scholarly journals Neomycin does not interfere with the inositol phospholipid metabolism, but blocks binding of α-thrombin to intact human platelets

1991 ◽  
Vol 273 (1) ◽  
pp. 241-243 ◽  
Author(s):  
O B Tysnes ◽  
E Johanessen ◽  
V M Steen
Keyword(s):  
Platelet Activation ◽  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Cellular Receptor ◽  
Inositol Phospholipid ◽  
Induced Changes

Neomycin was demonstrated to inhibit the binding of thrombin to intact human platelets. The effects of neomycin on both thrombin binding and thrombin-induced changes in inositol phospholipid metabolism could be reproduced by the thrombin antagonist hirudin. We propose that neomycin inhibits thrombin-induced platelet activation by interference with the cellular receptor.

scholarly journals Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium

1984 ◽  
Vol 219 (3) ◽  
pp. 833-842 ◽  
Author(s):  
W K Pollock ◽  
R A Armstrong ◽  
L J Brydon ◽  
R L Jones ◽  
D E MacIntyre
Keyword(s):  
Receptor Antagonist ◽  
Platelet Activation ◽  
Human Platelet ◽  
Thromboxane A2 ◽  
Receptor Occupancy ◽  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Thromboxane A2 Receptor ◽  
Agonist Concentration ◽  
Inositol Phospholipid

The inter-relationships between receptor occupancy, inositol phospholipid metabolism and elevation of cytosolic free Ca2+ in thromboxane A2-induced human platelet activation were investigated by using the stable thromboxane A2 mimetic, 9,11-epoxymethanoprostaglandin H2, and the thromboxane A2 receptor antagonist, EPO45. 9,11-Epoxymethanoprostaglandin H2 stimulated platelet phosphatidylinositol metabolism as indicated by the rapid accumulation of [32P]phosphatidate and later accumulation of [32P]phosphatidylinositol in platelets pre-labelled with [32P]Pi. These effects of 9,11-epoxymethanoprostaglandin H2 were concentration-dependent and half-maximal [32P]phosphatidate formation occurred at an agonist concentration of 54 +/- 8 nM. With platelets labelled with the fluorescent Ca2+ indicator quin 2, resting cytosolic free Ca2+ was 86 +/- 12 nM. 9,11-Epoxymethanoprostaglandin H2 induced a rapid, concentration-dependent elevation of cytosolic free Ca2+ to a maximum of 300-700 nM. Half-maximal stimulation was observed at an agonist concentration of 80 +/- 23 nM. The thromboxane A2 receptor antagonist EPO45 selectively inhibited 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and elevation of cytosolic free Ca2+, indicating that both events are sequelae of receptor occupancy. Human platelets contain a single class of stereospecific, saturable, high affinity (KD = 70 +/- 13 nM) binding sites for 9,11-epoxymethano[3H]prostaglandin H2. The concentration-response curve for receptor occupancy (9,11-epoxymethano-[3H]prostaglandin H2 binding) is similar to that for 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and for elevation of cytosolic free Ca2+. These observations indicate that human platelet thromboxane A2 receptor occupation is closely linked to inositol phospholipid metabolism and to elevation of cytosolic free Ca2+. Both such events may be necessary for thromboxane A2-induced human platelet activation.

Abstract 489: Differential Phosphoproteomic Analysis of Platelet Activation by Specific Oxidized Phospholipids and Thrombin Reveals Mechanisms of Platelet Activation in Hyperlipidemia

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Alejandro Zimman ◽  
Bjoern Titz ◽  
Evangelia Komisopoulou ◽  
Thomas G Graeber ◽  
Eugene A Podrez
Keyword(s):  
Signaling Pathways ◽  
Signaling Pathway ◽  
Platelet Activation ◽  
Scavenger Receptor ◽  
Human Platelets ◽  
Oxidized Phospholipids ◽  
Systematic Analysis ◽  
Bioinformatic Tools ◽  
Induced Changes

We previously showed that specific oxidized phospholipids (oxPC CD36 ) activate platelets via the scavenger receptor CD36 and promote platelet hyper-reactivity in hyperlipidemia, however the signaling pathway(s) induced in platelets by oxPC CD36 are not defined. We employed mass spectrometry-based phosphoproteomics for the unbiased analysis of changes in protein phosphorylation induced by oxPC CD36 and thrombin, a strong platelet agonist, in human platelets. oxPC CD36 induced changes in phosphorylation of 148 unique phosphorylation sites (116 proteins) while thrombin induced changes of 297 unique sites (181 proteins). Most of the changes in phosphorylation induced by oxPC CD36 and thrombin identified in our study have never been reported before in platelets and include high- and low-abundant proteins with diverse molecular functions located in the plasma membrane, cytosol, or cytoskeleton. Analysis using multiple bioinformatic tools identified protein interaction networks, signaling pathways, activated kinases, and enriched phosphorylation motifs. Comparison between platelet agonists revealed multiple differences including the specific activation of a signaling pathway involving Src-family kinases (SFK), SYK kinase, and PLCγ2 by oxPC CD36 . Subsequent biochemical studies in human platelets demonstrated that this pathway is critical for platelet activation by oxPC CD36 and is downstream of CD36. In conclusion, systematic analysis of platelet activation pathways provided novel insights into the mechanism of platelet activation and specific signaling pathways induced by oxidized phospholipids that modulate platelet function in vivo in hyperlipidemia.

