0.02 u ml-1) or arachidonic acid (AA) (0.1-0.2 mM) were markedly inhibited when conducted in the presence of unstimulated PMNs. 3. Platelet inhibition induced by PMNs was dependent on the number of PMNs and on the incubation time of the mixed cell suspension. 4. Platelet inhibition was not reversed in time when PMNs were depleted from the mixed-cell suspension. 5. PMN-mediated platelet-inhibition was not mediated by AA metabolites, oxygen reactive intermediates, nitric oxide or proteases. 6. The factor(s) accounting for the platelet inhibition mediated by PMNs are not yet characterized.     

Inhibition of platelet activation by tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers (tyrphostins) inhibit in a dose-dependent fashion thrombin-induced aggregation and serotonin release with IC50 values in the 10-35 microM concentration range. The inhibition of thrombin-induced aggregation correlates with their potency in inhibiting phosphorylation of proteins on tyrosine residues. Using metabolically 32P-labelled human platelets, it was found that the tyrphostins have no effect on the decrease in [32P]phosphatidylinositol bisphosphate but prevent the replenishment of [32P]polyphosphoinositide. Tyrphostins decreased [32P]phosphatidic acid production induced by thrombin, although never by more than 50%, and only delayed the peak of diacylglycerol, suggesting that phospholipase C was still activated. Tyrphostins inhibited the thrombin-elicited early phosphorylation of p43 and p20, substrates for protein kinase C (PKC) and myosin light chain kinase, respectively, at short times of activation. This inhibition, however, was overcome after 1 min of stimulation with thrombin. Tyrphostin AG213 also inhibited platelet aggregation and tyrosine protein phosphorylation induced by phorbol myristate acetate (PMA), but did not inhibit pleckstrin phosphorylation. These results suggest that thrombin induces the phosphorylation of proteins on tyrosine residues which most probably results in the activation of phosphoinositide kinases. The ability of tyrphostins to inhibit phosphorylation of p43 and p20 when induced by thrombin but not when induced by PMA confirms that PTKs may be involved subsequent to PKC activation.     

Inhibition of platelet activation and aggregation

It has recently been established that platelets are involved at all stages of atherosclerotic disease. A major platelet mediated process is the acute vessel closure at the site of atherosclerotic plaque rupture and there is emerging evidence for platelet adhesion to endothelial cells in the early stage of atherosclerotic disease. This, through engagement of other cells, leads to the development of the atherosclerotic plaque. Beside dietary, cholesterol- and lipid-lowering, and other pharmaceutical approaches antiplatelet therapy plays an important part in the treatment of atherosclerosis and its multifarious clinical manifestations. Antiplatelet therapy and the currently approved substances for oral (acetylsalicylic acid, dipyridamole, cilostazol, ticlopidin and clopidogrel) and parenteral (acetylsalicylic acid, abciximab, eptifibatide and tirofiban) administration are discussed in the following section. Attention is given to each single agent and its mechanism of action. Differences in pharmacodynamic and pharmacokinetic properties are elucidated and outlook on future antiplatelet strategies is discussed.     

Phospholipase A2 inhibitors p-bromophenacyl bromide and arachidonyl trifluoromethyl ketone suppressed interleukin-2 (IL-2) expression in murine primary splenocytes

Phospholipase A₂ (PLA₂) has been postulated to play a role in the regulation of cytokine expression. Therefore, the objective of the present study was to investigate the effects of PLA₂ inhibitors p-bromophenacyl bromide (BPB) and arachidonyl trifluoromethyl ketone (AACOCF₃) on interleukin-2 (IL-2) expression in murine primary splenocytes. Pretreatment of the splenocytes with both BPB and AACOCF₃ suppressed phorbol 12-myristate 13-acetate plus ionomycin-induced IL-2 secretion in a concentration-dependent manner. Inhibition >90% of IL-2 secretion was observed at 1 μM BPB and 10 μM AACOCF₃ compared to the respective vehicle control. Likewise, IL-2 steady-state mRNA expression was inhibited by both PLA₂ inhibitors in a concentration-dependent fashion with >90% inhibition at 1 μM BPB and 20 μM AACOCF_3. Taken together, these data demonstrated that PLA₂ inhibitors BPB and AACOCF₃ are robust inhibitors of IL-2 expression at both the mRNA and protein levels in murine splenocytes. Moreover, these findings suggest that drugs and chemicals which inhibit PLA₂ may have marked effects on T-cell function. Received: 27 July 1998 / Accepted: 24 November 1998     

Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G.

Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy.     

Inhibition of platelet activation by 5-aminosalicylic acid in inflammatory bowel disease

BACKGROUND: Platelets play an important role in inflammation and are activated in inflammatory bowel disease. Micro-vascular thrombosis in the gut wall leading to intestinal micro-infarction may be a pathogenic feature of Crohn's disease. 5-Aminosalicylic acid is an effective treatment for patients with inflammatory bowel disease. AIMS: To assess the effects of 5-aminosalicylic acid on platelet activation, when taken orally and in vitro by patients with inflammatory bowel disease. METHODS: Spontaneous and thrombin-induced platelet activation were studied using fluorescent antibodies to the activated platelet surface glycoprotein P-selectin and flow cytometry. RESULTS: Baseline platelet activation in inflammatory bowel disease was significantly greater than that in controls (P=0.0003). Independent of diagnosis or disease activity, spontaneous ex-vivo platelet activation was 50% lower in patients with inflammatory bowel disease taking 5-aminosalicylic acid orally than in those not on such treatment (P < 0.05). In vitro, 5-aminosalicylic acid significantly reduced both spontaneous (P < 0. 03 for >/=1 microM 5-aminosalicylic acid) and thrombin-induced platelet activation (P < 0.02 for >/= 1 microM 5-aminosalicylic acid). CONCLUSIONS: 5-Aminosalicylic acid given either orally or in vitro inhibits platelet activation. If this effect reflects an in vivo action in the gut, it could contribute to the beneficial actions of 5-aminosalicylic acid in inflammatory bowel disease.     

Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib, nilotinib and dasatinib

Imatinib, nilotinib and dasatinib are protein kinase inhibitors which target the tyrosine kinase activity of the Breakpoint Cluster Region-Abelson kinase (BCR-ABL) and are used to treat chronic myelogenous leukemia. Recently, using a chemical proteomics approach another tyrosine kinase, the collagen receptor Discoidin Domain Receptor1 (DDR1) has also been identified as a potential target of these compounds. To further investigate the interaction of imatinib, nilotinib and dasatinib with DDR1 kinase we cloned and expressed human DDR1 and developed biochemical and cellular functional assays to assess their activity against DDR1 and the related receptor tyrosine kinase Discoidin Domain Receptor2 (DDR2). Our studies demonstrate that all 3 compounds are potent inhibitors of the kinase activity of both DDR1 and DDR2. In order to investigate the question of selectivity among DDR1, DDR2 and other tyrosine kinases we have aligned DDR1 and DDR2 protein sequences to other closely related members of the receptor tyrosine kinase family such as Muscle Specific Kinase (MUSK), insulin receptor (INSR), Abelson kinase (c-ABL), and the stem cell factor receptor (c-KIT) and have built homology models for the DDR1 and DDR2 kinase domains. In spite of high similarity among these kinases we show that there are differences within the ATP-phosphate binding loop (P-loop), which could be exploited to obtain kinase selective compounds. Furthermore, the potent DDR1 and DDR2 inhibitory activity of imatinib, nilotinib and dasatinib may have therapeutic implications in a number of inflammatory, fibrotic and neoplastic diseases.     

Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase

Carbon monoxide (CO) inhibits human platelet aggregation triggered with threshold levels of agonists like arachidonate, ADP, collagen, thrombin, or the prostaglandin endoperoxide analogue U46619. This inhibition is counteracted by illumination with light above 400 nm indicating the involvement of a ferrous hemoprotein. An earlier suggestion that the mechanism of CO inhibition involves the cytochrome P450 protein thromboxane A2 synthase was ruled out as well as the involvement of the iron containing enzymes like cyclooxygenase or 12-lipoxygenase. In the presence of CO, no arachidonate was released from phospholipids, no increase of intracellular calcium levels was observed, and phospholipase C was not activated suggesting that the transducing mechanisms from the receptors to phospholipase C was effected in the presence of CO. cAMP levels were also unchanged but cGMP levels showed an increase of about 30%. By comparison with the guanylate cyclase stimulator nitroprusside, it was shown that such levels could block aggregation. In a 10,000 X g supernatant, CO enhanced guanylate cyclase activity 4-fold, supporting the view that CO acts by increasing platelet cGMP levels. With respect to the mechanism of guanylate cyclase action, the binding of CO to the regulatory subunit of guanylate cyclase must be responsible for the observed activation. It is concluded that cGMP is an important feedback regulator of the Pl response and that already a 25% increase in its steady state levels can cause inhibition of platelet aggregation.     

Nanoliposomal delivery of cytosolic phospholipase A2 inhibitor arachidonyl trimethyl ketone for melanoma treatment

Drug resistance and toxicity are major limitations of cancer treatment and frequently occurs during melanoma therapy. Nanotechnology can decrease drug resistance by improving drug delivery, with limited toxicity. This study details the development of nanoparticles containing arachidonyl trifluoromethyl ketone (ATK), a cytosolic phospholipase A₂ inhibitor, which can inhibit multiple key pathways responsible for the development of recurrent resistant disease. Free ATK is toxic, limiting its efficacy as a therapeutic agent. Hence, a novel nanoliposomal delivery system called NanoATK was developed, which loads 61.7% of the compound and was stable at 4^oC for 12 weeks. The formulation decreased toxicity-enabling administration of higher doses, which was more effective at inhibiting melanoma cell growth compared to free-ATK. Mechanistically, NanoATK decreased cellular proliferation and triggered apoptosis to inhibit melanoma xenograft tumor growth without affecting animal weight. Functionally, it inhibited the cPLA₂, AKT, and STAT3 pathways. Our results suggest the successful preclinical development of a unique nanoliposomal formulation containing ATK for the treatment of melanoma.     

银杏内酯B抑制血小板CD40Ligand表达的分子机制研究

目的探讨银杏内酯B(ginkgolide B)对活化血小板CD40 Ligand(CD40L)的影响以及相关的分子机制。方法取正常人血分离血小板,用不同浓度银杏内酯B孵育血小板5 min,然后用胶原(collagen)刺激血小板活化。用Westernblot分析PI3K表达,Akt磷酸化,CD40L的变化。结果①用collagen刺激血小板聚集,银杏内酯B(0.2、0.4、0.6 g.L-1)预处理血小板5 min,血小板聚集率明显降低,聚集率分别为77%,60%和48%。②Western blot结果显示胶原刺激血小板活化后CD40L表达明显增加,银杏内酯B以剂量依赖方式抑制了CD40L表达。③银杏内酯B对胶原刺激的血小板PI3K表达无明显影响。④collagen刺激血小板活化后Akt的磷酸化增加,银杏内酯B抑制了Akt磷酸化。结论银杏内酯B能够有效抑制collagen诱导的血小板聚集以及CD40L的表达,并明显抑制了Akt磷酸化,表明银杏内酯B能够通过PI3K/Akt信号传导通路抑制血小板活化。     

Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-kappaB activation

N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) is known to inhibit NF-kappaB activation and the expression of inflammation mediators in cultured cells. We measured the potential of TPCK to inhibit the pathogenesis of collagen-induced arthritis by blocking NF-kappaB activation. Arthritis was induced in DBA/1J mice by the injection of bovine type II collagen in adjuvant on days 0 and 14. Mice received either TPCK (3 or 10 mg/kg, i.p.) or vehicle three times a week for 3 weeks starting on day 21. TPCK moderately reduced clinical disease activity scores, whereas it markedly suppressed histological indications of joint destruction. In vitro production of tumor necrosis factor-alpha, interleukin-6, and monocyte chemotactic protein-1 from lipopolysaccharide-stimulated spleen cells was also reduced by in vivo treatment with TPCK. Proliferation of cells isolated from spleen or draining lymph nodes and production of interferon-gamma and interleukin-17 in response to stimulation with type II collagen was decreased by TPCK. Moreover, nuclear NF-kappaB activity induced by collagen immunization was significantly reduced in mice treated with TPCK. Finally, osteoclast differentiation of bone marrow cells induced by macrophage colony-stimulating factor and receptor activator of NF-kappaB ligand was completely inhibited by TPCK. These results indicate that TPCK attenuates collagen-induced arthritis and bone erosion by suppressing NF-kappaB activation and thus expression of inflammatory and osteoclastogenic genes.     

硝酸异山梨酯针剂对不稳定型心绞痛病人血小板活性的抑制作用

目的 :观察体内外不同浓度硝酸异山梨酯(ISDN)对不稳定型心绞痛 (UAP)病人血浆P 选择素、凝血烷B2 (TXB2 )、6 酮 前列腺素F1α(6 keto PGF1α)水平的影响及其量效关系。方法 :体内实验 ,4 5例UAP病人分为 3组 ,分别给予ISDN 2 ,4 ,8mg·h- 1静脉滴注。体外实验 ,16例UAP病人清晨空腹采静脉血与不同浓度ISDN体外培养。测定用药前后血中P 选择素 ,TXB2 ,6 keto PGF1α的含量 ,计算TXB2 / 6 keto PGF1α。健康对照 2 0人。结果 :体内实验中 ,P 选择素和TXB2 在各剂量组 ,用药后 2h和 8h均明显抑制 (P <0 .0 5或P <0 .0 1)。6 keto PGF1α 在 2mg·h- 1组用药后变化不明显(P >0 .0 5 ) ,其余均较用药前升高 (P <0 .0 5或P <0 .0 1)。体外实验中 30min时 12 5mg·L- 1组P 选择素和TXB2 表达下降 (P <0 .0 5和P <0 .0 1) ,2h时12 .5mg·L- 1及 12 5mg·L- 1组P 选择素和TXB2均表达下降 (P <0 .0 1)。 6 keto PGF1α无明显变化。结论 :ISDN在体内外均可抑制UAP病人血小板P 选择素和TXB2 的表达且存在量效依赖关系 ,体外起效浓度远远大于体内浓度。     

Inhibition of thrombin-induced platelet aggregation by uteroglobin

Uteroglobin, a steroid-dependent, small molecular weight (15K) protein in the rabbit, inhibited thrombin-induced aggregation of both rabbit and human gel-filtered platelets (GFP). GFP aggregation by arachidonic acid was not affected by uteroglobin. There were no effects of uteroglobin on thrombin-induced clotting of plasma or purified fibrinogen, or inhibition of thrombin by antithrombin III. Additionally, preliminary results suggest that uteroglobin does not interfere with binding of thrombin to platelets. We suggest that inhibition of platelet aggregation by uteroglobin may function in preventing thrombosis and ensuring free flow of blood through the microvasculature of the uterus and the placenta and may induce some of the antimotility effects of progesterone on the uterus.     

