The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets

1986 ◽  
Vol 64 (12) ◽  
pp. 1256-1261 ◽  
Author(s):  
Bonnie J. Weaver ◽  
Bruce J. Holub
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
The Other ◽  
Preferential Loss ◽  
Marked Selectivity ◽  
The Individual ◽  
Layer Chromatography

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA- versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.

Inhibition Of Platelet Aggregation, Malonyldialdehyde And Thromboxane Formation By Hydroperoxides Of Arachidonic Acid

1981 ◽  
Author(s):  
D Aharonv ◽  
J B Smith ◽  
M J Silver
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Lipoxygenase Pathway ◽  
Dose Dependent Fashion ◽  
Induced Aggregation ◽  
Layer Chromatography ◽  
Dose Dependent ◽  
Thromboxane Formation

The arachidonate hydroperoxides 12-HPETE and 15-HPETE were biosynthesized from arachidonic acid using partially purified human platelet lipoxygenase or soybean lipoxidase respectively, and isolated by thin layer chromatography. Both compounds inhibited the arachidonic acid- induced aggregation of washed human platelets, suspended in calcium-free Krebs Henseleit solution, in a dose dependent fashion at concentrations between 1 and 50 uM. No inhibition was seen with up to 100 uM of these hydroperoxides when platelet -rich plasma was used. 12-HPETE (in micromolar concentrations) inhibited the formation of both thromboxane B2 (radioimmunoassay) and malonyldialdehyde (spectrophotometrie assay) when washed platelets were incubated with arachidonic acid. The 12-hydroxide, 12-HETE also inhibited platelet aggregation and thromboxane formation, but was less potent than 12-HPETE. We suggest that arachidonate hydroperoxide generated in platelets via the lipoxygenase pathway modulates platelet aggregation induced by arachidonic acid by inhibiting thromboxane formation.

The thrombin-dependent enrichment of alkenylacyl ethanolamine phosphoglyceride with [14C]eicosapentaenoic and [3H]arachidonic acids in prelabelled human platelets

1987 ◽  
Vol 65 (4) ◽  
pp. 405-408 ◽  
Author(s):  
B. J. Weaver ◽  
B.J. Holub
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
Human Subjects ◽  
Human Platelets ◽  
Arachidonic Acids ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Individual Human

The thrombin-dependent enrichment of alkenylacyl ethanolamine phosphoglyceride in [14C]eicosapentaenoic acid ([14C]EPA) was demonstrated and compared with [3H]arachidonic acid ([3H]AA) following the simultaneous prelabelling of individual human platelet phospholipids with these two fatty acids. The alkenylacyl, diacyl, and alkylacyl classes of ethanolamine phosphoglycerides (PE) were separated by thin-layer chromatography as their acetylated derivatives after hydrolysis of the parent phospholipid with phospholipase C. The ratios of [3H]/[14C] for the increased radioactivity appearing in alkenylacyl PE following 60 and 120 s of thrombin stimulation were the same as the corresponding ratio (2.0) found in the choline phosphoglycerides (PC) from control (unstimulated) platelets. These results suggest no significant selectivity between EPA and AA in the thrombin-stimulated transfer of these fatty acids from diacyl PC to alkenylacyl PE. The present findings may possibly bear some relevance to the altered platelet reactivity and (or) decreased thromboxane A2 formation observed in human subjects following the ingestion of marine lipid containing EPA.

scholarly journals Thrombin induces a biphasic 1,2-diacylglycerol production in human platelets

1991 ◽  
Vol 275 (2) ◽  
pp. 355-361 ◽  
Author(s):  
S Nakashima ◽  
A Suganuma ◽  
A Matsui ◽  
Y Nozawa
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Myristic Acid ◽  
Time Course ◽  
Human Platelets ◽  
Second Phase ◽  
Mass Content ◽  
Small Production ◽  
Later Phase

