The coumarin derivative AD6 inhibits the release of arachidonic acid by interfering with phospholipase A2 activity in human platelets stimulated with thrombin

AD6 is a coumarin derivative which is able to inhibit platelet aggregation and release due to various agonists as adrenaline, PAF, Ca++ ionophore and others. It has been demonstrated that this compound reduces the production of free arachidonate and diglyceride from human platelets pulse-labeled with radioactive arachidonic acid thus suggesting a possible interference with the activity of phospholipase A2 and/or phospholipase C. The present report indicates that the drug has no effect on the increase of the labeling of phosphatidic acid which takes place when platelets pulse-labeled with arachidonic acid are stimulated with thrombin. Furthermore, AD6 is not able to cause changes on the metabolism of phosphoinositides monitored using platelets pre-labeled with [3H] inositol. These observations exclude the possibility that AD6 interferes with phospholipase C activity. Experiments with platelets pulse-labeled with arachidonate suggest that AD6 inhibits phospholipase(s) A2 activity or modulate negatively one or more processes involved in its activation.     

Verapamil inhibits phosphatidic acid formation and modifies phosphoinositide metabolism in stimulated platelets

Rabbit platelet-rich plasma was incubated with [32P]orthophosphate, after which the platelets were washed, further incubated in the absence or presence of verapamil and subsequently stimulated with PAF-acether or thrombin. In the absence of verapamil, a rapid increase in radioactivity in phosphatidic acid was observed in platelets stimulated with PAF-acether or thrombin. This was inhibited by verapamil over the concentration range 10(-7) to 10(-4) M, at which concentration the rise in phosphatidic acid was completely abolished. In unstimulated platelets, 10(-4) M verapamil induced an increase in radioactivity in polyphosphoinositides but not significantly in phosphatidylinositol. When these verapamil-treated platelets were stimulated with PAF-acether or thrombin, there was a rapid, sustained loss of the additional radioactivity induced in the polyphosphoinositides by verapamil. Polyphosphoinositide radioactivity remained unchanged in platelets stimulated in the absence of verapamil. Verapamil may stimulate formation of a separate pool of polyphosphoinositide which is susceptible to agonist-induced phospholipase C, and failure to re-synthesize this polyphosphoinositide could result from inhibition of phosphatidic acid synthesis.     

Stimulation of 12-HETE production in human platelets by an immunomodulator, LF 1695. Evidence for activation of arachidonate liberation coupled to cyclo-oxygenase inhibition

Upon incubation with human platelets previously labelled with [14C]arachidonic acid, a new immunomodulator, LF 1695, induced the accumulation of [14C]-12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). Although the time course of [14C]HETE accumulation was identical with 60 microM LF 1695 and calcium ionophore A23187, the latter compound also promoted the formation of 14C-labelled thromboxane B2 and 12-(S)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), whereas 12-HETE was the only arachidonic acid metabolite generated under the action of LF 1695, suggesting that the drug inhibited cyclo-oxygenase. This was further confirmed by the fact that LF 1695 inhibited the second wave of platelet aggregation induced by ADP as well as arachidonic acid effects. Cell lipid analysis revealed that arachidonic acid was liberated from both triacylglycerol and phosphatidylcholine. The effect was observed in the concentration range 15-90 microM, with a half-maximal effect at 30 microM for HETE production, 15 microM for triacylglycerol hydrolysis and 45 microM for phosphatidylcholine deacylation. Incubation of platelets with [14C]arachidonic acid in the presence of 60 microM LF 1695 resulted in a strong inhibition of arachidonic acid incorporation into the various cell lipids, indicating that arachidonic acid mobilization might be due to inhibition of reacylation processes. It is concluded that LF 1695 displays an original and complex effect on platelet lipid metabolism, resulting in the specific generation of lipoxygenase metabolites.     

