scholarly journals A role for cyclooxygenase products in the formation of phosphatidic acid in stimulated human platelets. Differential mechanisms of action of thrombin and collagen.

1983 ◽  
Vol 258 (8) ◽  
pp. 4683-4686 ◽  
Author(s):  
W Siess ◽  
P Cuatrecasas ◽  
E G Lapetina
Keyword(s):  
Phosphatidic Acid ◽  
Human Platelets ◽  
Mechanisms Of Action

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.

The paradox of tianeptine

1993 ◽  
Vol 8 (S2) ◽  
pp. 89s-93s ◽  
Author(s):  
M Ansseau
Keyword(s):  
Chronic Conditions ◽  
Depressive Disorders ◽  
Antidepressant Activity ◽  
Synaptic Cleft ◽  
Human Platelets ◽  
Mechanisms Of Action ◽  
Functional Deficit ◽  
Number Of Patients ◽  
Biochemical Studies ◽  
Antidepressant Efficacy

SummaryThe classical biochemical hypothesis of depression posits a functional deficit in central neurotransmitter systems, particularly serotonin (5-HT) and/or noradrenaline. The major support for this theory was that antidepressants increase the amount of neurotransmitters in the synaptic cleft, by inhibiting reuptake mechanisms (tricyclics) or inhibiting enzymatic catabolism (MAOIs). The major role suggested for 5-HT in this theory led to the development of a large number of compounds which selectively inhibit 5-HT reuptake, such as fluvoxamine, fluoxetine, citalopram, sertraline, paroxetine, etc. Numerous clinical studies have demonstrated the antidepressant activity of such types of agents, supporting 5-HT deficit as the main origin of depression. Tianeptine is active in classical animal models of antidepressants. Its antidepressant efficacy has been established in controlled trials involving a large number of patients. Several biochemical studies however demonstrated that tianeptine induces in acute as well as in chronic conditions, a presynaptic increase of 5-HT reuptake, both in animal and human platelets and animal CNS. Therefore, as a 5-HT reuptake enhancer, tianeptine exhibits a mechanism of action totally opposite to 5-HT reuptake blockers such as fluoxetine but, paradoxically, both mechanisms of action are associated with a therapeutic activity in depressive disorders. Several hypotheses to explain these paradoxical findings and different methodologies to test them clinically are proposed.

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.

scholarly journals Riluzole Stimulates BDNF Release from Human Platelets

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Patrick Türck ◽  
Marcos Emílio Frizzo
Keyword(s):  
Healthy Subjects ◽  
Direct Action ◽  
Human Platelets ◽  
Mechanisms Of Action ◽  
Bdnf Expression ◽  
Extracellular Glutamate ◽  
Low Concentrations ◽  
Treatment Of Depression ◽  
The Central Nervous System ◽  
Antidepressant Action

Brain-derived neurotrophic factor (BDNF) has several functions in the central nervous system, where it contributes to brain development and its functionality through affecting neuronal survival and activity and also modulating neurotransmitter levels. This neurotrophin is also found in the serum, but its origin and peripheral function remain unknown. Although the source of circulating BDNF is uncertain, it is stored in platelets and can be released through pharmacological treatment. Decreased levels of BDNF in the serum have been related to the pathophysiology of depression, and this relationship is reinforced by the reversal of this condition by treatment with antidepressants. Recently, riluzole has been proposed for the treatment of depression because it has the ability to lower extracellular glutamate levels and increase BDNF expression; and both mechanisms could be associated with its antidepressant action. Considering that riluzole enhances BDNF levels in the serum of patients, we investigated if treatment with this drug could stimulate the release of this neurotrophin from human platelets obtained from healthy subjects. When platelets were incubated with riluzole for 4 h, the basal value of BDNF (92.9±11.1 pg 10−6platelets) was significantly increased (P<0.05,n=27). This stimulatory effect was achieved at low concentrations of riluzole (from 10 µM) and was not observed when platelets were incubated with the drug for 24 h. The direct action of riluzole evoking BDNF release from human platelets at therapeutic concentrations is important and may contribute to the understanding of its mechanisms of action in the treatment of depression.

scholarly journals Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.

2010 ◽  
Vol 57 (3) ◽  
Author(s):  
Marek Krzystanek ◽  
Henryk I Trzeciak ◽  
Ewa Krzystanek ◽  
Andrzej Małecki
Keyword(s):  
Phosphatidic Acid ◽  
Rat Brain ◽  
Phospholipase D ◽  
Brain Cortex ◽  
Enzymatic Reaction ◽  
Biological Materials ◽  
Human Platelets ◽  
Choline Oxidase ◽  
Fluorometric Method ◽  
Platelet Protein

Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H₂O₂. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, in the presence of horseradish peroxidase, H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials.

