Human platelet phospholipase A2 activity is responsive in vitro to pH and Ca2+ variations which parallel those occurring after platelet activation in vivo (original) (raw)
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Journal of Biological Chemistry
The ability of epinephrine or ADP to cause an increase in the production of phospholipase C products (diacylglycerol and inositol phosphates) in human platelets is blocked by perturbants of Na+/H+ exchange, i.e. ethylisopropylamiloride, decreased extraplatelet pH, or removal of extraplatelet Na+. These perturbants do not, however, block inositol phosphate production in response to 0.2 unit/ml thrombin, indicating that inhibition of Na+/H+ exchange does not inhibit the phospholipase C enzyme directly. Since the cyclooxygenase inhibitor indomethacin and the endoperoxide/ thromboxane antagonist 8629548 block epinephrineand ADP-induced inositol phosphate production, it can be concluded that these agonists activate phospholipase C secondary to mobilization of arachidonic acid and production of cyclooxygenase products. This conclu-*ion is consistent with the observation that the endoperoxide analogue U46619 causes inositol phosphate production. Furthermore, the effect of U46619 is not blocked by inhibitors of Na+/H+ exchange. The initial pool of arachidonic acid mobilized by epinephrine can be measured using negative ion gas chromatography/ mass spectrometry and is sensitive to inhibition of Na+/ H+ exchange.
The Journal of biological chemistry, 1984
The present study compares the molecular mechanism by which thrombin, platelet-activating factor, and epinephrine induce platelet activation. Thrombin and platelet-activating factor induce an initial activation of phospholipase C, as measured by formation of 1,2-diacylglycerol and phosphatidic acid, during platelet shape change which is independent of and dissociated from metabolism of arachidonic acid. Phospholipase C activation and shape change are independent of extracellular Ca2+ and Mg2+. Formation of cyclooxygenase products occurs subsequent to the initial activation of phospholipase C and those metabolites are associated with platelet aggregation and further activation of phospholipase C. On the other hand, epinephrine is an unique platelet stimulus since it requires extracellular divalent cations and does not induce platelet shape change or activation of phospholipase C. Our results indicate that activation of phospholipase C may be a mechanism by which physiological agonist...
Purification and Characterization of Ca2+-Dependent Phospholipases A2from Rat Kidney
Archives of Biochemistry and Biophysics, 1996
Phospholipase A 2 (phosphatide 2-acyl hydrolase, EC 3.1.1.4.) (PLA 2) 5 specifically hydrolyzes the fatty acyl Three phospholipase A 2 (PLA 2) activities were ester bonds at the sn-2 position of sn-3 phosphoglyceridentified in rat kidney. In the particulate fraction a ides. Ca 2/-dependent PLA 2 is a heterogeneous family PLA 2 activity was present which was cross-reactive with polyclonal antibodies against the 14-kDa group of enzymes that is widely distributed in nature (1). II PLA 2. This PLA 2 was partially solubilized and puri-High concentrations of this enzyme are found in panfied to near homogeneity. The amino acid sequence creatic juice and in the venoms of snakes and bees (2). at the N-terminus of the purified enzyme was identi-Additional PLA 2 activities are found in trace amounts cal to that of the 14-kDa rat group II PLA 2 from rat in almost every cell type and with a diverse subcellular liver mitochondria, platelet, and spleen. The cytolocalization, including mitochondria, Golgi memsolic fraction of rat kidney contained at least two branes, plasma membranes, secretory granules, and PLA 2 activities which could be separated on a Mono cytosol (3). These intracellular PLA 2 s are involved in Q column. Upon gel filtration the activity that eluted the turnover of phospholipids and in the deacylationfrom the anion-exchange column in the salt gradient reacylation cycle, participating in the biosynthesis of behaved as a high molecular mass PLA 2 , exhibited a phospholipids with a specific acyl chain composition. preference for arachidonic acid at the sn-2 position Moreover these phospholipases can liberate arachiof glycerophospholipids, and was already optimally donic acid from the sn-2 position of glycerophospholipactive at submillimolar Ca 2/ concentrations. The cyids. This generation of free arachidonic acid is thought tosolic PLA 2 activity that did not bind to the anionto be the rate-limiting step in the biosynthesis of a exchange column was purified by gel filtration, imvariety of biologically active lipids like prostaglandins, munoaffinity chromatography using immobilized leukotrienes, thromboxane, and