Human Platelet Microsomes Research Articles

Abstract 9050: Drug-Drug Interactions Between Aspirin and NSAIDs are Predicted by Kinetics of Cyclooxygenase-1 Inhibition

Background and aims : Therapy with NSAIDs may interfere with antiplatelet activity of aspirin. The mechanism of this drug-drug interaction has not yet been established on the level of enzyme kinetics and quantitative structure-activity relationship (QSAR). Methods: Time-dependent kinetics (log K) for the onset of COX-1 inhibition was measured for 17 common NSAIDs using arachidonic acid-induced COX-1 activity (human platelet microsomes, bioluminescence). IC50 values of all NSAIDs were determined by arachidonic acid-induced platelet thromboxane (TX) formation in platelet rich plasma, as well as the in vitro interaction with aspirin. Dragon and SYSTAT softwares served to develop QSAR models in which dependent parameters were log K and IC50 and independent parameters were structural/physico-chemical parameters (MoR32m, G2v, L3S, representing the shape, size and polarizibility). Results: All NSAIDs could be classified into a time-dependent (slow onset) or a time-independent (fast onset) mode of inhibition. Time-dependent NSAIDs included diclofenac, flurbiprofen, indomethacin, ketoprofen and ketorolac. These NSAIDs did not decrease TX inhibition by aspirin. In contrast, all other studied NSAIDs were time-independent inhibitors and prevented inhibiton by aspirin. The inhibition kinetics (log K) and IC50 were correlated with MoR32m, G2v and L3S (equations 1 and 2). eq.1: -log IC50 = 4.54 (±0.99) MoR32m - 64.62 (±12.58) G2v + 3.41 (±0.56) L3S + 8.59; r = 0.91; r2 = 0.84; s = 0.41; F = 22.81; PRESS = 5.00. eq.2: -log K = 2.35 (±0.69) MoR32m - 16.50 (±8.68) G2v + 1.18 (±0.39) L3S + 1.61; r = 0.74; r2 = 0.55; s = 0.29; F = 5.49, PRESS= 3.01; r, correlation coefficient; s, standard deviation; F, Fisher’s test; PRESS, sum of square differences. Conclusions: NSAIDs decrease the antiplatelet activity of aspirin. This is highly dependent on the inhibition kinetics of platelet COX-1, whereas the inhibition kinetics and IC50 values for TX inhibition further depends on MoR32m, G2v and L3S of a particular NSAID. Time-independent NSAIDs decrease the anti-platelet activity of aspirin. Therefore, time-dependent NSAIDs should be preferred in patients on antiplatelet treatment with aspirin. The QSAR models may predict the kinetics and enzyme inhibition by NSAIDs.

Effect of bakuchiol on leukocyte functions and some inflammatory responses in mice.

The effects of bakuchiol, a meroterpenoid isolated from the leaves of Psoralea glandulosa L., on phospholipase A2 (PLA2) activity from different sources, human neutrophil responses, zymosan air pouch and topical inflammation in mice, were investigated. This natural product was a weak inhibitor of secretory and intracellular PLA2 but dose-dependently reduced the formation of LTB4 and TXB2 by human neutrophils and platelet microsomes, respectively. In addition, bakuchiol inhibited degranulation in human neutrophils, whereas superoxide generation was not affected. In mice, bakuchiol decreased cell migration, myeloperoxidase activity and eicosanoid levels in the air pouch inflammation induced by zymosan. After topical administration, this compound was effective as an inhibitor of oedema and myeloperoxidase activity in the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear oedema and significantly reduced the PGE2 content and ear oedema in the arachidonic acid-induced response. Bakuchiol is a natural anti-inflammatory agent able to control leukocytic functions such as eicosanoid production, migration and degranulation in the inflammatory site.

Pyrazolinone analgesics prevent the antiplatelet effect of aspirin and preserve human platelet thromboxane synthesis.