Microbubble-Induced Phospholipase C Activation Does not Correlate with Platelet Aggregation

1993 ◽  
Vol 69 (04) ◽  
pp. 394-396 ◽  
Author(s):  
R Malmgren ◽  
T Thorsen ◽  
A Nordvik ◽  
H Holmsen
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Human Platelets ◽  
Similar Extent ◽  
Arachidonic Acid Metabolites ◽  
Acid Metabolites ◽  
Rule Out ◽  
Stimulation Of

SummaryThe effect of nitrogen-(N2-)microbubbles on platelets resembles that of common platelet agonists with respect to aggregation and secretion, but is considerably slower and is poorly inhibited by aspirin. This paper reports the effect of microbubbles on platelet phospholipase C activity in gelfiltered human platelets prelabelled with [32P]Pi ([32P]-GFP). The experiments were run in the presence of an ADP scavenging system in order to rule out effects of ADP. Stimulation of [32P]-GFP for 30 min with microbubbles caused a significant reduction in single platelets (p <0.0004) and a significant increase in 32P-activity in the phosphatidic acid (PA) fraction (p <0.02). Epinephrine potentiated the microbubble-induced reduction in single platelets (p <0.05), but did not enhance the amount of 32P in the platelet [32P]PA fraction. The 32P-radioactivity in the PI-fraction increased with time to a similar extent when [32P]-GFP was stirred for 30 min in absence of microbubbles as it did after 30 min of agonist exposure. There were no significant changes in the [32P]PIP and [32P]PIP2 fractions. Aspirin abolished the microbubble-induced increase in 32P-activity in the PA fraction, but had no significant effect on the reduction in single platelets. Aspirin had a small but significant, reducing effect on platelet aggregation induced by a combination of epinephrine and microbubbles (p <0.05). With epinephrine, however, aspirin did not completely abolish the increase in [32P]-PA. It is concluded that microbubbles alone cause platelets to aggregate by a novel mechanism that operates independent of cyclooxygenase-dependent arachidonic acid metabolites and phospholipase C activation.

THROMBIN-INDUCED RELEASE OF INTRA-PLATELET CA2+ STORES IS INHIBITED BY PROSTACYCLIN, BUT GTP- AND IP3-INDUCED RELEASE IS UNAFFECTED

1987 ◽  
Author(s):  
Michael F Crouch ◽  
Roger D Nolan ◽  
Eduardo G Lapetina
Keyword(s):  
Phosphatidic Acid ◽  
External Medium ◽  
Inositol Phosphates ◽  
Human Platelets ◽  
Direct Application ◽  
Total Cell ◽  
Intracellular Camp ◽  
Dibutyryl Camp ◽  
Tubular System ◽  
Camp Levels

Alpha-thrombin induced the release of internal Ca2+ stores and the influx of Ca2+ in human platelets, as measured by quin-2 fluorescence. This was accompanied by a stimulated formation of inositol phosphates and phosphatidic acid. The Ca2+ responses were inhibited almost totally by pretreatment of cells with prostacyclin (PGI2). Epinephrine was able to restore the influx of Ca2+ from the external medium, but not the alpha-thrombin-induced release of internal Ca2+ stores. This was despite epinephrine restoring phosphatidic acid formation and, at least partially, the generation of inositol trisphosphate (IP3). This suggested that PGI2 was inhibiting the actions of IP3 in inducing release of Ca2+ from the dense tubular system. Since the effects of PGI2 are thought to be mediated by formation of cAMP, we examined whether cAMP could modulate the release of 45Ca2+ induced by IP3 from permeabilized platelets. IP3 induced about a 30% release of cellular 45Ca2+ over a 4 min period. However, neither pretreatment of cells with PGI2 nor the direct application of dibutyryl cAMP had any effect on the IP3-stimulated 45Ca2+ release. GTP, which released about 10% of total cell 45Ca2+, also was not affected by these agents. These results suggest either that permeabilization of platelets dilutes cytoplasmic components which are necessary for cAMP action, or that PGI2 is inhibiting the release of Ca2+ stores induced by thrombin, presumably via IP3, by a mechanism which is separate to the elevation of intracellular cAMP levels.

Mobilization of arachidonic acid in thrombin-stimulated human platelets

1990 ◽  
Vol 68 (2) ◽  
pp. 520-527 ◽  
Author(s):  
V. G. Mahadevappa ◽  
Frank Sicilia
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Selective Inhibition ◽  
Human Platelets ◽  
Phospholipase A ◽  
Serine Esterase ◽  
Membrane Phospholipids ◽  
Esterase Inhibitors ◽  
Hydrolysis Of

In the present work we investigated the effect of serine esterase inhibitors such as 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and phenylmethylsulfonyl fluoride (PMSF), as well as the effect of mepacrine on thrombin-induced mobilization of arachidonic acid (AA) in human platelets. The inhibitor NCDC (0.6 mM) completely abolished the thrombin-induced activation of phospholipase C, phospholipase A2, and transacylase enzymes, whereas the pretreatment of platelets with PMSF (2 mM) resulted in a highly selective inhibition of phospholipase A2 and transacylase activities, with no marked effect on thrombin-induced activation of phospholipase C. The thrombin-induced release of [3H]AA from phosphatidylcholine and phosphatidylinositol was reduced by 90 and 56%, respectively, in the presence of PMSF. This inhibitor also caused a parallel inhibition in the accumulation of [3H]AA (85%) with little effect on thrombin-induced formation of [3H]phosphatidic acid (5%), whereas mepacrine (0.4 mM) caused a selective inhibition of phospholipase A2 and transacylase activities with concomitant stimulation of [3H]phosphatidic acid formation in intact human platelets. These results demonstrate that NCDC and PMSF (serine esterase inhibitors) do not affect agonist-induced activation of phospholipases that mobilize arachidonic acid through a common site. Our results further demonstrate that the inhibition of [3H]AA release observed in the presence of NCDC, PMSF, and mepacrine is primarily due to their direct effects on enzyme activities, rather than due to their indirect effects through formation of complexes between inhibitors and membrane phospholipids. Based upon these results, we also conclude that the combined hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 serves as a major source for eicosanoid biosynthesis in thrombin-stimulated human platelets.Key words: deacylation, phospholipids, thrombin, platelets, phospholipase A2.

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