Inhibition of platelet thromboxane synthetase by sulfasalazine

Sulfasalazine is a potent antiinflammatory drug used in the treatment of ulcerative colitis. The mechanism of action of sulfasalazine is unknown but a recent study [W. F. Stenson and E. Lobos, J. clin. Invest. 69, 494 (1982)] demonstrated that sulfasalazine, at therapeutic concentrations, blocks human neutrophil lipoxygenase, suggesting that its antiinflammatory effects may be mediated in part by the inhibition of the synthesis of the chemotactic lipids 5-hydroxy-6,8,11, 14-eicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). In the present study the effect of sulfasalazine on metabolism of exogenous arachidonic acid by human platelets was investigated. Sulfasalazine inhibited platelet thromboxane synthetase (IC50 = 0.9 mM) and partially inhibited cyclooxygenase. A methylated analog of sulfasalazine also inhibited thromboxane synthetase (IC50 = 0.3 mM) and partially inhibited cyclo-oxygenase. Neither of the cleavage products of sulfasalazine (5-aminosalicylate and sulfapyridine) inhibited thromboxane synthetase although 5-aminosalicylate blocked cyclooxygenase (IC50 = 5 mM). Neither sulfasalazine nor the methylated analog nor the cleavage products inhibited platelet lipoxygenase. This is in contrast to the inhibitory effects of sulfasalazine on neutrophil 5-lipoxygenase. The concentration of sulfasalazine in the colons of treated patients is several-fold greater than the IC50 for thromboxane synthetase.     

Inhibition of mouse liver alcohol dehydrogenase by methyl N-butyl ketone

Methyl N-butyl ketone (MNBK) inhibited the activity of mouse liver alcohol dehydrogenase (LADH) in vitro. Ethanol elimination was reduced in MNBK-treated mice as compared to controls. Ethanol-induced induced loss of righting reflex was significantly prolonged in mice pretreated with MNBK.     

Inhibition of platelet aggregation by adenosine receptor agonists

2-(Ar)alkoxyadenosines, which are agonists selective for the A_2AAR in PC 12 cell and rat striatum membranes, are also agonists at the A₂AR coupled to adenylate cyclase (AC) that mediates the inhibition of platelet aggregation. A panel of twelve well-characterized adenosine analogues stimulated human platelet AC and inhibited ADP-induced platelet aggregation at sub- to low-micromolar concentrations with a potency ranking CGS 21680 < adenosine < R-PIA. There were significant correlations between the ECso of anti-aggregatory activity and either the ECso of stimulation of platelet and PC 12 cell AC (r ² = 0.66 and 0.67, respectively) or the K₁ of inhibition of [³H]NECA binding to the rat striatum membranes (r ² = 0.75). Likewise, platelet AC stimulation correlated well with stimulation of PC 12 cell AC and with [³H]NECA binding (r ² = 0.94 and 0.91, respectively). Ten 2-(ar)alkoxyadenosines stimulated platelet AC at EC₅₀s ranging between 0.16 and 2.3 μM and inhibited platelet aggregation at EC₅₀s ranging between 2 and 30 μM. There were no correlations between the EC₅₀s of anti-aggregatory activity and either the EC₅₀s of the stimulation of platelet or PC 12 AC (r ² = 0.08 and 0.06, respectively) or with the K₁ of the inhibition of [³H]NECA binding to the A_2aAR in rat striatum (r ² = 0.02). The EC₅₀s of the stimulation of platelet AC correlated with those of the stimulation of PC 12 AC (r ² = 0.48), and also with the K₁ of [³H]NECA binding (r ² = 0.71). Each of the 23 adenosines completely inhibited platelet aggregation and thus, functionally, all behaved as full agonists. As stimulants of PC 12 cell AC, Group A and B analogues were equally efficacious. As stimulants of platelet AC, however, the efficacy relative to NECA ( = 1.0) of Group B analogues was significantly less than that of Group A analogues, 0.49 ± 0.2 vs. 0.72 ± 0.05, P±0.01. The partial agonist activity of Group B analogues at the platelet A2AR but full agonist activity at the PC 12 cell A_2aAR, as well as the relatively low correlations between platelet AC stimulation and other indices of A_2aAR agonist actlVlty, suggest the platelet receptor is not a typical A_2aAR. Further, the lack of a correlation between the platelet anti-aggregatory and AC stimulatory activity suggests that (a) the 2-(ar)alkoxyadenosines might affect platelet aggregation by mechanisms other than AC stimulation or (b) that the stimulation of the platelet membrane AC by 2-(ar)alkoxy-adenosines does not correspond to the accumulation of cyclic AMP in intact platelets.