Initiation of phospholipase A2 activity in human platelets by the calcium ion ionophore A23187

Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.

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DIRECT STIMULATIONOF CA2+-ACTIVATED HUMAN PLATELET PHOSPHOLIPASE A2 BY DIACYLGLYCEROL

10.1055/s-0038-1644673 ◽

1987 ◽

Author(s):

D Deykin ◽

R M Karmer

Keyword(s):

Enzymatic Activity ◽

Phospholipase A2 ◽

Conformational Changes ◽

Cellular Membrane ◽

Human Platelets ◽

Stable Form ◽

Membrane Phospholipids ◽

Kinase C ◽

Snake Venom Phospholipase ◽

Phospholipase A2 Activity

These studies examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of octylglucoside and its enzymatic activity determined using sonicated arachidonoyl phosphatidylcholine (PC) as substrate. (Kramer RM, et al: BBA 878:394, 1986) Phospholipase A2 activity was increased when dioleoylglycerc_ was incorporated into the substrate arachidonoyl-PC. In the presence of 1 uM (29 mol %) sn-1,2-dioleoylglycerol the enzymatic activity was stimulated 4.1-fold. Exogenously added sn-l-oleoyl-2-acetoylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 uM. Comparative studies conducted with pancreatic, bee-venom and snake venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but the stimulation was less than observed for platelet phospholipase A2. We conclude that in stimulated platelets Ca2+-activated phospholipase A2 may be regulated by newly generated diacylglycerols, not only via protein kinase C-mediated events, but also directly through conformational changes imposed by the diglycerides on cellular membrane phospholipids.

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

Agents and Actions ◽

10.1007/bf01966469 ◽

1990 ◽

Vol 29 (3-4) ◽

pp. 364-373 ◽

Cited By ~ 19

Author(s):

S. Porcellati ◽

V. Costantini ◽

M. Prosdocimi ◽

M. Stasi ◽

R. Pistolesi ◽

...

Keyword(s):

Arachidonic Acid ◽

Phospholipase A2 ◽

Coumarin Derivative ◽

Human Platelets ◽

Phospholipase A2 Activity

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Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport

Biochemical Journal ◽

10.1042/bj1840651 ◽

1979 ◽

Vol 184 (3) ◽

pp. 651-661 ◽

Cited By ~ 56

Author(s):

J E B Fox ◽

A K Say ◽

R J Haslam

Keyword(s):

Calcium Ion ◽

Prostaglandin E1 ◽

Plasma Membranes ◽

Human Platelets ◽

Particulate Fraction ◽

Ionophore A23187 ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Calcium Ion Transport

Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.

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Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet .beta.-cells

Biochemistry ◽

10.1021/bi00052a041 ◽

1993 ◽

Vol 32 (1) ◽

pp. 327-336 ◽

Cited By ~ 89

Author(s):

Richard W. Gross ◽

Sasanka Ramanadham ◽

Kelly K. Kruszka ◽

Xianlin Han ◽

John Turk

Keyword(s):

Phospholipase A2 ◽

Beta Cells ◽

Adenosine Triphosphate ◽

Pancreatic Islet ◽

Calcium Ion ◽

Islet Cells ◽

Pancreatic Islet Cells ◽

Islet Beta Cells ◽

Hydrolysis Of ◽

Phospholipase A2 Activity

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Diradylglycerols stimulate phospholipase A2 and subsequent exocytosis in ram spermatozoa. Evidence that the effect is not mediated via protein kinase C

Biochemical Journal ◽

10.1042/bj2970225 ◽

1994 ◽

Vol 297 (1) ◽

pp. 225-232 ◽

Cited By ~ 28

Author(s):

E R S Roldan ◽

C Fragio

Keyword(s):

Arachidonic Acid ◽

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase A2 ◽

Kinase Inhibitor ◽

Ionophore A23187 ◽

Kinase C ◽

Acrosomal Exocytosis ◽

Phospholipase A2 Activity

We tested the hypothesis that the role of diacylglycerol (DAG) in sperm acrosomal exocytosis is related to the activation of phospholipase A2, and that this effect is not mediated via protein kinase C. Treatment of [14C]arachidonic acid-labelled ram spermatozoa with Ca2+ and the ionophore A23187 stimulated both liberation of arachidonic acid and acrosomal exocytosis. No changes in [14C]DAG or [14C]monoacylglycerol were found after stimulation of spermatozoa, thus suggesting that arachidonic acid may be released exclusively via phospholipase A2. An increase in the endogenous levels of diradylglycerols (DRGs), resulting from exposure either to the DAG kinase inhibitor R 59022 or to exogenous 1-oleoyl-2-acetyl-sn-glycerol or 1,2-dioctanoyl-sn-glycerol, led to an increase in both phospholipase A2 activity and exocytosis when cells were stimulated with A23187 and Ca2+. Addition of DRGs that do not stimulate protein kinase C(1,3-dioctanoylglycerol, 1-O-hexadecyl-2-acetyl-rac-glycerol) also resulted in an increase in phospholipase A2 activity and exocytosis. On the other hand, phorbol esters (phorbol 12,13-dibutyrate; phorbol 12-myristate 13-acetate) did not enhance enzyme activity or exocytosis. Finally, exposure to 1-O-hexadecyl-2-O-methyl-rac-glycerol, a compound known to inhibit protein kinase C, did not affect phospholipase A2 activity or acrosomal exocytosis. We therefore conclude that in spermatozoa the messenger role of DAG is related to the activation of phospholipase A2, which in turn would generate an array of metabolites directly or indirectly involved in bringing about exocytosis of the acrosome.

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