RELEASE AND TRANSACYLATION OF ARACHIDONATE FROM A COMMON POOL OF 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE IN HUMAN PLATELETS

1987 ◽  
Author(s):  
A D Purdon ◽  
J B Smith
Keyword(s):  
Molecular Species ◽  
Specific Activity ◽  
Gel Filtration ◽  
Human Platelets ◽  
Time Interval ◽  
Resting Cells ◽  
Ether Phospholipid ◽  
Before And After ◽  
Layer Chromatography ◽  
Derivatives Of

We have previously shown that the main source of arachidonate in thrombin-stimulated human platelets is 1-acyl-2-arachidonoyl (AA) glycerophosphocholine (GPC) and release of 3H-AA from this phospholipid also was correlated with increased 3H-AA in ether phospholipid. This ATP independent transfer of 3H-AA from 1,2 diacyl GPC to ether phospholipid (transacylation) also occurs in resting cells. Human platelets in 1/10 volume of plasma (ACD anticoagulant, pH 6.5) were radiolabelled with 3H-AA for 60 min at 37°C and then exogenous 3H-AA was removed by gel filtration into Tyrode's buffer, pH 7.4, 0.2% albumin. These radiolabelled cells were incubated in the absence of exogenous 3H-AA for four hours followed by Bligh and Dyer extraction and thin layer chromatography purification of phospholipids. 3H-AA in 1,2 diacyl GPC was found to decrease by over 20% and increase substantially in 1-0-alkyl-2-acyl GPC and 1-0-alk-1'-enyl-2-acyl glycerophospho ethanolamine (GPE), In this same time interval the mass of AA released by thrombin (5 U/ml, 10 min, 37°C, no stirring)in the presence of BIT 775C and measured by GLC, stayed the same (30 nmoles/109 cells), however, the specific activity decreased. Using reverse phase HPLC to resolve diradylglycerobenzoate derivatives of phospholipids: acylation, deacylation, and transacylation were observed for individual AA-containing molecular species of phospholipid, including those with an unsaturated fatty acid at sn-1. In particular the radiolabellinq of the 1-unsaturate-2-arachidonoyl GPC correlated with the specific activity of the 3H-AA released by stimulation with thrombin. Furthermore, 1-arachidonoyl-2-3H-arachidonoyl GPC was completely deacylated while 50 % of its mass remained. This contrasted with 16:0, and 18:0-2-arachidonoyl GPC in which the specific activity remained the same before and after deacylation. We conclude that deacylation of AA-containing molecular species of 1,2 diacyl GPC in stimulated cells includes molecular species which are also a source of arachidonic acid for transacylation reactions.

ISOLATION OF A SOLUBLE PHOSPHOLIPASE A2 FROM HUMAN PLATELETS ACTIVE AGAINST 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE

1987 ◽  
Author(s):  
A D Purdon ◽  
J B Smith
Keyword(s):  
Phospholipase A2 ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Human Platelets ◽  
Phospholipid Bilayer ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Enzyme Substrate ◽  
Membrane Bound ◽  
Layer Chromatography

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.

scholarly journals Potassium uptake and release by human blood platelets

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 185-197
Author(s):  
JS Wiley ◽  
J Kuchibhotla ◽  
CC Shaller ◽  
RW Colman
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Lactic Dehydrogenase ◽  
Human Platelets ◽  
First Order Kinetics ◽  
A Minor ◽  
Human Blood Platelets

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

scholarly journals Potassium uptake and release by human blood platelets

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 185-197 ◽  
Author(s):  
JS Wiley ◽  
J Kuchibhotla ◽  
CC Shaller ◽  
RW Colman
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Lactic Dehydrogenase ◽  
Human Platelets ◽  
First Order Kinetics ◽  
A Minor ◽  
Human Blood Platelets

Abstract Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

The incorporation of [3H]glycerol and 1-[14C]acyl-sn-glycero-3-phosphocholine into different molecular species of phosphatidylcholine in human platelets