Anti-inflammatory analgesics, including ibuprofen and naproxen, are known to interfere with the antiplatelet effect of aspirin, presumably as a result of a drug-drug interaction at the level of platelet cyclooxygenase-1 (COX-1). We studied whether dipyrone, which has recently been reported to inhibit COX isoforms by a mechanism different from conventional non-steroidal anti-inflammatory drugs (NSAIDs), also interferes with the antiplatelet effect of aspirin. Arachidonic acid- and collagen-induced aggregation, as well as thromboxane formation, were measured in human platelet-rich plasma. Platelet P-selectin expression was determined by flow cytometry and cell-free COX enzyme activity was quantified by luminol-enhanced luminescence of human platelet microsomes. In addition, computerized docking was performed based on the crystal structure of COX-1. 4-Methylaminoantipyrine (MAA), the active metabolite of dipyrone, largely attenuated or even completely abolished the inhibition of arachidonic acid-induced platelet aggregation, thromboxane formation and P-selectin expression by aspirin. Similar results were obtained for other pyrazolinones, as well as for the conventional NSAIDs ibuprofen and naproxen. Moreover, MAA attenuated the effect of aspirin on COX activity of platelet microsomes, suggesting a competition with aspirin at the COX-1 enzyme. This was confirmed by docking studies, which revealed that MAA forms a strong hydrogen bond with serine 530 within the COX-1, thereby preventing enzyme acetylation by aspirin. This study demonstrates for the first time that dipyrone and other pyrazolinones have a high potential to attenuate or prevent the antiplatelet effect of aspirin. This should be considered if pyrazolinone analgesics are administered to patients with cardiovascular disease requiring antiplatelet aspirin therapy.

Purification and characterization of diacylglycerol lipase from human platelets.

Diacylglycerol lipase (DGL) was solubilized from human platelet microsomes with heptyl-beta-D-thioglucoside, and purified to homogeneity on SDS-PAGE using a combination of chromatographic and electrophoretic methods. The molecular mass of the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as determined by Immobiline isoelectro-focusing. The enzymatic activity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2 inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) enhanced it. In addition, the enzymatic activity was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, serine and histidine residues are required for the enzymatic activity of DGL. DGL was optimally active in the pH range of 7-8 and its activity did not change significantly in the presence of various calcium concentrations, even in the presence of 2 mM EGTA. This indicates that DGL can hydrolyze substrates with a basal cytosolic free Ca2+ level in the physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent manner with an IC50 (the concentration required for 50% inhibition) of about 5 microM. Unexpectedly, several phospholipase A2 (PLA2) inhibitors were potent inhibitors of DGL activity (IC50<5 microM), suggesting that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glycerol, 2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most potent inhibitor.

Whole-blood assays for evaluation of thromboxane synthase inhibition.

AbstractResearchers in the eicosanoid field have frequently applied whole blood assays (1–3) for evaluation of enzymic inhibitors in preclinical and clinical studies. In numerous investigations of antiplatelet agents, the capacity of blood to generate thromboxane A2 (TXA2) in response to an agonist has been assessed, and similar assays figure importantly in the current clinical trials (4) of cyclooxygenase-2 (COX-2) inhibitors. Early work by Sun (5), carried out with human platelet microsomes, established the reaction conditions for studies of thromboxane synthase, and demonstrated that thromboxane synthase is not inhibited by non steroidal anti-inflammatory agents, such as aspirin. It now seems clear that other blood elements, notably monocytes, also contain thromboxane synthase. When the objective is to manipulate, pharmacologically, arachidonic acid (AA) metabolism, or to track routes of transcellular metabolism of AA (discussed in Chapter 16), convenient assays are often set up to measure stable products, such as 6-keto-PGF1α and TXB2; hydrolysis products of prostacyclin (PGI2) and TXA2, respectively. Depending on the specific purpose, different agonists (e.g., thrombin, collagen, endotoxin) may be used to elicit formation of eicosanoids by blood elements.KeywordsBlood ElementHuman Lysed PlateletSerum TXB2Plasma TXB2TXB2 FormationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Enzyme self-inactivation is a main limitation of the preparation of eicosanoids. Enzymatic synthesis of thromboxane and 12(S)-hydroxytetraenoic acid.

