scholarly journals Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

1990 ◽  
Vol 1 (13) ◽  
pp. 1027-1041 ◽  
Author(s):  
J R Coorssen ◽  
M M Davidson ◽  
R J Haslam
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Inositol Phosphate ◽  
Human Platelets ◽  
Response Curves ◽  
Factors Affecting ◽  
Dense Granules ◽  
High Concentrations ◽  
Granule Secretion ◽  
Gamma S

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.

scholarly journals Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators

1994 ◽  
Vol 303 (2) ◽  
pp. 391-400 ◽  
Author(s):  
L F Lau ◽  
K Pumiglia ◽  
Y P Côté ◽  
M B Feinstein
Keyword(s):  
G Protein ◽  
Phosphatidyl Inositol ◽  
Human Platelets ◽  
Complex Model ◽  
Intrinsic Activity ◽  
Thrombin Receptor ◽  
Proteolytic Activation ◽  
Cytoplasmic Phospholipase A2 ◽  
High Concentrations ◽  
Granule Secretion

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5′-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5′-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors.

THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION

1987 ◽  
Author(s):  
C T Poll ◽  
J Westwick
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Free Calcium ◽  
Fluorescent Properties ◽  
Fura 2 ◽  
Stimulus Response ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

scholarly journals Collagen-induced platelet activation mainly involves the protein kinase C pathway

1990 ◽  
Vol 268 (2) ◽  
pp. 325-331 ◽  
Author(s):  
A Karniguian ◽  
F Grelac ◽  
S Levy-Toledano ◽  
Y J Legrand ◽  
F Rendu
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Membrane Receptors ◽  
Lag Phase ◽  
Response Curves ◽  
Kinase C ◽  
Metabolic Event ◽  
Granule Secretion

This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage.

Stimulation by G Protein βγ Subunits of Phospholipase C β Isoforms in Human Platelets

1998 ◽  
Vol 79 (05) ◽  
pp. 1008-1013 ◽  
Author(s):  
Yoshiko Banno ◽  
Tomiko Asano ◽  
Yoshinori Nozawa
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Human Platelet ◽  
Minor Component ◽  
Similar Efficacy ◽  
Human Platelets ◽  
Bovine Brain ◽  
Γ Subunit ◽  
A Minor

SummaryDifferent phospholipase C (PLC) isoforms were located in human platelet cytosol and membranes. PLCγ2 and PLCβ3b were mainly located in the cytosol and PLCβ2 and PLCβ3a were in both cytosol and membranes by using specific antibodies against PLC isozymes (Banno Y, Nakashima S, Ohzawa M, Nozawa Y. J Biol Chem 1996; 271: 14989-94). Three PLC fractions activated by G protein βγ subunits were purified from human platelet cytosol and membrane fractions. Two PLC fractions from membranes were identified as PLCβ2 and PLCβ3a, and one from cytosol was PLCβ3b. These PLCβ isoforms were activated by the purified βγ subunits of brain G proteins in the order PLCβ3b > PLCβ3a > PLCβ2. Western blot analysis of γ subunits of the purified platelet G proteins with antibodies against various standard γ subunits revealed that the major component of the γ subunit of Gi2 and Gq was γ5, and that γ7 was a minor component. Studies using various subtypes of βγ subunits, βγ2, βγ3, and βγ7 purified from bovine brain, βγ5 from bovine lung, or βγ12 from bovine spleen, failed to show differences in their ability to stimulate the isolated platelet PLCβ isoforms. These results suggest that the βγ subunits of Gi2 and Gq have similar efficacy in regulation of effectors in human platelets.

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

2011 ◽  
Vol 436 (2) ◽  
pp. 469-480 ◽  
Author(s):  
Knut Fälker ◽  
Linda Haglund ◽  
Peter Gunnarsson ◽  
Martina Nylander ◽  
Tomas L. Lindahl ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Human Platelets ◽  
Dense Granule ◽  
Receptor Internalization ◽  
Functional Responses ◽  
Protease Activated Receptors ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Aggregation

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both Gα12/13 and Gαq signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2+ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y12 receptor-induced Gαi signalling accounted for the loss of the aggregation response, as mimicking Gαi/z signalling with 2-MeS-ADP (2-methylthioadenosine-5′-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

Pertussis toxin sensitive G-protein coupling of HDL receptor to phospholipase C in human platelets

1992 ◽  
Vol 67 (5) ◽  
pp. 559-567 ◽  
Author(s):  
H. Nazih ◽  
D. Devred ◽  
F. Martin-Nizard ◽  
V. Clavey ◽  
J.C. Fruchart ◽  
...  
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Pertussis Toxin ◽  
Human Platelets ◽  
G Protein Coupling ◽  
Protein Coupling

scholarly journals G-proteins are not directly involved in the CD3-antigen-mediated production of inositol phosphates in HPB-ALL T-leukaemia cells expressing phospholipase C isoforms γ1 and β3