1984 ◽  
Vol 62 (9) ◽  
pp. 827-830 ◽  
Author(s):  
Vhundi G. Mahadevappa ◽  
Bruce J. Holub
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Rat Liver ◽  
Molecular Species ◽  
De Novo ◽  
Human Platelet ◽  
Human Platelets ◽  
Argentation Thin Layer Chromatography ◽  
Acyltransferase Activity ◽  
Layer Chromatography

The molecular species of phosphatidylcholine which are formed by de novo synthesis and by the acylation of 1-acyl-sn-glycero-3-phosphocholine were characterized and compared in human platelets. For this purpose, intact human platelets were incubated in the presence of [3H]glycerol or 1-[14C]palmitoyl-sn-glycero-3-phosphocholine and the newly formed radioactive phosphatidylcholine was isolated by thin-layer chromatography. The labelled phosphatidylcholines were converted to their 1,2-diacylglycerol acetate derivatives and fractionated into their various chemical classes (saturates, monoenes, dienes, trienes, tetraenes, and >tetraenes) by argentation thin-layer chromatography. Regardless of the precursor used, the radioactivity distributions among the various classes did not correspond closely to that of the mass. The highest percentage of the radioactivity incorporated from [3H]glycerol was found in the saturates (25% of total), followed closely by the tetraenes (21%) and monoenes (18%), with lesser amounts in the dienes (15%), pentaenes plus hexaenes (14%), and trienes (7%). These results indicate that the de novo pathway is capable of substantial synthesis of tetraenoic (1-acyl 2-arachidonoyl) phosphatidylcholine in human platelets in contrast to previous observations in rat liver. In close agreement with work in rat liver, 59% of the radioactivity in the [14C]phosphatidylcholine derived from 1-[14C]palmitoyl-sn-glycero-3-phosphocholine was found in the tetraenoic species. The present results, together with the existence of phospholipase A2 and acyltransferase activity in platelets, support the potential importance of a deacylation–acylation cycle for the enrichment of human platelet phosphatidylcholine in arachidonoyl molecular species.

Uptake of 32P-orthophosphate and incorporation into phospholipids in Tetrahymena pyriformis W exposed to phenothiazine derivatives

1968 ◽  
Vol 46 (4) ◽  
pp. 331-339 ◽  
Author(s):  
Charles G. Rogers
Keyword(s):  
Thin Layer ◽  
Specific Activity ◽  
Tetrahymena Pyriformis ◽  
Exponential Phase ◽  
Phosphorus Compounds ◽  
Resting Cells ◽  
Lipid Phosphorus ◽  
Phenothiazine Derivatives ◽  
Inhibition Of Growth ◽  
Layer Chromatography

Partial inhibition of growth of Tetrahymena pyriformis W by promazine or chlorpromazine resulted in a 60% increase in phospholipid concentration during the exponential phase. Age or drug treatment did not affect the pattern of phospholipids as shown by thin-layer chromatography. At least 8 phospholipid components were recognized, of which phosphatidylethanolamine accounted for almost 50% of the total lipid phosphorus and phosphatidylcholine for about 20%. Uptake of 32P-orthophosphate by resting cells from cultures in the exponential phase was inhibited by chlorpromazine, promazine, promethazine, trifluoperazine, prochlorperazine, acetophenazine, and 2,4-dinitrophenol.Chlorpromazine and 2,4-dinitrophenol differed from one another in their effect on the distribution of 32P-orthophosphate among the principal phosphorus compounds of the cell; in the pattern of labelling of phospholipids with 32P, as shown by autoradiography of thin-layer chromatograms; and in the magnitude of specific activity values for individual phospholipids. These findings showed that the mechanism(s) by which phenothiazine derivatives alter the distribution of absorbed 32P and its incorporation into phospholipids in Tetrahymena differs from that of 2,4-dinitrophenol and that this may be associated with the ability of the tranquilizing drugs to inhibit nutrient uptake by the cells.

