Release of a 6-Oxoprostaglandin E1-like Substance from Human Platelets

1983 ◽  
Vol 64 (1) ◽  
pp. 63-68 ◽  
Author(s):  
F. J. Lofts ◽  
P. K. Moore
Keyword(s):  
Biological Activity ◽  
Smooth Muscle ◽  
Thin Layer ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Prostaglandin I2 ◽  
Gastrointestinal Smooth Muscle ◽  
Layer Chromatography

1. Human platelet-rich plasma incubated at 37°C generates an antiaggregatory prostaglandin which is spasmogenic on gastrointestinal smooth muscle. 2. Platelet-rich plasma from female donors generated more biological activity and was more sensitive to the anti-aggregatory activity of added prostaglandin I2 (PGI2) compared with that from age-matched male controls. 3. After thin-layer chromatography of extracted platelet-rich plasma, biological activity was detected in a zone which co-chromatographed with 6-oxoprostaglandin E1. 4. Neither extracted platelet-rich plasma nor authentic 6-oxoprostaglandin E1 were inactivated following incubation with purified 15-hydroxyprostaglandin dehydrogenase. 5. The relevance of these findings for regulating platelet reactivity is discussed.

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.

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.

Inhibition of ADP-Induced Responses in Human Platelets by Agents Elevating the Cyclic AMP Level: Comparison of Aggregation and Shape Change

1984 ◽  
Vol 52 (03) ◽  
pp. 333-335 ◽  
Author(s):  
Vider M Steen ◽  
Holm Holmsen
Keyword(s):  
Inhibitory Action ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Early Step ◽  
Stimulus Response ◽  
Sequential Relationship

SummaryThe inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses.Human platelet-rich plasma was preincubated for 2 min at 37° C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors.These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.

Endotoxin-Induced Platelet Activation in Human Whole Blood In Vitro

1988 ◽  
Vol 59 (03) ◽  
pp. 378-382 ◽  
Author(s):  
Gyorgy Csako ◽  
Eva A Suba ◽  
Ronald J Elin
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Human Platelets ◽  
Lag Phase ◽  
Human Whole Blood ◽  
Dose Dependent

SummaryThe effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 μg/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand, endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by “solubilized” endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

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.

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 Fluorescein colchicine. Synthesis, purification, and biological activity.

1978 ◽  
Vol 76 (3) ◽  
pp. 619-627 ◽  
Author(s):  
J I Clark ◽  
D Garland
Keyword(s):  
Spectral Analysis ◽  
Biological Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Fluorescein Isothiocyanate ◽  
Significant Loss ◽  
Colchicine Derivative ◽  
Layer Chromatography ◽  
Kinetics Of ◽  
Brain Tubulin

The synthesis of a fluorescent colchicine derivative permits the localization of colchicine-binding receptors in cells. Fluorescein colchicine (FC) was prepared by the addition of fluorescein isothiocyanate to deacetyl colchicine. The product, FC, was separated from the reactants by thin-layer chromatography (TLC). The purity of FC was demonstrated by TLC, UV spectral analysis, and analysis of the kinetics of photodecomposition. FC inhibited [3H] colchicine binding to purified brain tubulin. The biological activity of FC was compared to the activity of unlabeled colchicine on mitosis, motility, secretion, and myogenesis. The effects of FC were identical to those of unlabeled colchicine in all biological systems tested. The results demonstrate that FC may be substituted for colchicine in biological experiments without significant loss in specificity or effectiveness.

Platelet Factor 3 Activity in Washed Platelets

1973 ◽  
Vol 30 (03) ◽  
pp. 557-566 ◽  
Author(s):  
S Renaud ◽  
P Gautheron ◽  
H Rosenstein
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Slow Speed ◽  
Platelet Factor ◽  
Platelet Poor Plasma ◽  
Platelet Counts ◽  
Edta Solution ◽  
Layer Chromatography

SummaryPlatelets collected with an EDTA solution and simply washed in an incomplete Tyrode’s presented clotting times in the recalcification (man and rat) and the Stypven (rat) tests that were practically identical to those of the PRP when slow speed centrifugation was used (800 G in man, 1000 G in rat). This was demonstrated, in 6 pools of 5 rats each and in 6 men, by comparing the clotting activity of the citrated platelet-rich plasma to that of the platelets washed and resuspended in the citrated platelet-poor plasma, for platelet counts ranging from 1 × 105 to 10 × 105/mm3. In contrast, centrifugation of platelets at 3000 G markedly affected these clotting activities, as was shown in an additional study comprising 6 pools of 3 rats.Finally, the clotting activity of platelets totally disrupted by sonication appears to be identical quantitatively in both man and rats to that of the total phospholipids extracted from these platelets and separated from the other lipids by thin-layer chromatography and resuspended in plasma by sonication.

scholarly journals Cross Linking of Human Platelet Membrane Aminophospholipids:Changes Induced by Thrombin

1977 ◽  
Author(s):  
P. K. Schick ◽  
P. Arosteguy
Keyword(s):  
Thin Layer ◽  
Human Platelet ◽  
Phosphatidyl Serine ◽  
Cross Linking ◽  
Lipid Phosphorus ◽  
Membrane Bilayer ◽  
Intact Cells ◽  
Platelet Membranes ◽  
Cross Links ◽  
Layer Chromatography

The proximity of the aminophospholipids, phosphatidylethanolamine (PE) and phosphatidyl-serine (PS), in platelet membranes was probed with difluorodinitrobenzene (D) which reacts with and cross-links aminophospholipids when they are less than 5 A° apart. D can penetrate intact cells and labels lipids on either side of the membrane bilayer. The incubation of mixtures of pure PE and PS can result in the formation of the following derivatives: PE-D-PE, PE-D-PS, PE-D, PS-D-PS, PS-D. Following incubation of washed platelets with D, lipids and derivatives were extracted, separated by thin-layer chromatography, and quantitated by lipid phosphorus. The incubation of platelets with D (125μM) for 2 hours resulted in derivatization of 49% PE and 34.6%PS. When platelets were treated with thrombin (0.1U/ml) for 5 minutes prior to incubation with D, 71.4% PE and 53.8% PS reacted. Results are shown in Table; %PE(PS)=% total platelet PE(PS).In controls significant amounts of only PE-D-PE and PE-D-PS were detected and thus are intimately associated within platelet membranes. In thrombin-treated platelets significant amounts of PS-D-PS were formed which did not occur in controls. However, there was no increase in PE-D-PE and PE-D-PS. Also, in thrombin-treated platelets additional PE and PS reacted but were not close enough to be cross-linked. The study indicates that incubation of platelets with thrombin results in the unmasking or rearrangement of specific compartments of PE and PS within platelet membranes which may be critical for platelet hemostatic activities.

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