1993 ◽  
Vol 289 (2) ◽  
pp. 387-394 ◽  
Author(s):  
M Biffen ◽  
M Shiroo ◽  
D R Alexander
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
G Protein ◽  
G Proteins ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cross Linking ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Inositol Phosphate Production

The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.

scholarly journals ASK1, a Novel Regulator of Thrombosis, Is Activated Downstream of G-Protein Coupledreceptors in Human Platelets

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4163-4163
Author(s):  
Randall Derstine ◽  
Meghna Ulhas Naik ◽  
Ramya Turaga ◽  
Ulhas P Naik
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
Platelet Activation ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Kinase Signaling ◽  
Dense Granules ◽  
Map Kinase Signaling

Abstract In the event of vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins such as collagen and Von Willebrand factor and activate to form a stable hemostatic platelet plug. Defects in the molecular mechanisms dictating platelet plug formation are responsible for numerous thrombotic disorders. Elucidating the signaling pathways and molecular mechanisms of platelet activation is paramount to the development of safer and more effective anti-thrombotic drugs. While it is known that MAP-Kinase signaling participates in platelet activation, it is unknown how MAP-Kinase signaling specifically mediates platelet activation. Our laboratory has identified the presence and activation of a MAP-Kinase Kinase Kinase known as Apoptosis Signal Regulating Kinase 1 (ASK1). We have demonstrated using an ASK1 knockout mouse model that ablation of ASK1 leads to a significantly increased (p = .0003) time of vessel occlusion associated with unstable thrombus formation following a carotid artery injury induced by 10% FeCl3. Furthermore, ASK1 knockout mice display protection from pulmonary thromboembolism induced by an intravenous injection of collagen and epinephrine. In order to determine the kinetics of ASK1 activation by physiological agonists, washed human platelets (4 x 108 platelets/mL) were treated with 0.1 U/mL of thrombin for 30”, 1’, 3’, 5’, and 8’. Robust activation of ASK1 by thrombin occurred as early as 30 seconds up until 5 min, after which ASK1 activation decreased sharply. Platelets treated with 100 µM of PAR1 (SFLLRN) or PAR4 (AYPGKF) peptides resulted in strong ASK1 activation, suggesting that both the PAR1 and PAR4 receptors lead to ASK1 activation. Inhibition of Src family kinases by PP2 or PI3K by wortmannin or Rho kinase by Y-27632 had no effect on thrombin-induced ASK1 activation. However, inhibition of PLC-β2, a mediator of platelet activation downstream of the PAR1/4 receptors, strongly inhibited ASK1 activation by thrombin. We next determined whether TxA2 generation was responsible for ASK1 activation by thrombin. Washed platelets were pre-treated with 1 mM aspirin to block TxA2 generation, followed by treatment with 0.1 U/mL of thrombin. It was found that blocking TxA2 generation eliminated ASK1 activation by thrombin at 30” and 1’, but not at a later time point, suggesting there may be an additional pathway contributing to ASK1 activation. The observation that TxA2 generation contributes to ASK1 activation by thrombin seemed to correlate with the finding that treatment of platelets with 1 µM of the TxA2 mimetic U46619, which activates the TP-α receptor, could also activate ASK1. We also determined whether ADP released from dense granules, which would activate the P2Y1 and P2Y12 receptors, leads to ASK1 activation. To test this, washed platelets were pre-treated with 1 U/mL of apyrase to hydrolyze secreted ADP. It was found that apyrase treatment completely eliminates ASK1 activation by thrombin, suggesting a strong dependency of thrombin-induced ASK1 activation on ADP release from dense granules. To further investigate this possibility, washed platelets were pre-treated with 50 µM of the P2Y1 antagonist MRS2179 or P2Y12 antagonist 2-MeSAMP, followed by treatment with 0.1 U/mL of thrombin. Antagonism of the P2Y12 receptor and not P2Y1 receptor severely diminished ASK1 activation by thrombin. This indicates that ASK1 activation by thrombin is also dependent on ADP released from dense granules and subsequent activation of the P2Y12 receptor. Surprisingly, collagen, a strong activator of platelets, was unable to activate ASK1 in washed platelets at a concentration of 2 µg/mL. Similarly, 2 µM epinephrine treatment also had no effect. However, when washed platelets were treated with 2 µg/mL collagen and 2 µM epinephrine together, a strong ASK1 activation was observed (p=.0012). This suggests the existence of a novel mechanism for ASK1 activation by simultaneous stimulation of the collagen receptors GPVI/α2β1 and epinephrine receptor α2A. The finding that ASK1 activation occurs downstream of TP-α, P2Y12, and possibly α2A receptors highlights the importance of ASK1 in regulation of these G-Protein Coupled Receptors in platelet activation. In conclusion, our data indicates ASK1 to be a key mediator in platelet activation and represents a novel target for anti-thrombotic drug therapy. Disclosures No relevant conflicts of interest to declare.