Inhibitory effect of various traditional Chinese medicines on rabbit platelet phospholipase A2 in vitro and suppressive effect of t?ki-syakuyaku-san on increased aggregability in hypercholesterolemic rabbit ex vivo

The inhibitory mechanism of 6 traditional Chinese medicines on rabbit platelet aggregation in vitro, and the suppressive effect of oral administration of T?ki-syakuyakusan on hyper-aggregability of the platelet from rabbit fed high cholesterol diet for 2 months, were investigated. Collagen-induced aggregation was inhibited by Keisi-bukury?gan, Kami-sy?y?-san, Dai-saiko-t?, T?ki-syakuyaku-san, Hatimi-zi?-gan and Sy?-saiko-t? in their lower concentrations than those inhibiting arachidonic acid- and thrombin-induced aggregation. These traditional Chinese medicines inhibited the release of [3H]arachidonic acid from membrane phospholipids by phospholipase A2, in [3H]arachidonic acid-labelled platelets under stimulation with collagen and thrombin in the concentration ranges that inhibited each aggregation. In their higher concentrations to inhibit arachidonic acid-induced aggregation, they suppressed the conversion of arachidonic acid to thromboxane A2 by about 50%. However, they had no effect on diacylglycerol formation induced by thrombin. The oral administration of T?ki-syakuyaku-san depressed the increased aggregability of platelets from rabbit fed high cholesterol diet by 20-40% at the period of 1-2 months of feeding, without affecting plasma and platelet cholesterol level. These results indicate that the traditional Chinese medicines used here have an inhibitory effect on platelet phospholipase A2 activation, rather than on cyclooxygenase, and therefore inhibit platelet activation in vitro and ex vivo.     

Effect of a stimulant of guanylate cyclase, sin 1, on calcium movements and phospholipase C activation in thrombin-stimulated human platelets

The effects of sin 1, a metabolite of an antianginal agent, molsidomine, were investigated on human platelet activation induced by thrombin. This drug promoted a slight inhibition of serotonin release in a medium containing 1 mM Ca2+ or 1 mM EGTA (from 63% to 46% and from 57% to 41% of total serotonin secretion, respectively, with the highest dose used). Under these conditions, Ca2+ movements, monitored by quin 2 fluorescence, were markedly impaired. The most pronounced effect was towards Ca2+ influx, which presented a rapid inhibition with low doses. In the presence of external calcium, thrombin raised cytoplasmic free Ca2+ concentration from 100 nM to 1277 nM. This was reduced to 466 nM and 175 nM with 10(-7) M and 10(-4) M sin 1, respectively. Ca2+ mobilization from internal stores was less inhibited, since cytoplasmic free Ca2+ movements, sin 1 was tested on [32P] phosphatidic acid synthesis resulting from phospholipase C activation induced by thrombin. Phosphatidic acid labelling displayed a maximal inhibition of 43-50% with the highest doses of sin 1 (10(-4) M-10(-3) M) with or without Ca2+ in the incubation medium. However, this effect appeared much more sensitive to sin 1 in the presence of external Ca2+ (25% at 10(-7) M sin 1 with external Ca2+ against 12% at the same sin 1 concentration with EGTA). This discrepancy might be explained by the difference of cGMP level obtained when platelets were treated by sin 1 in the presence or in the absence of Ca2+ in the medium. This study shows that the major target of sin 1 via cGMP is not platelet phospholipase C as previously described, but inhibition of Ca2+ influx through plasma membrane.     

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.     

Effects of oral hypoglycaemic agents on platelet functions.

The in vitro effects of three oral hypoglycaemic agents, gliclazide (1-(4-methylbenzensulfonyl)-3-[3-azabicylo(3,3,0)octyl]urea) , glibenclamide (1-[4-[2-(chloro-2-methoxybenzamide)-ethyl]-phenyl- sulfonyl]-3-cyclohexyl-urea) and glimepiride (1-[4-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-carboxamide)- ethyl]-phenylsulphonyl]3-(4-methylcyclohexyl)-urea), on functions of human platelets were evaluated. None of these agents up to a concentration of 40 microM inhibited platelet aggregation induced by thrombin. Glibenclamide and glimepiride in the range of 20-40 microM suppressed Ca2+ release from internal Ca2+ stores induced by thrombin. Gliclazide showed no effect on arachidonic acid metabolism of human platelets. Glimepiride selectively inhibited the cyclooxygenase pathway, while the activities of 12-lipoxygenase and phospholipase A2 were unaffected. Glibenclamide inhibited both the cyclooxygenase and 12-lipoxygenase pathways. It also attenuated arachidonic acid release from phospholipase A2. Oral hypoglycaemic agents with inhibitory effects on arachidonic acid metabolism may prove useful for the treatment of diabetic patients with enhanced platelet functions.     