Synthesis of prostanoids is accompanied by various processes reducing the product yield. These processes are also known to affect syntheses of thromboxane (TX) and 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-HETE). Partially purified preparations of TX synthase and prostaglandin (PG) synthase were used to optimize TX synthesis with respect to concentrations of the enzymes and eicosapolyenoic acid (EPA). Conditions for the maximum product yield and the minimum consumption of enzymes were determined. Consumption of the TX synthase was large owing to its inactivation during the reaction and the nonenzymatic destruction of the intermediate product PG-endoperoxide. Separate addition of PG and TX synthases increased the product yield by preventing EPA sorption on ballast proteins. Microsomal 12-lipoxygenase (12-LO) was also shown to be inactivated during the reaction, and this process was the major limitation of 12-HETE synthesis. Lipoxygenase reaction in the presence of some reducing agents led to a considerable increase of the 12-HETE yield, supposedly by preventing further oxidation of the 12-LO reaction product 12-hydroperoxy derivative of eicosatetraenoic acid. The possibility of using human blood platelet microsomes for preparation of some derivatives of EPAs is discussed.

Thromboxane A 2 synthase inhibition and thromboxane A 2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC 50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 μg/mL)-, AA (5C μM)-, and A23187 (2 μM)-induced aggregation were 36.2 ± 1.5, 6.7 ± 0.7, 35.4 ± 1.7, and 93.1 ± 1.4 μM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 μM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A 2 (TXA 2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA 2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA 2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H 2 to TXA 2. Similarly, NQ-Y15 selectively inhibited the TXA 2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A 2, cyclooxygenase, and PGI 2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA 2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA 2 synthase inhibition with TXA 2 receptor blockade, and that it may be useful as an antithrombotic agent.

Sodium nonivamide acetate: A non-pungently antinociceptive capsaicin derivative with unusual anti-inflammatory properties

1. 1. Bradykinin-induced vascular pain in conscious rats, hyperalgesia in the rat hind paw, rat hind paw edema induced by compound 48/80 and carrageenin and dye exudation induced by intraperitoneal injection of 0.7% acetic acid in mice were all inhibited by sodium nonivamide acetate (SNA). 2. 2. Collagen and arachidonic acid-induced rabbit platelet aggregations were inhibited by SNA and capsaicin. In human platelet microsomes, prostaglandin E 2 formation in arachidonic acid metabolite was not inhibited by SNA but was inhibited by capsaicin and indomethacin; thromboxane B 2 formation and its synthetase activity were inhibited by SNA and capsaicin. 3. 3. In the extracellular recording, SNA could not decrease the action potential amplitude of the vagus nerve. 4. 4. The motor activity of mice induced by caffeine (1.0 mg/kg) was inhibited by SNA and capsaicin.

Effects of marine 2-polyprenyl-1,4-hydroquinones on phospholipase A 2 activity and some inflammatory responses

Three 2-polyprenyl-1,4-hydroquinone derivatives (2-heptaprenyl-1,4-hydroquinone: IS1, 2-octaprenyl-1,4-hydroquinone: IS2 and 2-[24-hydroxy]-octaprenyl-1,4-hydroquinone: IS3) isolated from the Mediterranean sponge Ircinia spinosula, were evaluated for effects on phospholipase A 2 activity of different origin ( Naja naja venom, human recombinant synovial fluid and bee venom), as well as on human neutrophil function and mouse ear oedema induced by 12- O-tetradecanoylphorbol 13-acetate (TPA). IS1 interacted minimally with these responses. In contrast, IS2 and IS3 inhibited human recombinant synovial phospholipase A 2 in a concentration-dependent manner, with minor effects on the rest of the enzymes. Both compounds slightly affected superoxide generation and degranulation in human neutrophils, whereas they decreased thromboxane B 2 and leukotriene B 4 synthesis and release in a mixed suspension of human platelets and neutrophils stimulated by ionophore A23187, with IC 50 values in the μM range. IS3 was the most effective inhibitor of the synthesis of thromboxane B 2 by human platelet microsomes and of leukotriene B 4 by high speed supernatants from human neutrophils. IS2 and IS3 showed topical anti-inflammatory activity against the TPA-induced ear inflammation in mice, with similar effects on oedema and a higher inhibition of IS3 on leukocyte migration, estimated as myeloperoxidase activity in supernatants of ear homogenates. Some structure-activity relationships were established since differences in the prenylated chain attached to the hydroquinone moiety result in important modifications of these inflammatory responses.