lipoxins (3-5). polyclonal antibodies to group I PLA 2 , and C18 re-Based on the molecular mass, phospholipases A 2 can versed-phase chromatography. Immunological propbe divided into two classes: the low molecular mass erties and N-terminal sequence analysis identified enzyme (14 kDa) (3) and the high molecular mass enthis enzyme as rat group I PLA 2. Rat glomerular meszyme (85 kDa) or cytosolic phospholipase A 2 (cPLA 2) angial cells contained only group II and high molecu-(6-8). The low molecular mass enzymes can be divided lar mass PLA 2 enzymes. ᭧ 1996 Academic Press, Inc. into group I (pancreatic-type) and group II (non-pancre-Key Words: calcium; phospholipase A 2 ; rat; kidney. atic-type) phospholipases A 2 (9) and both enzymes require millimolar concentrations of Ca 2/ in the catalytic step (1, 10). On the other hand, cPLA 2 needs micromolar concentrations of the divalent cation for its translo
Hypersensitivity of phospholipase C in platelets of spontaneously hypertensive rats
Hypertension, 1987
Thrombin-induced aggregation and serotonin release were markedly enhanced in platelets from spontaneously hypertensive rats (SHR) when compared with those from normotensive Wistar-Kyoto rats (WKY). Since phosphoinositides are involved in calcium-mediated platelet responses, the metabolism of these lipids was investigated in SHR and WKY by using 32P-labeled quiescent platelets. In unstimulated cells, both the rate and extent of 32P incorporation into individual inositol-containing phospholipids and phosphatidic acid were identical in SHR and WKY. This finding suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced phosphoinositide metabolism, when monitored as changes in [32P]phosphatidic acid, was significantly higher in SHR than in WKY. For example, a 20-second exposure to thrombin, 0.3 U/ml, induced the formation of 1.6 times more [32P]phosphatidic acid in SHR than in WKY. These results provide evidence for a leftward shift of the dose-response and time-course curves of thrombin-induced [32P]phosphatidic acid formation in SHR. Moreover, the extent of the difference between SHR and WKY was independent of the extracellular calcium concentration. Following thrombin stimulation, [32P]phosphatidic acid formation likely reflects the initial agonist-receptor interaction; therefore, these results suggest that phospholipase C activity is enhanced in platelets of SHR and that the hypersensitivity of phospholipase C in SHR may play a role in the overall alteration of cell calcium handling and, hence, in the platelet responses of SHR.
Effect of ethanol on platelet phospholipase A2
Lipids, 1992
Platelet aggregation is known to be inhibited by ethanol, and this has been suggested to be one of the attenuating effects of ethanol in cardiovascular disease. Recent studies have implicated an inhibition of phospholipase A2 induced arachidonic acid release, since the production of prostanoids that are formed from arachidonic acid and are involved in the aggregation process has been shown to be diminished by ethanol. Phospholipase A2 is found in platelets in both a cytosolic form, from where it may translocate to the plasma membrane to release arachidonic acid, and in a secretory form which is released extracellularly upon activation. In the present study, the effect of ethanol on the secretion of phospholipase A2 and on its activity was determined. It was found that ethanol inhibited phospholipase As secretion but not its activity. By contrast, the activity of the cytosolic form of phospholipase A2 was inhibited by ethanol. Lipids 27, 255-260 (1992).
A radioenzymatic assay to identify three groups of phospholipase A2 in platelets
Prostaglandins, Leukotrienes and Essential Fatty Acids, 2012
Phospholipases A 2 (PLA 2 ) are key enzymes in membrane metabolism. The release of fatty acids and lysophospholipids by PLA 2 activates several intra-cellular second messenger cascades that regulate a wide variety of physiological responses. The aim of the present study is to describe a radioenzymatic assay to determine the activity of three main PLA 2 subtypes in platelets, namely extracellular calciumdependent PLA 2 (sPLA 2 ) and intracellular calcium-dependent (cPLA 2 ) and calcium-independent PLA 2 (iPLA 2 ). The differentiation of these distinct PLA 2 subtypes was based on the enzyme substrate preference (arachdonic acid or palmitoyl acid) and calcium concentration. Our results indicate that this new assay is feasible, precise and specific to measure the activity of the aforementioned subtypes of PLA 2 . Therefore, this protocol can be used to investigate modifications of PLA 2 homeostasis in distinct biological models addressing the pathophysiology of many medical and neuropsychiatric disorders such as schizophrenia and Alzheimer's disease.