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.

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.

scholarly journals The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes

1986 ◽  
Vol 238 (2) ◽  
pp. 425-436 ◽  
Author(s):  
C J Meade ◽  
G A Turner ◽  
P E Bateman
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Cytochalasin B ◽  
Polymorphonuclear Leucocyte ◽  
Limiting Factor ◽  
Polymorphonuclear Leucocytes ◽  
Hydrolysis Of

Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products of their breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a ‘second signal’ (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.

scholarly journals Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP

1984 ◽  
Vol 222 (1) ◽  
pp. 103-110 ◽  
Author(s):  
S E Rittenhouse
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Human Platelet ◽  
Human Platelets ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Free Arachidonic Acid ◽  
Stimulation Of

Human platelets exposed to the Ca2+ ionophore A23187 form cyclo-oxygenase metabolites from liberated arachidonic acid and secrete dense granule substituents such as ADP. I have shown previously that A23187 causes activation of phospholipase A2 and some stimulation of phospholipase C. I now report that, in contrast to the case for thrombin, the activation of phospholipase C in response to ionophore is completely dependent upon the formation of cyclo-oxygenase products and the presence of ADP. The addition of A23187 to human platelets induces a transient drop in the amount of phosphatidylinositol 4,5-bisphosphate, a decrease in the amount of phosphatidylinositol, and the formation of diacylglycerol and phosphatidic acid. In addition, lysophosphatidylinositol and free arachidonic acid are produced. The presence of cyclo-oxygenase inhibitors or agents which remove ADP partially impairs these changes. When both types of inhibitor are present, the changes in phosphatidylinositol 4,5-bisphosphate and the formation of diacylglycerol and phosphatidic acid are blocked entirely, whereas formation of lysophosphatidylinositol and free arachidonic acid are relatively unaffected. The prostaglandin H2 analogue U46619 activates phospholipase C. This stimulation is inhibited partially by competitors for ADP. I conclude that phospholipase C is not activated by Ca2+ in the platelet, and suggest that stimulation is totally dependent upon a receptor coupled event.

scholarly journals Effect of Phospholipase C Hydrolysis of Membrane Phospholipids on Membranous Enzymes

1972 ◽  
Vol 247 (9) ◽  
pp. 2835-2841 ◽  
Author(s):  
Richard D. Mavis ◽  
Robert M. Bell ◽  
P. Roy Vagelos
Keyword(s):  
Phospholipase C ◽  
Membrane Phospholipids ◽  
Hydrolysis Of

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 Activation of phospholipase C and prostaglandin synthesis by [arginine]vasopressin in cultures

1984 ◽  
Vol 223 (3) ◽  
pp. 855-859 ◽  
Author(s):  
J Pfeilschifter ◽  
A Kurtz ◽  
C Bauer
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Prostaglandin E2 ◽  
Phospholipase C ◽  
Arginine Vasopressin ◽  
Mesangial Cells ◽  
Significant Loss ◽  
Prostaglandin Synthesis ◽  
Phosphatidylinositol 4 ◽  
In Cells

[Arginine]vasopressin (AVP) stimulates maximal prostaglandin E2 production in cultured rat renal mesangial cells within 2 min. As early as 10s after addition of AVP (10(-6)M) a significant loss of radioactivity from phosphatidylinositol 4,5-bisphosphate but not from phosphatidylinositol 4-phosphate and phosphatidylinositol was observed in cells prelabelled with 32Pi. Cells labelled with [14C]arachidonic acid showed an increase of label in 1,2-diacylglycerol after 15 s and in phosphatidic acid after 30 s upon stimulation with AVP. Pretreatment of the cells with indomethacin (10(-5)M) did not abolish the effect of AVP on the increased labelling of phosphatidic acid.

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.

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