Primary stimuli of icosanoid release inhibit arachidonoyl‐CoA synthetase and lysophospholipid acyltransferase

Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.

The relative utilization of eicosapentaenoic and arachidonic acids by the 1-acylgycerophosphorylcholine and the 1-acylglycerophosphorylinositol acyltransferase pathways in human platelet microsomes

The relative entry of eicosapentaenoic acid (EPA) and arachidonic acid (AA) via 1-acylglycerophosphorylcholine and 1-acylglycerophosphorylinositol acyltransferase activity into phosphatidylcholine (PC) and phosphatidylinositol (PI), respectively, was studied in human platelets. A known mixture of [ 14C]EPA and [ 3H]AA along with an acyl-CoA generating system and lyso-acceptor phospholipids were incubated with a platelet mixed membrane preparation. The radioactivities in the products were determined following lipid extraction and thin-layer chromatographic separations. The [ 3H]AA/[ 14C]EPA ratios in the products, PC and PI, indicated some preference of both acyltransferase pathways for EPA over AA at 50 μM exogenously-added lysoPC or lysoPI. Altering the concentration of lysoPC (10–50 μM) did not significantly affect the selectivity for EPA. In contrast, reducing the concentration of lysoPI from 50 μM to 10 μM changed the selectivity of the acyltransferase pathway from favouring EPA to slightly favouring AA. The presence of a mixture of lysoPC and lysoPI resulted in a partitioning of EPA more towards PC and less towards PI which may account, in some part, for the greater AA/EPA mass ratio observed in the PI from human subjects consuming fish oil. The magnitude of the differences between PC and PI in their AA/EPA mass ratios observed in vivo, however, cannot be explained by the selectivity demonstrated herein by the acyltransferase pathways.

Thromboxane synthase catalyses hydroxylations of prostaglandin H2 analogs in the presence of iodosylbenzene

Human platelet microsomes supplemented with iodosylbenzene converted the stable prostaglandin H2 analog 15(S)-hydroxy-11 alpha,9 alpha-epoxymethano-5(Z),13(E)-prostadienoic acid (U46619) into three metabolites (17.5% yield) which were not formed in the presence of specific thromboxane synthase inhibitors. The same three products were also formed among others by incubation of U46619 with liver microsomes from phenobarbital-pretreated rats with NADPH/O2 or with iodosylbenzene. The NADPH-supported metabolism of U46619 was suppressed in the presence of carbon monoxide. Combined gas chromatography/negative-ion chemical ionization mass spectrometry analysis revealed for all three compounds the incorporation of one oxygen atom which according to the electron impact fragmentation pattern had to be introduced either at the 9-methylene group or at the cyclopentane ring. The identification of these metabolites as 9 beta,15(S)-dihydroxy-11 alpha,9 alpha-epoxymethano-5(Z),13(E)-prostadienoic acid and the R and S isomer of 15(S)-hydroxy-11 alpha,9 alpha-(C-hydroxy-epoxymethano)-5(Z),13(E)-prostadienoic acid is only tentative since no reference compounds were available, but clearly thromboxane synthase was acting as an oxene transferase in this reaction. In contrast to U46619, its 9,11-epoxymethano isomer U44069 was found to be only a poor substrate for the oxene transferase activity of thromboxane synthase (1% yield) which indicates a preference for the 9-methylene group of U46619 which is orientated close to the heme iron of thromboxane synthase as evidenced by spectroscopic studies. Low-level chemiluminescence detected following incubation of iodosylbenzene with partly purified thromboxane synthase is in agreement with the formation of an activated (FeO)3+ oxygen species. In summary, these results point to a common role of the thiolate ligand in the oxygen activation mechanism by thromboxane and prostacyclin synthase and liver cytochrome P-450 monooxygenases.

