scholarly journals Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets

1984 ◽  
Vol 222 (2) ◽  
pp. 389-394 ◽  
Author(s):  
C Solberg ◽  
C Little ◽  
S Holme ◽  
S E Aakre
Keyword(s):  
Plasma Membrane ◽  
Lactate Dehydrogenase ◽  
Bacillus Cereus ◽  
Phospholipase C ◽  
Lipid Extraction ◽  
Human Platelets ◽  
Platelet Concentrate ◽  
Membrane Phospholipids ◽  
Phospholipid Hydrolysis

Phospholipase C (from Bacillus cereus) was used to study fresh and stored human platelets. Provided that the enzyme was inactivated before lipid extraction, no significant degradation of phospholipid in fresh cells was noted, even when platelets were activated or induced to change shape by ADP, collagen or thrombin. With platelets isolated from concentrates stored for transfusion for 4 days at 22 degrees C, membrane phospholipids were degraded by the enzyme to an extent depending on the pH in the platelet concentrate at day 4 of storage. The extent of phospholipid hydrolysis in platelets correlated well with the extent of release of lactate dehydrogenase during storage, with both being minimal for platelets from concentrates of final pH 6.5-6.9. Under non-lytic conditions, phosphatidylcholine was the phospholipid most degraded (40%), with no significant degradation of phosphatidylserine being detected. Storage does not seem to alter the distribution of phospholipids at the external leaflet of the plasma membrane.

Effect of Phospholipase C on Human Platelets

1975 ◽  
Author(s):  
A.-B. Otnœss
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Electron Micrographs ◽  
Gel Filtration ◽  
Outer Part ◽  
Human Platelets ◽  
Scanning Electron Micrographs ◽  
Platelet Factor ◽  
Asymmetrical Distribution ◽  
Scanning Electron

The effect on human platelets of phospholipase C (Bacillus cereus) has been studied. Platelets prepared by gel filtration lost 20-30% of their phospholipids when incubated with phospholipase C for 20 min. Phosphatidylethanolamine (PE) was reduced by about 50%, whereas phosphatidylcholine and phosphatidylserine were reduced each by about 20%. Sphingomyelin was not reduced.These data suggest an asymmetrical distribution of phospholipids in the platelet membrane, PE being more accessible and therefore probably mainly located in the outer part of the membrane.The loss of phospholipids was not accompanied by aggregation, nor did the platelets lose their ability to aggregate with thrombin or ADP. Data on release of serotonin, platelet factor 3 and 4 and scanning electron micrographs of treated platelets will be given.

Platelet Activation : Role of Exogenous and Endogenous Phospholipases

1979 ◽  
Author(s):  
B. Perret ◽  
G. Mauco ◽  
M.F. Simon ◽  
H. Chap ◽  
L. Douste-Blazy
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Alternative Pathway ◽  
Hydrolysis Product ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Platelet Response ◽  
Phospholipid Hydrolysis ◽  
Outer Leaflet

Phosoholipase A2 from bee venom induces aggregation of human platelets, provided that phospholipid hydrolysis is enabled by simultaneous incubation with sphingomyelinase C. Inhibition of the platelet response by indomethacin indicates that aggregation is due to arachidonic acid release. On another hand, this model allows to describe an asymmetrie distribution of arachidonic acid, whose only 6% is located in the outer leaflet of the plasma membrane.During platelet aggregation by phospholipase C, the diacylglycerol and its hydrolysis product 2-acyl-glycerol are phosphorylated into phosphatide and lysophosphatidic acids, respectively. As the same kinds of changes occur in the presence of thrombin, a unifying hypothesis for platelet activation is proposed, involving the stimulation of an endogenous phospholipase C, whose some properties will be reported (neutral optimal pH, Ca-requlrement, phosphatidylinositol specificity and cytosol-localization). This model can be related to the recent finding that phosphatide acid behaves as a calcium-ionophore (Gerrard, J.M. et al., Prostaglandins Med., 1978, 1, 387) and provides an alternative pathway for arachidonic acid mobilization.