The 1,2-diacylglycerol (DAG) mass content was measured in thrombin-stimulated human platelets. Thrombin stimulates a biphasic accumulation of DAG, with an early phase reaching a peak at 10 s and a later phase reaching a peak at 2-3 min. The time course of first-phase DAG production corresponded well to that of Ins(1,4,5)P3 formation, which was rapid and transient. The second phase of DAG accumulation occurred after the level of Ins(1,4,5)P3 returned to nearly basal. Thrombin stimulated the decrease in PtdIns and phosphatidylcholine contents. The source of second-phase DAG was examined in platelets prelabelled with three radioactive fatty acids, i.e. arachidonic, palmitic and myristic. Thrombin stimulated the increase in radioactivity of DAG with decline of PtdIns in platelets labelled with [3H]arachidonic acid or [3H]palmitic acid, in which PtdIns was considerably labelled. In contrast, significant accumulation of [3H]DAG was not observed in [3H]myristic acid-labelled platelets, in which PtdIns was poorly labelled. In platelets prelabelled with [3H]inositol, an increase in InsP in response to thrombin was seen for more than 5 min. In contrast, upon stimulation, significant increases in [3H]phosphocholine and [3H]choline were not observed in [methyl-3H]choline-labelled platelets. Thrombin induced a small production of phosphatidylethanol, when ethanol was present during stimulation. However, the formation of DAG and phosphatidic acid was not significantly affected by ethanol. These results suggest that thrombin stimulates a biphasic accumulation of DAG, initially from PtdInsP2 and later from PtdIns in human platelets.

Adenine Nucleotide Metabolism of Human Platelets

1971 ◽  
Vol 25 (02) ◽  
pp. 223-233
Author(s):  
M Murakami ◽  
K Odake
Keyword(s):  
Adenine Nucleotide ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Nucleotide Metabolism ◽  
The Other ◽  
Supernatant Fraction ◽  
Two Dimensional ◽  
Adenine Nucleotide Metabolism ◽  
Layer Chromatography

SummaryAfter platelet-rich plasma was incubated with radioactive adenine, radioactive adenine nucleotides in platelets were separated by two-dimensional thin-layer chromatography.Radioactive adenine was selectively incorporated into adenine nucleotides. Gradual decomposition of labelled nucleotides was observed after longer period of incubation. Radioactive ATP, ADP, AMP, IMP, and hypoxanthine were separated from PCA extract of platelets. On the other hand, radioactive adenine and hypoxanthine were separated from platelet-poor plasma.After thrombin treatment, radioactive ATP in platelets broke down rapidly, while radioactive ADP decreased more slowly. Radioactive AMP increased at first in the cellular and supernatant fractions, and then decreased gradually. The accumulation of radioactive hypoxanthine was observed in the supernatant fraction. Released radioactive ATP and ADP were 23% and 22% of the initial radioactive ATP and ADP in platelets, respectively.

Diacylglycerol Biosynthesis in Everted Sacs of Rat Intestinal Mucosa

1975 ◽  
Vol 53 (11) ◽  
pp. 1170-1183 ◽  
Author(s):  
W. C. Breckenridge ◽  
A. Kuksis
Keyword(s):  
Fatty Acids ◽  
Phosphatidic Acid ◽  
Thin Layer ◽  
Free Fatty Acids ◽  
Thin Layer Chromatography ◽  
Intestinal Mucosa ◽  
Gas Liquid Chromatography ◽  
Acid Pathway ◽  
Layer Chromatography ◽  
Everted Sacs

