Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets (original) (raw)
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The Journal of biological chemistry, 1983
Human platelets prelabeled with (32P)orthophosphate or [14C]arachidonic acid (AA) were stimulated with collagen or thrombin, and platelet activation (shape change, aggregation, and release of serotonin) was determined in parallel to the formation of 32P- or 14C-labeled phosphatidic acid (PA). The results show a close correlation between the degree of platelet activation and the amount of PA formed. Activation of platelets and formation of PA induced by collagen (2 to 20 micrograms/ml) was blocked by pretreatment of platelets with trifluoperazine, indomethacin, aspirin, or N-methylimidazole. This suggests that the formation of AA by phospholipase A2 and its subsequent metabolism by cyclooxygenase and thromboxane synthetase are required for the collagen-induced formation of PA. Endoperoxide analog U-44069 induces formation of PA in human platelets that have been pretreated with or without aspirin. The action of thrombin does not follow the same pattern of collagen. Low concentrations ...
Thrombosis and haemostasis, 2000
Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2- and OH(0), respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2- and OH was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2- and OH(0) formation. To further analyze this relationship, O2- and OH(0) formation was measured during AA-stimulated platelet aggregation (PA); we observed that O2- and OH(0) release were dependent upon AA concentration. Furthermore, we found that the incubation of...
Journal of Lipid Research
Lipid hydroperoxides have been reported to regulate cell function and eicosanoid formation. The aim of the present study was to determine the effect of 12(S)-hydroperoxyeicosatetraenoic acid [12(S)-HPETE], the platelet 12-lipoxygenasederived hydroperoxide of arachidonic acid (AA), on the availability of nonesterified AA, which represents a rate-limiting step in the biosynthesis of eicosanoids. The coincubation of human platelets with concentrations of 12(S)-HPETE below 50 nM and subthreshold concentrations (STC) of collagen (less than 0.24 g/ml) significantly enhanced platelet aggregation and the formation of thromboxane B 2 , the stable catabolite of the potent aggregating agent thromboxane A 2. In addition, the nonesterified endogenous AA concentration increased by 3-fold. Arachidonoylcontaining molecular species concentrations of 1,2-diacyl-glycero-3-phosphocholine, 1-alkyl-2-acyl-glycero-3-phosphocholine, and 1-alkenyl-2-acyl-glycero-3-phosphoethanolamine decreased specifically in response to 12(S)-HPETE, whereas no significant changes were observed within 1,2-diacyl-glycero-3-phosphoethanolamine and 1,2-diacyl-glycero-3-phosphoinositol molecular species. The 12(S)-HPETE-induced increase in nonesterified AA was fully prevented by arachidonoyl trifluoromethyl ketone, an inhibitor of cytosolic phospholipase A 2 (cPLA 2), and cPLA 2 was translocated to membranes and phosphorylated in platelets incubated with 12(S)-HPETE. In conclusion, these results indicate that nanomolar concentrations of 12(S)-HPETE could play a significant role in controlling the level of endogenous AA and the formation of thromboxane, thereby potentiating platelet function.-Calzada, C., E. Véricel, B. Mitel, L. Coulon, and M. Lagarde. 12(S)-Hydroperoxy-eicosatetraenoic acid increases arachidonic acid availability in collagen-primed platelets.
Agricultural and Biological Chemistry, 1986
Monodansylcadaverine (250jum), an inhibitor of transglutaminase, completely inhibited aggregation of human platelets induced by 2^ig/ml of collagen. Platelet responses necessary for aggregation (shape change, serotonin release, and phosphorylation of the 47,000-Da protein) were also inhibited by this reagent. However, incorporation of [3H]putrescine into platelet proteins was very low and independent of the stimulation by collagen. It seems unlikely, therefore, that the transglutaminase reaction participates in platelet aggregation. Monodansylcadaverine inhibited the formation of thromboxane A2 with a similar median inhibitory concentration (about 120 /im) for aggregation. Whenplatelets prelabeled with [14C]arachidonic acid were stimulated by collagen, the radioactivities in thromboxane B2, hydroxyheptadecatrienoic acid, and hydroxyeicosatetraenoic acid increased. The increase was completely suppressed by the addition of monodansylcadaverine. Radioactivity in free arachidonic acid was very low throughout the experiments. Fromthese results, i t was concluded that monodansylca'daverine inhibited platelet aggregation by suppressing the release of arachidonic acid from phospholipids during platelet activation by collagen.