Ca2+ channel blockers distinguish between G protein-coupled pharmacomechanical Ca2+ release and Ca2+ sensitization

1991 ◽  
Vol 260 (2) ◽  
pp. C364-C370 ◽  
Author(s):  
S. Kobayashi ◽  
M. C. Gong ◽  
A. V. Somlyo ◽  
A. P. Somlyo
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Smooth Muscles ◽  
Channel Blockers ◽  
Alpha Toxin ◽  
Adrenergic Agonist ◽  
Pharmacomechanical Coupling ◽  
Gamma S ◽  
G Protein Coupled ◽  
Ca2 Channels

The effects of Ca2+ channel blockers on two modes of G protein-mediated pharmacomechanical coupling, Ca2+ release and modulation of Ca2+ sensitivity of the contractile apparatus, were investigated. Smooth muscles were permeabilized with Staphylococcal alpha-toxin or with beta-escin to avoid effects due to block of sarcolemmal Ca2+ channels, while retaining receptor/G protein coupling. In permeabilized portal vein smooth muscle, verapamil and nifedipine inhibited Ca2+ release induced by an alpha 1-adrenergic agonist (phenylephrine) and by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not that induced by inositol 1,4,5-trisphosphate (InsP3). These Ca2+ channel blockers also did not block the phenylephrine- or GTP gamma S-induced force development at constant cytoplasmic Ca2+ ("Ca2+ sensitization"). An alpha 1-blocker (prazosin) inhibited both the Ca2(+)-releasing and Ca2(+)-sensitizing effects of phenylephrine, but not those of GTP gamma S, nor did it block InsP3-induced Ca2+ release. We conclude that Ca2+ channel blockers selectively uncouple the Ca2(+)-releasing, but not the Ca2(+)-sensitizing, component of pharmacomechanical coupling. These findings raise the possibility that pharmacomechanical Ca2+ release may be modulated by dihydropyridine binding proteins at the level of G proteins/phospholipase C and also indicate a divergence of the Ca2(+)-releasing and Ca2(+)-sensitizing effects at some step prior to phospholipase C.

scholarly journals Stimulated cholesterol-enriched platelets display increased cytosolic Ca2+ and phospholipase A activity independent of changes in inositol trisphosphates and agonist/receptor binding

1990 ◽  
Vol 265 (3) ◽  
pp. 747-754 ◽  
Author(s):  
A Sorisky ◽  
G L Kucera ◽  
S E Rittenhouse
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Receptor Binding ◽  
Choline Chloride ◽  
Surface Membrane ◽  
Human Platelets ◽  
Phospholipase A ◽  
Release Mechanism ◽  
Membrane Microviscosity ◽  
Substrate Interaction

To investigate the mechanism of enhanced responsiveness of cholesterol-enriched human platelets, we compared stimulation by surface-membrane-receptor (thrombin) and post-receptor (AlF4-) G-protein-directed pathways. Platelets were labelled with [32P]Pi and [methyl-3H] choline chloride, incubated with sonicated lipid dispersions of various ratios of cholesterol and phospholipid, and loaded with the fluorescent Ca2+ indicator fura-2. We report the following. (1) Cholesterol enrichment enhances cytosolic Ca2+ accumulation and phospholipase A activation in response to both receptor-directed and post-receptor-directed agonists. No enhancement by cholesterol of phospholipase A activity at fixed Ca2+ concentrations is observed in lysed platelets, implying that no perturbation by cholesterol of phospholipase A/substrate interaction occurs in our preparations. (2) In both normal and cholesterol-enriched platelets, Ca2+ mobilization is promoted by a factor(s) apart from InsP3 that appear(s) to be modulated by cholesterol. A disproportionate increase in cytosolic Ca2+ relative to [32P]InsP3 is observed with increasing doses of thrombin in normal, and to a larger extent in cholesterol-enriched, platelets. When AlF4- is the agonist, there is no cholesterol-associated enhancement in [32P]InsP3 to account for the heightened Ca2+ rise seen with cholesterol enrichment. (3) Enhanced phospholipase A activation is not necessarily proportional to cytosolic Ca2+ increase. The magnitude of the increase in phospholipase A activity for a given rise in cytosolic Ca2+ is greater in cholesterol-enriched platelets that are stimulated by AlF4- than in those stimulated by thrombin. We conclude that increased membrane microviscosity associated with cholesterol enrichment may promote G-protein/phospholipase A interaction as well as the Ca2(+)-release mechanism, without significantly altering G-protein/phospholipase C interaction.

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