The molecular specificity in the biosynthesis of diacylglycerols by rat intestinal mucosa was examined by means of radioactive markers, thin-layer chromatography with silver nitrate and gas-liquid chromatography with radioactivity monitoring. Bile salt micelles of alternately labeled monoacylglycerols and free fatty acids were incubated with everted sacs of intestinal mucosa for various periods of time and the diacylglycerols were isolated by solvent extraction and thin-layer chromatography. Stereospecific analyses of the X-1,2-diacylglycerols labeled from 2-monoacylgiycerols showed that the sn-1,2-isomers (45–55%) were slightly in excess of the sn-2,3-isomers (34–45%) with the X-1,3-diacylglycerols accounting for the rest of the radioactivity (5–10%). This suggests that racemic diacylglycerols may be intermediates in the resynthesis of dietary fat in rat intestinal mucosa. Detailed analyses of the molecular species of the sn-1,2-diacylglycerols labeled from free fatty acids revealed that 10–45% of the total did not contain the acid present in the 2-monoacylglycerol supplied, and therefore had originated from the phosphatidic acid pathway. These findings are at variance with those obtained in isolated microsomes, which have suggested an inhibition of the phosphatidic acid pathway by monoacylglycerols as well as have given evidence of an exclusive synthesis of sn-1,2-diacylglycerols from 2-monoacylglycerols.

Fluoride, An Internally Acting Platelet Activator, Causes Phosphorylation Of Platelet Phosphatidic Acid And 20K And 47K Proteins

1981 ◽  
Author(s):  
E H Mürer ◽  
E Siojo ◽  
J L Daniel
Keyword(s):  
Phosphatidic Acid ◽  
Platelet Activation ◽  
Great Increase ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Human Platelets ◽  
Silica Gels ◽  
Late Step ◽  
Aluminum Plates ◽  
Chloroform Methanol

The effects of fluoride, which is transported into platelets in order to induce secretion, are compared with known effects of thrombin, which acts via external sites. Thus, the changes related to transmission of signal through the platelet membrane will not be common to the two activators, only those changes which are subsequent to the internal triggering of platelet activation. Human platelets were prepared by collection in EDTA and washing in saline-EDTA or by gel filtration of citrated platelet-rich plasma. The two methods gave similar results. Platelets prelabeled in plasma with 32P and them separated were incubated at 37°C with 10 mM fluoride at pH 7.4, and samples removed at intervals. (1) The protein was precipitated with HC104, then solubilized by sonication with SDS buffer and the protein bands separated by acrylamide slab gel electrophoresis. The 20K and 47K bands showed 100 to 200% increase in label, with maximum at 8 min incubation (50% secretion) and a great increase seen already at 3 min incubation, where little secretion is observed. (2) Samples were extracted with chloroform-methanol, evaporated to dryness under N2, redissolved in chloroform and applied on thinlayer silica gels on aluminum plates. Two different systems for separating phosphatidic acid (PA) were used. No significant increase in 32P radioactivity was seen in PA the first 3 min. The label at 20 min was 3x that at 8 min. Thus the labeling related to contractile events, a late step in secretion, precedes the labeling of PA, suggesting that the major part of this labeling is not related to the initial phase of platelet activation.

Depressed Responsiveness To Adrenaline In Platelets From Apparently Normal Human Donors – A Familial Trait

1981 ◽  
Author(s):  
M C Scrutton ◽  
K R Bruckdorfer ◽  
R A Hutton
Keyword(s):  
Adenylate Cyclase ◽  
Platelet Function ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
The Other ◽  
Stimulus Response ◽  
A 23187 ◽  
Phospholipid Classes ◽  
Normal Human

Decreased responsiveness to adrenaline has been observed in 5 out of approximately 150 apparently normal unrelated human donors. In 4 donors, familial studies have shown that this trait is inherited. Three of the donors, as well as their affected relatives, exhibit depressed responsiveness to collagen and vasopressin but normal responsiveness to ADP and thrombin in association with the decreased responsiveness to adrenaline. The other two affected donors exhibit normal responsiveness to most other agonists although in one instance depression of responsiveness to vasopressin and absence of a secretory response to ADP may be associated with the decreased adrenaline response.Normal responsiveness can be restored in all instances either by incubating the platelet-rich plasma at 20°C or by addition at 37°C of a low concentration of the divalent cation ionophore, A-23187. No such effect results from addition of an adenylate cyclase inhibitor. All affected platelets have normal ATP and ADP contents, cholesterol to phospholipid ratios, and composition of the phospholipid classes. Mixing experiments demonstrate the absence of a circulating inhibitor of platelet function and suggest that the defect resides in the platelets. We conclude that depressed responsiveness of human platelets to adrenaline may result from a defect in Ca2+ mobilisation to the cytosol. The observed selectivity in the agonists affected may indicate that the stimulus-response coupling pathways converge at the level of an increase in cytosolic Ca2+ concentration.