Effects of vinblastine on malondialdehyde formation, serotonin release and aggregation in human platelets

Effects of the microtubular agent vinblastine on human platelet malondialdehyde formation, [14C]serotonin release and aggregation were studied in suspensions of [14C]serotonin-labelled platelets. Vinblastine caused dose-dependent inhibition of malondialdehyde formation and aggregation in platelet suspensions stimulated with thrombin, ADP or palmitaldehyde acetal phosphatidic acid (PGAP). Malondialdehyde formation, aggregation and [14C]serotonin release caused by threshold doses of thrombin were reduced but not abolished by 100 muM vinblastine; 30-100 muM vinblastine abolished ADP- and PGAP-induced malondialdehyde formation and [14C]serotonin released and transformed ADP- and PGAP-induced irreversible aggregation to a diminished reversible response. Arachidonate conversion to malondialdehyde catalysed by human platelet microsomes was inhibited by vinblastine and the cyclooxygenase inhibitors indomethacin and aspirin, but not by salicylate. Vinblastine inhibited the microsome-catalysed formation of malondialdehyde from prostaglandin H2. It is concluded that vinblastine inhibits the thromboxane pathway of arachidonate metabolism in stimulated platelets, consequently inhibiting release and aggregation, and that this effect of vinblastine may be, at least in part, independent of its antimicrotubular actions.     

Increase of phosphatidylinositol arachidonic acid incorporation induced by mepacrine

In view of the fact that mepacrine (Mp) is usually used as an inhibitor of the endogenous phospholipase A2, and since this enzyme produces the release of arachidonic acid (AA) from membrane phospholipids, we studied the effect of different concentrations of Mp on the mobilization of [1-14C]AA in rat renomedullary phospholipids. During the acylation period, 0.1 mM Mp did not produce any significant change in the incorporation of [1-14C]AA into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and only a slight increase in phosphatidylinositol (PI). Higher concentrations of Mp (0.5 to 1.0 mM) produced a decrease of radioactivity in PE and PC with an increase in PI. Using prelabeled slices, a dose-dependent decrease in the 14C-radioactivity in PE and PC was observed, with a parallel increase in PI. This effect of Mp persisted even in the presence of a physiological activator of phospholipase A2, bradykinin (BK). No change in the net amount of phospholipids was observed at any of the Mp concentrations used. The results of this study show that Mp, at concentrations generally used to inhibit phospholipase A2, produced a transfer of arachidonic acid from PE and PC to PI, rather than a blockade in the release of AA from membrane phospholipids.     

Cationic amphiphilic drugs as a potential tool for modifying phospholipids of tumor cells. An in vitro study of chlorpromazine effects on Krebs II ascites cells

[³²P]orthophosphate and [U-¹⁴C]glycerol incorporation into Krebs ascites cell lipids was studied in vitro in the presence of chlorpromazine (CPZ)¹. At concentrations not exceeding 0.1 mM, the drug produced no cell damage within 3 hr incubation. Under these conditions, CPZ inhibited the incorporation of [³²P]orthophosphate into phosphatidylcholine and phosphatidylethanolamine and of [U-¹⁴C]glycerol into phosphatidylcholine and triglycerides, in a dose-dependent manner. On the other hand, synthesis of phosphatidic acid and phosphatidylglycerol was greatly enhanced, whereas phosphatidylinositol showed no major change. These results are compatible with an inhibition of phosphatidate phosphohydrolase, redirecting phospholipid biosynthesis towards the anionic classes. In vitro treatment of cells for 3 hr induced profound changes of phospholipid composition, which displayed a relative increase of phosphatidylglycerol and phosphatidic acid at the expense of phosphatidylcholine and phosphatidylethanolamine. The use of amphipathic cationic drugs can thus offer an interesting model for studying the relationship between cell proliferation and membrane phospholipid biosynthesis.     