Role of arachidonic acid metabolism in human platelet activation and irreversible aggregation
American Journal of Hematology, 1985
Previous studies from our laboratory have demonstrated that the aggregation response of platelets inhibited by agents blocking cyclooxygenase activity could be restored to a normal state of sensitivity by prior stimulation of a-adrenergic receptors. Since cyclooxygenase activity and thromboxane synthesis are not absolutely required for irreversible platelet aggregation, it is important to define precisely what role this pathway serves in platelet physiology. The present study has evaluated the influence of agents that selectively block arachidonic acid conversion at different steps of synthesis. Inhibition of peroxidase, cyclooxygenase, lipoxygenase, and thromboxane synthetase blocked the second wave response of platelets to several agonists, but did not cause dissociation of aggregates preformed by prior exposure to arachidonate (AA) or adenosine diphosphate. Phospholipase (A*/C) inhibitors, similar to prostaglandin inhibitors, blocked the second wave response of platelets to the action of agonists and, in addition, caused dissociation of aggregates induced by aggregating agents. Results of our study demonstrate that when single agonists are tested at threshold concentrations, products of arachidonate metabolism may play a role in the activation process. However, continued generation of these metabolites does not appear to be essential for the maintenance of irreversible aggregation. When a combination of agents or high concentration of physiological agonists are used, both activation and irreversible aggregation can be secured independent of prostaglandin synthesis or the release reaction.
The Journal of biological chemistry, 1983
Exogenous unlabeled arachidonic acid (AA) added to human platelets prelabeled with [3H]AA induces breakdown of [3H]phosphatidylinositol and the rapid and transient formation of [3H]1,2-diacylglycerol and [3H]phosphatidic acid (PA), indicating activation of phosphatidylinositol-specific phospholipase C. Formation of [3H]1,2-diacylglycerol and [3H]PA is inhibited by pretreatment of platelets with aspirin, which suggests that endoperoxides or thromboxane A2 are responsible for AA-induced stimulation of phospholipase C. Exogenous unlabeled AA also induces the formation of [32P]PA or [14C]PA in platelets that have been prelabeled with 32Pi or [14C]AA, respectively. Increased radioactivity in PA reflects increased content of PA as measured by the fatty acid composition of PA. The relation of PA production, which reflects stimulation of phospholipase C, to specific platelet responses was further investigated. Low concentrations of AA (0.05-0.2 microM) induces platelet shape change in paral...
Thrombosis Research, 1985
Platelet reduced glutathione ( GSH ) is completely depleted by l-chloro-2,4_dinitrobenzene ( CDNB ), which is a substrate for GSH-S-transferase. GSH-depleted platelets: a) aggregate normally at high inducer concentration; b) respond with increased ( after arachidonic acid ) or depressed ( after collagen ) aggregability at low inducer concentration; c) show almost no arachidonic acid-induced stimulation of the hexose monophosphate shunt; d) are sensitized to oxidant agents such as diamide, which elicits a faster cytoskeletal protein oxidative polymerization and reversible aggregation. Our results suggest that GSH acts as a reducing cofactor and/or free radical scavenger in the PG-hydroperoxidase step of the cyclooxygenase pathway; moreover, GSH protects membrane and cytoskeletal protein -SH groups from oxidation.
Journal of Clinical Investigation, 1973
A B ST R A CT In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This conce-ntration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the al(I) and a2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 AM and 55 AM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 AM al(I) and 160 AM a2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1985
Three icosaenoic acids (20: 3(n-6), 20: 5(n-3) and 20: 3(n-9)) which may arise in platelet phospholipids under certain dietary conditions and which may affect platelet functions have been taken up by human platelets. Each acid was pre-coated onto delipidated albumin and then incubated with platelets isolated from their plasma. The distribution study of each acid in cellular lipids revealed that around 80% of the acid taken up was located in phospholipids, of which the bulk was in phosphatidylcholine. The percentage incorporation of each acid into the different glycerophospholipids was similar to their endogenous percentage profiles, therefore simulating the in vivo situation. The icosaenoic acids then incorporated were liberated from phospholipids when platelets were incubated with thrombin or calcium ionophore A23187 and subsequently oxygenated through the cyclooxygenase and/or lipoxygenase pathway. Whereas 20: 3(n-6) was readily converted into cyclooxygenase products, 20: 5(n-3) was more specifically converted into lipoxygenase products, and this latter conversion was comparable to that of 20: 3(n-9) which is not a prostanoid precursor. Finally, only 20:3(n-6)-or 20:5(n-3)-rich platelets exhibited a reduced availability of endogenous arachidonic acid from phospholipids when induced by thrombin. It is concluded that inhibitory polyunsaturated fatty acids (20: 3(n-6) and 20: S(n-3)) could act both by reducing prostaglandin H,/thromboxane A, production from endogenous arachidonic acid and in generating platelet inhibitory substances (cyclooxygenase and/or lipoxygenase products of 20: 3(n-6) and 20: 5(n-3)). On the other hand, 20:3(n-9), a fatty acid which potentiates platelet aggregation through its lipoxygenase end product, could produce sufficient amounts of this compound to enhance the aggregation when platelets are triggered with inducers of phospholipase activity such as thrombin or calcium ionophore.
Biochimica et biophysica acta, 1985
The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1...