Effect of Heparin on Aggregation, Release Reaction and Thromboxane A2synthesis in Human Platelets

1979 ◽  
Author(s):  
H.Y.K. Chuang ◽  
S.F. Mohammad ◽  
R.G. Mason
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Thromboxane A2 ◽  
Rabbit Aorta ◽  
Human Platelets ◽  
Appreciable Effect ◽  
Layer Chromatography ◽  
Aggregation Of Platelets ◽  
Platelet Functions

Studies on the effect of heparin on platelet functions have resulted in conflicting observations: heparin has been reported to cause aggregation of platelets, potentiate aggregation induced by various aggregating agents, or cause inhibition of aggregation. Using paritally purified heparin (beef lung or porcine mucosa) we observed that addition of heparin to citrated platelet rich plasma(C-PRP)potentiated the aggregation of platelets induced by ADP, epinephrine, or arachidonic acid. Presence of heparin in C-PRP results in complete inhibition of thrombin induced effects and partial inhibition of platelet aggregation induced by collagen. Presence of heparin in C-PRP also resulted in release of significantly higher concentrations of 14C-serotonin when platelets were challenged by appropriate aggregating agents. Those concentrations of heparin that resulted in potentiation of aggregation had no appreciable effect on c-AiMP or c-GMP levels of platelets. However, the presence of heparin results in a significant elevation of thromboxane A2 as determined by contraction of rabbit aorta or after conversion to thromboxane B2 by thin layer chromatography. These observations are of interest since increased production of thromboxane A2 in the presence of heparin may explain in part, the potentiation of platelet aggregation in vitro or thrombocytopenia observed frequently in patients receiving heparin intravenously Supported in part by grants HL22583 & 20679 from NHLBI of NIH.

scholarly journals Evidence from studies employing radioactively labelled fatty acids that the stimulation of flux through the diacylglycerol pool is an early action of vasopressin on hepatocytes

1987 ◽  
Vol 245 (1) ◽  
pp. 211-216 ◽  
Author(s):  
L B Pickford ◽  
A J Polverino ◽  
G J Barritt
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Isolated Hepatocytes ◽  
Maximal Increase ◽  
Maximal Stimulation ◽  
Early Action ◽  
Palmitic Acids ◽  
Stimulation Of ◽  
In Cells

1. In isolated hepatocytes prelabelled with [14C]-arachidonic, -stearic, -linoleic, -oleic or -palmitic acids, vasopressin increased the amount of radioactivity present in diacylglycerols. The largest increase was observed in cells labelled with arachidonic or stearic acids. 2. In cells prelabelled with [14C]- or [3H]-arachidonic acid, the onset of the increase in radioactivity in diacylglycerols induced by vasopressin was slow, the increase was partly dependent on the presence of extracellular Ca2+, and was associated with an increase in radioactivity present in phosphatidic acid which was more rapid in onset. Vasopressin decreased the amount of [3H]arachidonyl-phosphatidylinositol 4,5-bisphosphate, but the magnitude of this decrease was less than 10% of the observed increase in radioactivity in [3H]arachidonyl-diacylglycerol. 3. The concentration of vasopressin which gave half-maximal increase in [14C]arachidonyl-diacylglycerol at low extracellular Ca2+ was 10-fold higher than that which gave half-maximal stimulation of 45Ca2+ efflux. Phenylephrine, but not glucagon, also increased the amount of [14C]arachidonyl-diacylglycerol. 4. It is concluded that an early action of vasopressin on the liver cell is to increase the flux of carbon from phospholipids, including the phosphoinositides, to diacylglycerols.

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