Partial purification and characterization of thrombolamban, A 22,000 dalton cAMP-dependent protein kinase substrate in platelets

In preparations of human platelet microsomes, cyclic AMP-dependent protein kinase induced the rapid phosphorylation of a single protein that was electrophoretically identical to the 22,000 dalton protein (P22) phosphorylated by cAMP in intact platelets. Phosphorylation of the microsomal protein was maximal at one minute and was followed by slow dephosphorylation. Although the protein was associated with a microsomal fraction, it could be separated from the membrane by 2 M NaCl indicating that it was a peripheral protein. Molecular weight was estimated by NaDodSO 4-PAGE and by gel filtration chromatography. The molecular weight estimated by NaDodSO 4-PAGE was 22,400 daltons and was somewhat larger than the 16,000 molecular weight estimated by gel filtration in the presence of NaDodSO 4. In the absence of NaDodSO 4, the protein chromatographed as a 36,000 dalton form. The presence of the 36,000 dalton form was not dependent on the phosphorylation state of the protein. The partially purified protein contained phosphoserine, but no phosphothreonine or phosphotyrosine. Two dimensional NaDodSO 4-PAGE and isoelectric focusing of the phosphorylated protein revealed isomers with pl values of 5.9 and 6.3. These studies indicate that the 22 kDa microsomal protein and P22 in intact platelets are the same protein and that the 22 kDa protein is tightly bound to the microsomal membrane although the nature of this binding and the microsomal component(s) to which it is bound remain to be determined. We conclude that the 22 kDa protein in platelet microsomes is structurally distinct from, but functionally similar to, phospholamban, the cAMP-dependent protein kinase substrate in muscle, and may play a similar role in calcium transport. Based on this similarity, it is proposed that the 22 kDa protein in platelets be called thrombolamban.

Vinca alkaloids inhibit conversion of arachidonic acid to thromboxane by human platelet microsomes: comparison with other microtubule-active drugs

The Vinca alkaloid vinblastine causes dose-dependent inhibition of malondialdehyde formation and aggregation in activated human platelets as a result of inhibition of arachidonic acid metabolism via the thromboxane pathway (Brammer, J.P., Kerecsen, L. and Maguire, M.H. (1982) Eur. J. Pharmacol. 81, 577). The nature of the inhibition by vinblastine has been investigated with human platelet microsomes, measuring conversion of arachidonic acid to malondialdehyde and thromboxane B 2 via spectrophotometric assay and RIA, respectively, determining arachidonate oxygenation by monitoring oxygen consumption, and identifying metabolites formed from [1- 14C]arachidonic acid. Vinblastine was compared with other Vinca alkaloids and with structurally unrelated microtubule-active drugs. Vinca alkaloids were unique in causing dose-dependent inhibition of both malondialdehyde and thromboxane B 2. Order of potency was vinblastine = vincristine = vindesine > leurosine > vinepidine. Inhibition of malondialdehyde and thromboxane B 2 by 50 μM vinblastine was at least 60%. Microsomal cyclooxy genese was not inhibited by 200 μm vinblastine. Inhibition by vinblastine of [1- 14C]arachidonic acid conversion to thromboxane B 2 was associated with a 4-fold increase in prostaglandin E 2 formation. Thromboxane B 2, but not malondialdehyde, formation was inhibited by colchicine < nocodazole ⪡ vinblastine. Results indicate that microsomal thromboxane synthetase is inhibited by Vinca alkaloids and other tubulin-binding drugs, and suggest that the action of vinblastine in inhibiting thromboxane synthesis, aggregation and release in intact platelets is not dependent upon its antimicrotubular actions.

The formation of lysophosphatidylinositol phosphate in human platelet microsomes.