Suppressive effect of biscoclaurine alkaloids on agonist-induced activation of phospholipase A2 in rabbit platelets

The effect of biscoclaurine (bisbenzylisoquinoline) alkaloids on phospholipase A2 and C activation in signal transduction system of rabbit platelet was studied. Isotetrandrine, cepharanthine and berbamine inhibited the aggregation induced by collagen but not by other stimuli such as thrombin and arachidonic acid, while tetrandrine equally inhibited the aggregation by any of these agonists. All these four alkaloids suppressed arachidonic acid liberation in response to collagen or thrombin, but not diacylglycerol formation and increase in cytoplasmic Ca2+ concentration in response to thrombin or arachidonic acid. In saponin-permeabilized platelets, they also suppressed arachidonic acid liberation induced by an addition of both GTP gamma S and Ca2+, whereas the liberation induced by an addition of Ca2+ alone was not prevented by them. These data suggest that isotetrandrine, cepharanthine and berbamine have a rather specific potency to suppress the phospholipase A2 activation by a mechanism other than direct inhibition of the enzyme or interference with the ligand-receptor interaction. They seem, at least in part, to exert the effect on the GTP-binding protein-phospholipase A2 complex in the platelet signal transduction system. In contrast, tetrandrine appears to inhibit a step following an increase in cytosolic free Ca2+ concentration in the agonist-induced signal transduction system, in addition to suppressing the phospholipase A2 activation.     

Identification of small peptide analogues having agonist and antagonist activity at the platelet thrombin receptor

Two tripeptide analogues (N-[3-methyl-1-S[[2-S [(methyl-amino)carbonyl]-1-pyrrolidinyl] carbonyl]butyl-D-analine) (SC40476) and N-[3-methyl-S-(1-pyrrolidinylcarbonyl)butyl]-D-alanine, ethyl ester, hydrochloride (SC42619], inhibit aggregation of, and secretion from, human platelets induced by thrombin but cause no significant inhibition of esterolysis or fibrin formation catalysed by this enzyme. Inhibition by SC40476 of the aggregatory response induced by thrombin is incomplete. Neither peptide analogue inhibits aggregation induced by ADP, collagen, vasopressin or 11,9-epoxymethanoprostaglandin H2 (U-46619). Enhancement of the response is observed when nonsaturating concentrations of these agonists are employed. SC42619 causes a parallel shift to the right in the concentration-response curve describing aggregation induced by thrombin. The Schild plot of these data has a slope of 1.05 and the pA2 is 2.9 +/- 0.1. Both SC40476 and SC42619 induced a small but significant decrease in the single platelet content of platelet suspensions. Neither peptide analogue increases platelet cytosolic [Ca2+] measured using quin 2 or Fura 2. Both analogues cause inhibition of the increase in cytosolic [Ca2+] induced by thrombin. Inhibition by SC42619 is competitive with respect to thrombin when the extracellular [Ca2+] is reduced to less than 0.1 microM but is non-competitive in the presence of 1 mM Ca2+. SC42619 also inhibits the increase in cytosolic [Ca2+]induced by ADP in the presence of 1 mM Ca2+ but not the smaller increase caused by this agonist when the medium contains less than 0.1 microM Ca2+. SC42619 inhibits Mn2+ influx induced by thrombin and ADP. SC40476 and SC42619 inhibit the enhanced incorporation of [32P] into phosphatidic acid observed on stimulation by thrombin of platelets pre-labelled with [32P]-phosphate. Addition of the peptide analogues alone fails to increase significantly the 32P content of phosphatidate, phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. SC40476 causes no detectable hydrolysis of glycoprotein V as detected by release of the proteolytic product (glycoprotein VFR). The results indicate that SC40476 and SC42619 interact selectively with the platelet thrombin receptor. Both peptide analogues act as effective antagonists for this receptor but also possess weak agonist activity which may also result from interaction with the thrombin receptor. The molecular basis for this latter activity has not been defined. SC42619 non-selectively inhibits Ca2+ influx induced by several agonists but this effect does not appear to contribute to the observed inhibition of the aggregatory and secretory responses.     