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.

scholarly journals Inhibition of Bacillus cereus phospholipase C by univalent anions

1982 ◽  
Vol 203 (3) ◽  
pp. 799-801 ◽  
Author(s):  
S E Aakre ◽  
C Little
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Bovine Brain ◽  
Erythrocyte Ghosts ◽  
Substrate Effects ◽  
Phospholipid Hydrolysis ◽  
Marked Inhibition ◽  
Soluble Substrate

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.

scholarly journals Utilization of phosphatidylcholine and production of diradylglycerol as a consequence of sphingomyelin synthesis

1998 ◽  
Vol 331 (1) ◽  
pp. 251-256 ◽  
Author(s):  
Daniel J. SILLENCE ◽  
David ALLAN
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Bacillus Cereus ◽  
Phospholipase C ◽  
De Novo ◽  
Total Cell ◽  
Baby Hamster Kidney ◽  
Similar Composition ◽  
Bhk Cells ◽  
Human Leukaemia

1. After the degradation of cell-surface sphingomyelin (SM) by exogenous sphingomyelinase (SMase), the resynthesis of SM by baby-hamster kidney (BHK) and human leukaemia-60 (HL-60) cells was examined in relation to utilization of substrate phosphatidylcholine (PtdCho) and generation of the expected product, diradylglycerol (DRG). Using [3H]choline-labelled BHK cells incubated in non-radioactive medium, SMase caused a release of phosphocholine, which was derived approximately equally from SM and PtdCho, consistent with the anticipated resynthesis of SM at the expense of PtdCho. However, with choline-labelled cells incubated in radioactive medium or [14C]acetate-labelled cells treated with SMase, no loss of radioactivity from PtdCho or accumulation of labelled DRG was observed, suggesting that any DRG produced as a consequence of SM synthesis must have been rapidly converted back into PtdCho. In contrast, SMase treatment of HL-60 cells caused more than a doubling of DRG levels at the expense of PtdCho, and this appears to be the first demonstration of a rise in DRG related to the synthesis of SM. The DRG produced consisted of about 80% 1,2-diacylglycerol and 18% 1-O-alkyl-2-acylglycerol species, a similar composition to that of the DRG backbone of total cell PtdCho. 2. The requirement for cell-surface PtdCho in the biosynthesis of SM by BHK cells was also investigated. Treatment of [3H]choline-labelled BHK cells with Bacillus cereus PtdCho-specific phospholipase C (PLC) rapidly degraded about 6% of the total PtdCho, which was assumed to represent the cell-surface pool. This did not appear to be the pool of PtdCho required for SM synthesis, since (a) the released phosphocholine was additional to that derived from PtdCho in cells treated with SMase and (b) treatment with PLC did not affect SM synthesis, either de novo or in response to degradation of cell-surface SM by SMase. These findings suggest either that there is no SM synthase in the plasma membrane or, if it is present, then it does not utilize cell-surface PtdCho as a substrate.

scholarly journals Activation of V1-receptors by vasopressin stimulates inositol phospholipid hydrolysis and arachidonate metabolism in human platelets

1986 ◽  
Vol 233 (1) ◽  
pp. 83-91 ◽  
Author(s):  
W Siess ◽  
M Stifel ◽  
H Binder ◽  
P C Weber
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Second Messengers ◽  
Inositol Trisphosphate ◽  
Time Lag ◽  
Human Platelets ◽  
Phospholipid Hydrolysis ◽  
Low Concentrations ◽  
Inositol Phospholipid ◽  
High Concentrations