Epinephrine stimulates inositol phospholipid metabolism by activating alpha-2 adrenergic receptors in human platelets

Life Sciences ◽  
1989 ◽  
Vol 44 (11) ◽  
pp. 741-747 ◽  
Author(s):  
Hideki Mori ◽  
Masahiko Mikuni ◽  
Tsukasa Koyama ◽  
Itaru Yamashita
Keyword(s):  
Adrenergic Receptors ◽  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Inositol Phospholipid

Application Of High Performance Liquid Chromatography (HPLC) To The Study Of Platelet Phospholipid Metabolism

1981 ◽  
Author(s):  
I Alam ◽  
J B Smith ◽  
M J Silver
Keyword(s):  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Platelet Activating Factor ◽  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Two Dimensional ◽  
Platelet Phospholipid ◽  
Fraction Collector ◽  
Induced Changes ◽  
Layer Chromatography

A method for the separation of phospholipids using HPLC has been developed. Different radioactive prostaglandins and phospholipids are detected by passing a portion of the effluent from the HPLC column directly through a Radio Flow-1 detector. The remainder of the effluent is collected for phosphate and fatty acid determinations using a fraction collector. This system eliminates the solventquenching limitation associated with U.V. detection and offers many advantages over thin layer chromatography (TLC) which frequently requires two dimensional development and scraping of zones from the TLC plate. Separations of phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, spingomyelin and platelet activating factor (PAF) have been achieved. The system has been applied to the study of thrombin-induced changes in the phospholipids of human platelets prelabeled with 14C-arachidonic acid. After incubation of prelabeled platelets for 10 min. at 37° with 5 U/ml thrombin, a 30% decrease in radioactive phosphatidylcholine was detected.

Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

1987 ◽  
Vol 927 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Didier de Chaffoy de Courcelles ◽  
Peter Roevens ◽  
Jos Wynants ◽  
Herman Van Belle
Keyword(s):  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Inositol Phospholipid

Inositol phospholipid metabolism in human platelets stimulated by ADP

1990 ◽  
Vol 193 (2) ◽  
pp. 521-528 ◽  
Author(s):  
John D. VICKERS ◽  
Raelene L. KINLOUGH-RATHBONE ◽  
Marian A. PACKHAM ◽  
J. Fraser MUSTARD
Keyword(s):  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Inositol Phospholipid

scholarly journals Thrombin-Induced Changes in Phosphatidic ACID (PA) and Phosphoinositides

1977 ◽  
Author(s):  
N. L. Leung ◽  
R. L. Kinlough-Rathbone ◽  
J. F. Mustard
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Thin Layer ◽  
Phospholipid Metabolism ◽  
Rabbit Platelets ◽  
Inositol Phospholipid ◽  
Induced Changes ◽  
Layer Chromatography ◽  
Stimulation Of

Thrombin-induced changes in PA, monophosphoinositide (MPI) , diphosphoinositide (DPI) and triphosphoinositide (TPI) of washed rabbit platelets have been examined. Platelets prelabeled by incubation with 32P-orthophosphate, 3H-glycerol, 3H-inositol or 14C-arachidonate were exposed to 0.33 u/ml thrombin for one minute, and the phospholipids extracted and separated by thin layer chromatography. Measurement of absolute amounts of PA and MPI by phosphorus assay showed that PA increased by 180% while MPI decreased by 15%.The changes in MPI with 3H-G, 3H-I and 14C-AA and in 1,2-diacyl glycerol (DG) indicate that some MPI may be converted to PA via DG. Changes in the 3H-G and 3H-I labeling of DPI and TPI suggest that the changes observed with 32P-Iabeled platelets are a result of the turnover of the phosphorylinositol moiety. The increase in AA with 14C-AA indicates that some of the decrease in MPI may be due to the formation of lyso MPI and free fatty acid. These results indicate that thrombin stimulation of platelets may affect inositol phospholipid metabolism through three pathways: (1) involving PA, DG and MPI; (2) the cleavage of free fatty acids from MPI; (3) turnover of the ester phosphates on DPI and TPI.

scholarly journals Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism

1988 ◽  
Vol 253 (2) ◽  
pp. 581-586 ◽  
Author(s):  
V M Steen ◽  
O B Tysnes ◽  
H Holmsen
Keyword(s):  
Narrow Range ◽  
Phospholipid Metabolism ◽  
Human Platelets ◽  
Dense Granule ◽  
Phosphoinositide Metabolism ◽  
Inositol Phospholipids ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Changes ◽  
Induced Aggregation

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.

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