NMDA receptor activation stimulates phospholipase A2 and somatostatin release from rat cortical neurons in primary cultures.

We have recently shown that glutamate exerts a stimulatory action on somatostatin secretion in cortical neurons essentially through NMDA receptor sites. Here, we investigated whether arachidonic acid release could be modified after NMDA receptor activation in cortical neurons in primary culture. We also studied whether pharmacological manipulation of phospholipase A2 could modify somatostatin release. We found that both glutamate and NMDA (N-methyl-D-aspartate) stimulated [3H]arachidonic acid release. NMDA-evoked arachidonic acid release was inhibited by MK-801 and TCP (two NMDA receptor-type antagonists), or by mepacrine, an inhibitor of phospholipase A2. NMDA-induced somatostatin release was inhibited by MK-801, mepacrine and by another phospholipase A2 inhibitor, p-bromophenacylbromide (pBPB). However, responses to NMDA were unaffected by H7, NDGA (nordihydroguaiaretic acid), indomethacin or by RHC 80267 (inhibitors of protein kinase C, lipooxygenase, cyclooxygenase and diacylglycerol lipase, respectively). Mepacrine (greater than or equal to 100 microM) decreased NMDA-stimulated phosphatidylinositol (PI) hydrolysis and at higher concentrations (250 microM) was also able to inhibit basal release whereas pBPB had no effect in the range of concentrations tested. Neomycin (which inhibits phosphatidylinositol metabolism by binding strongly and selectively to inositol phospholipids) reduced by 30% the NMDA-stimulated somatostatin release, although chronic treatment of neurons with the phorbol ester 12-myristate, 13-acetate (PMA) had no effect on this response. Melittin, an activator of phospholipase A2, was able to stimulate both arachidonic acid release and somatostatin secretion. High-performance liquid chromatography (HPLC) analysis of tritiated metabolites released from cortical neurons under basal or NMDA-stimulated conditions revealed that [3H]arachidonic acid was the only metabolite detectable. Furthermore, external addition of arachidonic acid increased somatostatin secretion. Our results show a correlation between the two parameters studied.     

Neuronal "nucleotide" receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP.

We have investigated the characteristics of the receptor for ATP on neuronal cells and the involvement of phospholipase C and phospholipase D in the effector mechanisms, using PC12 rat phaeochromocytoma cells in culture. We show that the cells respond, with generation of total inositol phosphates, to ATP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) but not to 2-methylthioadenosine5'-triphosphate (2MeSATP), beta,gamma-methylene ATP, or adenosine 5'-O-(2-thiodiphosphate) (ADP beta S). The largest response to ATP gamma S was mainly independent of extracellular calcium, had an EC50 of 7.93 +/- 0.76 microM, and was competitively inhibited by the nonspecific antagonist suramin. The pyrimidine nucleotide UTP also elicited a response in these cells. Measurement of [3H]inositol triphosphate showed a rapid rise to maximum (10-15 sec) in response to both ATP gamma S and UTP but no response to 2MeSATP. Cells prelabeled with 32Pi and stimulated in the presence of 50 mM butanol responded to ATP gamma S, ATP, and UTP with enhanced formation of [32P]phosphatidylbutanol as well as [32P]phosphatidic acid, indicating that agonist-stimulated phosphatidic acid occurs by both phospholipase D and phospholipase C activity. The stimulation of phospholipase D was inhibited by the presence of a protein kinase C inhibitor, Ro 31-8220. The dose-response curve for the stimulation by ATP gamma S of phospholipase C was shifted to the right by the presence of UTP, indicating that both compounds act on the same receptors. The data provide the first evidence for the existence of a nucleotide receptor on neuronal cells (insensitive to both purines and pyrimidines) and show that this receptor is linked to both phospholipase C and phospholipase D.     