The activation of platelet V1-receptors by vasopressin (0.01-1 microM) induces the rapid formation of inositol phosphates, 1,2-diacylglycerol and phosphatidic acid, indicating inositol phospholipid hydrolysis by phospholipase C. Vasopressin immediately induces the formation of inositol bisphosphate and inositol trisphosphate. Accumulation of inositol 1-monophosphate and inositol 4-monophosphate occurs later after a time lag of 15 s. Low concentrations (10-100 nM) of vasopressin only activate phospholipase C, whereas high concentrations (1 microM) induce activation of phospholipase C and subsequently the production of arachidonate metabolites. Cyclo-oxygenase metabolites are associated with further activation of phospholipase C, release reaction and irreversible platelet aggregation. Vasopressin requires for its action extracellular Mg2+, but not Ca2+. The described platelet changes are not induced by 1-desamino-[8-D-arginine]vasopressin, a V2-receptor agonist, and are blocked by a specific V1-receptor antagonist. The results indicate that platelets possess a V1-receptor that is coupled to polyphosphoinositide hydrolysis by phospholipase C, leading to the formation of 1,2-diacylglycerol and inositol trisphosphate. Those compounds may act as second messengers for platelet responses induced by vasopressin, whereas endoperoxides and thromboxane A2 stimulated by vasopressin may serve as amplifiers for platelet activation.

scholarly journals Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1331
Author(s):  
Ana Bura ◽  
Antonija Jurak Begonja
Keyword(s):  
Plasma Membrane ◽  
Membrane Lipids ◽  
Methodological Approach ◽  
Lipid Extraction ◽  
Human Platelets ◽  
Antibody Staining ◽  
Cell Compartments ◽  
Staining Protocol ◽  
Phosphatidylinositol 4 ◽  
Wall Injury

Phosphoinositides (PIs) are phosphorylated membrane lipids that have a plethora of roles in the cell, including vesicle trafficking, signaling, and actin reorganization. The most abundant PIs in the cell are phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P2 and PI4P are well established, is the broadly accepted methodological approach for their immunocytochemical visualization in different cell compartments in several cell lines. However, not much is known about these PIs in platelets (PLTs), the smallest blood cells that detect vessel wall injury, activate, and stop the bleeding. Therefore, we sought to investigate the localization of PI(4,5)P2 and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P2 and PI4P can be detected by the established staining protocol, and these pools can be modulated by inhibitors of OCRL phosphatase and PI4KIIIα kinase. However, although resting PLTs readily stain for the plasma membrane (PM) pools of PI(4,5)P2 and PI4P, just a few activated cells were stained with the established protocol. We show that optimized protocol allows for the visualization of PI(4,5)P2 and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P2 and PI4P are more sensitive to lipid extraction by permeabilizing agents in activated than in resting human PLTs, which suggests their different roles during PLT activation.

Inositol lipids and TRPC channel activation

2007 ◽  
Vol 74 ◽  
pp. 37-45 ◽  
Author(s):  
James W. Putney
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Transient Receptor Potential ◽  
Calcium Signalling ◽  
Receptor Potential ◽  
Breakdown Product ◽  
Channel Activation ◽  
Inositol Lipids ◽  
Transient Receptor ◽  
Secondary Mechanism

The original hypothesis put forth by Bob Michell in his seminal 1975 review held that inositol lipid breakdown was involved in the activation of plasma membrane calcium channels or ‘gates’. Subsequently, it was demonstrated that while the interposition of inositol lipid breakdown upstream of calcium signalling was correct, it was predominantly the release of Ca2+ that was activated, through the formation of Ins(1,4,5)P3. Ca2+ entry across the plasma membrane involved a secondary mechanism signalled in an unknown manner by depletion of intracellular Ca2+ stores. In recent years, however, additional non-store-operated mechanisms for Ca2+ entry have emerged. In many instances, these pathways involve homologues of the Drosophila trp (transient receptor potential) gene. In mammalian systems there are seven members of the TRP superfamily, designated TRPC1–TRPC7, which appear to be reasonably close structural and functional homologues of Drosophila TRP. Although these channels can sometimes function as store-operated channels, in the majority of instances they function as channels more directly linked to phospholipase C activity. Three members of this family, TRPC3, 6 and 7, are activated by the phosphoinositide breakdown product, diacylglycerol. Two others, TRPC4 and 5, are also activated as a consequence of phospholipase C activity, although the precise substrate or product molecules involved are still unclear. Thus the TRPCs represent a family of ion channels that are directly activated by inositol lipid breakdown, confirming Bob Michell's original prediction 30 years ago.

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