The formation of lysophosphatidylinositol phosphate (lysoPIP) from lysophosphatidylinositol (lysoPI) via kinase activity was studied in microsomal preparations from human platelets. For this purpose, [3H]lysoPI or [3H]phosphatidylinositol ([3H]PI) was prepared and incubated in the presence or absence of ATP, MgCl2 and Triton X-100, and the appearances of radioactivity in [3H]lysoPIP and [3H]phosphatidylinositol phosphate ([3H]PIP), respectively, were monitored using thin layer chromatography. Both lysoPI and PI phosphorylations were completely dependent upon the presence of ATP and MgCl2 in the incubation medium; Triton X-100 addition stimulated both reactions, with the stimulation of PI conversion being considerably greater than that for lysoPI can be converted to lysoPIP by phosphorylation in human platelet microsomes. The potential significance of this enzymatic reaction in stimulated cells is discussed in relation to the generation of inositol-1,4,5-trisphosphate, an important intracellular second messenger.

Acetyltransferase activity in human platelet microsomes and washed platelets

It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collegen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [ 3H]acetyl-CoA to lyso-PAF (1-alkyl- sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionphore A-23187. This stimulation resulted in a 2–4 fold increase in acetyltransferase activity over unstimulated platelets. Acetyltranferase activity was also demonstrated by incubating [ 3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets.

Differential effects of diuretics on eicosanoid biosynthesis

The selective influences of hydrochlorothiazide (HCT), furosemide (FUR), spironolactone (SPI) and indapamide (IND) on arachidonic acid (AA) metabolism were assessed in sheep seminal vesicle and in human platelet microsomes. All four compounds augmented the synthesis of prostaglandins (PGs) D2 (HCT,FUR), E2 (SPI) and I2 (FUR,IND), probably through facilitated reorientation of endoperoxide biotransformation. With the exception of HCT, the other drugs also suppressed the production of thromboxane A2 (IND > SPI > FUR); lipoxygenase formation of hydroxyeicosatetraenoic acid was also enhanced by SPI and IND. The antihypertensive efficacy of these diuretics could, in part, be related to their selective effects on AA metabolism which favor the net formation of depressor PGs.

Stimulation of prostacyclin synthesis by nizofenone

The effect of nizofenone on prostacyclin synthesis was investigated using rat arterial walls. Incubation of arterial walls with [ 14C] arachidonic acid resulted in a time-dependent formation of prostacyclin, which was radiochromatographically detected as the stable breakdown product, 6-keto prostaglandin F 1α. The addition of nizofenone dose-dependently stimulated the prostacyclin formation, and significant increases of 47 and 106% were observed at 0.1 and 0.3 mM, respectively. No stimulation of prostaglandin E 2 and thromboxane A 2 synthesis was observed in the experiments with ram seminal vesicle microsomes and human platelet microsomes. These findings suggest that nizofenone has a selective stimulatory action on prostacyclin synthesis.

Efect of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine on calcium fluxes by human platelet microsomes

Under conditions where optimal concentrations of arachidonic acid, phosphatidic acid, or the calcium ionophore A23187 caused release of 50–95% of calcium from preloaded platelet microsomes, basophil platelet activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) did not cause the release of calcium at concentrations as high as 2 × 10 −5 M. The failure to stimulate calcium release was not due to metabolism or inactivation of AGEPC. These results show that AGEPC is not a calcium ionophore and is unable to directly effect the release of calcium from microsomes by mechanisms other than ionophoric action. The increase in intracellular levels that occurs during AGEPC-induced platelet aggregation must be an indirect effect of the AGEPC.

Nicotine inhibits vascular prostacyclin but not platelet thromboxane formation

1. 1. The conversion of [ 14C]arachidonic acid to [ 14C]thromboxane B 2 in rabbit or human platelet microsomes is unaffected by nicotine. 2. 2. Nicotine dose-relatedly inhibits the amount of prostacyclin-like activity formed in incubations of rabbit aortic rings or human veins. 3. 3. Cigarette smoking markedly inhibits prostaglandin dependent reactive hyperemia in the human forearm. 4. 4. It is concluded that nicotine acts on vascular prostacyclin but not on platelet thromboxane formation. Such a selective action by nicotine may facilitate platelet aggregability and counteract PG-dependent reactive hyperemia. These actions by nicotine may consitute a biochemical link between tobacco smoking and cardiovascular disease.