Lipid peroxidation, phosphoinositide turnover and protein kinase C activation in human platelets treated with anthracyclines and their complexes with Fe(III).

The effects of the antitumor drugs daunorubicin, doxorubicin and their complexes with Fe(III) on phosphoinositide hydrolysis, lipid peroxidation and protein kinase C (PKC) activation were measured in intact human platelets. Doxorubicin and the Fe(III) complexes of both doxorubicin and daunorubicin quickly induced lipid peroxidation [as measured by the thiobarbituric acid (TBA) assay], phosphorylation of the 40 K substance of PKC, and increased levels of phosphatidic acid and inositol phosphates. Fe(III) alone or complexed to acetohydroxamic acid induced high levels of TBA-reactive material but did not affect either PKC activation or phosphoinositide turnover. In contrast, daunorubicin, which was ineffective per se, inhibited all these doxorubicin- and anthracyclines/Fe(III)-induced biochemical events. We suggest that phosphoinositide hydrolysis determined by anthracyclines, and consequently PKC activation, could be due to lipid peroxidation, thus triggering the activity of phospholipase C.     

Multiple effects of a new anti-inflammatory agent, timegadine, on arachidonic acid release and metabolism in neutrophils and platelets

Casein-elicited rat peritoneal polymorphonuclear leukocytes (PMNL) and rabbit platelets were prelabelled with [1-14C]arachidonic acid, and the effect of timegadine, a new anti-inflammatory agent, on the release and metabolism of arachidonic acid induced by A23187 (PMNL) and thrombin (platelets) was studied and compared with the effect of other compounds reported to affect these enzymatic mechanisms. Timegadine inhibited arachidonic acid release from both cells (IC50 = 2.7 X 10(-5) M), the lipoxygenase activity in PMNL (IC50 = 4.1 X 10(-5) M) and the cyclooxygenase activity in platelets (IC50 = 3.1 X 10(-8) M). By these mechanisms, PMNL leukotriene B4 formation was inhibited by 50% at 2.0 X 10(-5) M, platelet thromboxane B2 at 3.2 X 10(-8) M, and platelet 12-HETE at 4.9 X 10(-5) M. These effects might add to the understanding of the anti-inflammatory properties of timegadine.     

Triphenyltin fluoride in vitro inhibition of rabbit platelet collagen-induced aggregation and ATP secretion and blockade of arachidonic acid mobilization from membrane phospholipids

Recent studies have demonstrated that triphenyltin fluoride (TPTF) inhibits collagen-induced aggregation and ATP secretion of rabbit platelets in vivo [S. Manabe and O. Wada, J. Toxic. Sci. 6, 236 (1981)]. The aim of the present investigation was to test the effects in vitro of TPTF on platelet aggregation and to elucidate the mechanism of the inhibitory action by studying the release and metabolism of arachidonic acid and the cyclic AMP contents of rabbit platelets treated in vitro with TPTF. Although no inhibitory effect of TPTF was found on sodium arachidonate-induced platelet aggregation and ATP secretion, TPTF inhibited both reactions induced by collagen. Triphenylarsine and triphenylantimony did not inhibit, even at a concentration of 10(-3) M. The anti-aggregating concentration (IC50) of TPTF was 6.0 x 10(-6) M against collagen. TPTF had no inhibitory effect on the conversion of exogenous arachidonic acid to malondialdehyde (MDA) by platelets, while the collagen-induced production of arachidonate metabolites [MDA, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2] was remarkably inhibited by TPTF. Furthermore, TPTF apparently inhibited the collagen-induced release of arachidonic acid from platelets, although the formation of phosphatidic acid was not inhibited. Total cyclic AMP content after TPTF exposure was not changed significantly. These results indicate that TPTF inhibited the collagen-induced arachidonic acid release from platelet phospholipids, presumably by acting on phospholipase A2. Furthermore, it seems unlikely that the inhibition of arachidonic acid release by TPTF can be explained by the level of cyclic AMP in platelets.     