The entry into the brain of large molecules derived from dietary protein

1978 ◽  
Vol 200 (1139) ◽  
pp. 175-192 ◽  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Immune Serum ◽  
The Body ◽  
Brain Extract ◽  
Blood Contamination ◽  
Adult Rats ◽  
Large Molecules ◽  
Derivatives Of ◽  
The Brain

When bovine IgG, haemoglobin, or α-gliadin extracted from wheat, labelled with radio-iodine, are fed by stomach tube to suckling or adult rats, significant amounts of protein-bound radioactivity are found in extracts of whole brain. These amounts are greatly in excess of what can be accounted for by blood contamination: the specific activity of the brain is of the order of that of other tissues of the body, suggesting that it is permeated with protein derivatives of the fed material. This is borne out by differential centrifugation of brain macerates which shows sub­stantial protein-bound radioactivity in the cell sap fraction. Ultracentrifugation studies reveal the presence of high molecular mass material in the cell sap, some of greater sedimentation velocity than the original protein fed, suggesting complexing of derivatives of this with native components of the brain. This is confirmed by gel filtration studies. Immunological studies on the brain extract show that a high proportion of the protein-bound radioactivity of the brain retains the ability to be precipitated by specific immune serum, when α-gliadin or bovine IgG has been fed.

Ether Phospholipid Molecular Species in Human Platelets

1989 ◽  
Vol 105 (2) ◽  
pp. 168-172 ◽  
Author(s):  
Hitoshi Takamura ◽  
Ken-ichi Tanaka ◽  
Takao Matsuura ◽  
Makoto Kito
Keyword(s):  
Molecular Species ◽  
Human Platelets ◽  
Ether Phospholipid ◽  
Phospholipid Molecular Species

scholarly journals Characterization of phosphoinositide-specific phospholipase C from human platelets

1987 ◽  
Vol 243 (3) ◽  
pp. 763-771 ◽  
Author(s):  
V Manne ◽  
H F Kung
Keyword(s):  
Phospholipase C ◽  
Specific Activity ◽  
Human Platelet ◽  
Gel Filtration ◽  
Sodium Deoxycholate ◽  
Human Platelets ◽  
Ras Proteins ◽  
Ph Optimum ◽  
Pi Hydrolysis

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

1994 ◽  
Vol 71 (01) ◽  
pp. 091-094 ◽  
Author(s):  
M Cattaneo ◽  
B Akkawat ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
C Cimminiello ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Before And After ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Extraction of Protein-Bound ATP and ADP from Human Platelets in Plasma

1990 ◽  
Vol 63 (02) ◽  
pp. 286-290 ◽  
Author(s):  
Christina Beurling-Harbury ◽  
Pehr B Harbury
Keyword(s):  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Binding Protein ◽  
Human Platelets ◽  
Luciferase Assay ◽  
First Time ◽  
Plasma Extraction

SummaryActin is the major ATP and ADP binding protein in platelets, 0.9–1.3 nmol/108 cells, 50–70% in the unpolymerized state. The goal of these experiments was to develop a method for extracting all protein-bound ATP and ADP from undisturbed platelets in plasma. Extraction of actin-bound ADP is routine while extraction of actin-bound ATP from platelets in buffer has been unsuccessful. Prior to extraction the platelets were exposed to 14-C adenine, to label the metabolic and actin pools of ATP and ADP. The specific activity was determined from the actin-bound ADP in the 43% ethanol precipitate. Sequential ethanol and perchlorate extractions of platelet rich plasma, and the derived supernatants and precipitates were performed. ATP concentrations were determined with the luciferase assay, and radioactive nucleotides separated by TLC. A total of 1.18 nmol/108 cells of protein-bound ATP and ADP was recovered, 52% ATP (0.61 nmol). The recovery of protein-bound ADP was increased from 0.3 to 0.57 nmol/108 cells. This approach for the first time successfully recovered protein bound ATP and ADP from platelets in a concentration expected for actin.

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