Dantrolene and mepacrine antagonize the hemolysis of human red blood cells by halothane and bee venom phospholipase A2

Dantrolene is an effective antagonist of anesthesia-induced malignant hyperthermia due to a poorly understood action on skeletal muscle. The present study examines whether the red blood cell can be used as a model to investigate the mechanism of dantrolene action. Halothane (4.7 mM) caused 9% hemolysis of red blood cells. Phospholipase A2 (1 microM) alone caused less than 2% hemolysis, despite high levels (54%) of phosphatidylcholine hydrolysis. Incubation of red blood cells with halothane and phospholipase A2 caused 72% hemolysis. Halothane addition caused 100% hydrolysis of all diacylphosphoglycerides by phospholipase A2, suggesting a mutual potentiation. The major products of phospholipase A2 activity, arachidonic acid and lysophosphatidylcholine, when exogenously added, also greatly increased hemolysis induced by halothane, with arachidonic acid most closely resembling the synergism observed with phospholipase A2. Dantrolene (10 microM) and mepacrine (10 microM) significantly antagonized hemolysis induced by halothane and phospholipase A2 or halothane and exogenously added arachidonic acid and lysophosphatidylcholine. Dantrolene and mepacrine did not antagonize phospholipid hydrolysis or free fatty acid levels. Dantrolene and mepacrine antagonized the synergism between halothane and phospholipase A2 most likely by reducing the lytic action of halothane in the presence of arachidonic acid. The red blood cell is a useful model for studying the antagonism of halothane and phospholipase A2 toxicity by dantrolene and mepacrine.     

Organization and role of platelet membrane phospholipids as studied with phospholipases A2 from various venoms and phospholipases C from bacterial origin

Phospholipases A₂ from various snake or bee venoms and phospholipases C secreted as exotoxins by several bacteria have been used to study the transverse distribution of phospholipids in the platelet plasma membrane and their role in platelet activation. An asymmetric distribution was described for phospholipids, characterized by a preferential localization of sphingomyelin and phosphatidylcholine in plasma membrane outer leaflet, whereas the inner half contains almost all of the anionic procoagulant phosphatidylserine and phosphatidylinositol. Such a distribution might explain the latency of procoagulant activity in resting platelets and implies an intracellular localization of arachidonic acid, the precursor of prostaglandins and thromboxanes. The external arachidonic acid is involved in phospholipase A₂-induced aggregation, whereas phospholipase C from Clostridium welchii stimulates platelets through a thromboxane-independent pathway. The latter one is directly linked to the formation of phosphatidic and lysophosphatidic acids, which are able to activate cells through calcium mobilization. So, phospholipase C represents an interesting tool for studying the biochemical processes accompanying stimulation, since it is shown that it mimicks the effects of an intracellular phospholipase C, the role of which in platelet activation is discussed.     

Inhibition of collagen-induced platelet activation by arachidonyl trifluoromethyl ketone

Collagen-induced platelet activation is associated with, and markedly potentiated by, the release of arachidonic acid and its subsequent conversion to thromboxane A₂. The precise mechanism of arachidonic acid release is unknown. An inhibitor of isolated cytosolic phospholipase A₂ (cPLA₂), arachidonyl trifluoromethyl ketone (AACOCF₃), was used to examine the role that cPLA₂ plays in this process. AACOCF₃ inhibited platelet aggregation in response to collagen and arachidonic acid but not to thrombin, calcium ionophore, phorbol ester, or a thromboxane mimetic. Thromboxane formation stimulated by thrombin or collagen was inhibited by AACOCF₃. However, AACOCF₃ did not inhibit collagen-induced [¹⁴C]arachidonic acid release. These data are consistent with the inhibitory effects of AACOCF₃ on collagen-induced aggregation involving an action on the conversion of arachidonic acid to thromboxane.