Comparative study of platelet dense granule constituents

1982 ◽  
Vol 243 (3) ◽  
pp. R454-R461 ◽  
Author(s):  
K. M. Meyers ◽  
H. Holmsen ◽  
C. L. Seachord
Keyword(s):  
Comparative Study ◽  
Gel Filtration ◽  
Human Platelets ◽  
Dense Granule ◽  
Releasable Pool ◽  
Rabbit Platelets ◽  
Granule Secretion

Cat, cattle, dog, horse, human, mink, pig, and rabbit platelets were separated from plasma by gel filtration. The gel-filtered platelets (GFP) were treated with thrombin to induce maximal granule secretion and the potential dense granule constituents ATP, ADP, serotonin (5-HT), Ca2+, and Mg2+ were measured in GFP and in the control and thrombin-treated platelets and in the respective supernatants. The amount of Ca2+, Mg2+, 5-HT, ATP, and ADP within the nonreleasable pool for all species varied between 3.1 and 10.0 mumol/10(11) platelets for Ca2+ and Mg2+ was less than 1.5 mumol/10(11) platelets for ADP and 5-HT and was between 2.0 and 5.0 mumol/10(11) platelets for ATP. Marked differences were observed in the releasable fraction. Human platelets were characterized by the largest releasable Ca2+ pool (greater than 10 mumol/10(11) platelets), the smallest secretable 5-HT and Mg2+ pool (less than 0.5 mumol/10(11) platelets), and the lowest ATP-to-ADP ratio (greater than 1.0). Pig platelets had the highest amount of releasable Mg2+ (approximately 8.0 mumol/10(11) platelets). Rabbits platelets released the most 5-HT (greater than 3.0 mumol/10(11)) and had the highest ATP/ADP (greater than 5.0). The releasable pool of Ca2+, Mg2+, ATP, and ADP in the remaining species varied in mumol/10(11) platelets from approximately 1.5-4.0, approximately 1.0-3.0, 0.5-3.5, and approximately 0.5-1.5, respectively.

INHIBITION BY NEOMYCIN OF AGONIST-INDUCED POLYPHOSPHOINOSITIDE METABOLISM AND RESPONSES IN HUMAN PLATELETS

1987 ◽  
Author(s):  
Holm Holmsen ◽  
Ole-Bjørn Tysnes ◽  
Adrie J M Verhoeven ◽  
Vidar M Steen ◽  
Lindsey J Moore ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Human Platelets ◽  
Dense Granule ◽  
Stimulus Response ◽  
P Content ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Phosphatidylinositol 4 ◽  
Induced Aggregation ◽  
Aggregation Of Platelets

Signal processing in platelets seems to involve polyphosphoinositide (PPI) metabolism, although direct coupling between PPI metabolism and responses has not been proved. Neomycin binds tightly to PPIs and has been used to probe the involvement of PPI metabolism and responses in platelets. Neomycin(SO4)3 powerfully inhibited ADP- and adrenaline-induced aggregation of platelets in PRP. This was partly due to the sulphate anion; the chloride form was therefore prepared. Platelets were prelabelled in PRP with 32P-Pi. and transferred by gel filtration to a calcium-free Tyrode’s solution (GFP). Increasing concentrations (2-5 mM) of neomycinCl6 caused progressive inhibition of thrombin-induced aggregation, dense granule secretion, acid hydrolase secretion and formation of 32P-phosphatidic acid (PA); the inhibition was immediate, not affected by aspirin and counteracted by increasing thrombin concentrations. Incubation of neomycin (up to 5 mM) with this GFP or with P-Pi. On GFP prepared from unlabelled PRP had no effect on the P content of ATP, phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4,5-bisphosphate (PIP ). Increasing neomycin concentrations caused progressive inhibition of the thrombin-induced init^l (10 sec) decrease, but not of the late (90 sec) incg^ase i-n P-PIP2 while they enhanced the increase in P-PIP. Similar results were obtained ^th collagen and PAF. Both the increase in cytosolic Ca and pH (measured by INDO-I and BCECF, respectively) induced by thrombin were inhibited progressively by increasing concentrations of neomycin. These results are in support for a direct involvement of PPI metabolism in the stimulus-response coupling below the receptor level. However, the failure of neomycin to affect turnover of PIP and PIP2 in nonstimulated platelets suggests that the aminoglycoside does not penetrate the membrane, and only become available to PPI during stimulation.

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 Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

1979 ◽  
Vol 182 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Holm Holmsen ◽  
Linda Robkin ◽  
H. James Day
Keyword(s):  
Shape Change ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Antimycin A ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Dense Granule Secretion ◽  
Granule Secretion

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [14C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [14C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected 14C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, β-N-acetylglucosaminidase, β-glucuronidase and β-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)–response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.

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.

scholarly journals Thrombopoietin potentiates activation of human platelets in association with JAK2 and TYK2 phosphorylation

1996 ◽  
Vol 316 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Belén RODRÍGUEZ-LIÑARES ◽  
Steve P. WATSON
Keyword(s):  
Shape Change ◽  
Physiological Role ◽  
Human Platelets ◽  
Dense Granule ◽  
Functional Responses ◽  
Dramatic Effect ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Dense Granule Secretion ◽  
Granule Secretion

Thrombopoietin (TPO), also known as the c-mpl ligand, stimulates rapid tyrosine phosphorylation of multiple proteins in human platelets including the Janus family kinases JAK2 and TYK2. On its own, TPO has no effect on platelet aggregation and dense-granule secretion but induces a general potentiation of these responses by other stimuli. The most dramatic effect is observed against threshold concentrations of agonists for aggregation. Shape change or weak reversible aggregation induced by low concentrations of thrombin, collagen and the thromboxane mimetic, U46619, are converted into irrreversible aggregation in the presence of TPO. A similar result is obtained in the presence of the ADP scavenger apyrase and cyclo-oxygenase inhibitor indomethacin. TPO also induces potentiation of dense-granule secretion measured through release of 5-hydroxy[3H]tryptamine. This effect is most striking against low concentrations of stimuli and is independent of aggregation as it is observed in the presence of chelation of extracellular Ca2+ with EGTA. TPO potentiates activation of phospholipase C and elevation of intracellular Ca2+, providing a molecular explanation for potentiation of functional responses. TPO may have an important physiological role in priming platelet activation in thrombocytopenia, an action that may help to compensate for the reduced platelet density.

scholarly journals iPLA2β Contributes to Activation of Human and Mouse Platelets Via ITAM Receptors Fcγ RIIA and GPVI

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2339-2339
Author(s):  
Shaji Abraham ◽  
Pravin Patel ◽  
Ulhas P. Naik ◽  
Steven Edward McKenzie
Keyword(s):  
Transgenic Mouse ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Significant Inhibition ◽  
Dense Granule ◽  
Secretory Cells ◽  
Western Blots ◽  
Granule Secretion ◽  
Human And Mouse

Platelet activation by ITAM receptors contributes to hemostasis, thrombosis, vascular integrity and host defense. In the course of our studies of FcγRIIA-mediated platelet activation, we became interested in those mechanisms that require neither full Syk activation nor changes in intracellular calcium. Calcium-independent phospholipase family member iPLA2β has been observed to modulate phospholipid remodeling and second messenger generation in human platelets (Beckett, Thromb Res 2007; Duvernay, Biochem 2015), while iPLA2γ has been studied in knockout mouse platelets (Yoda, JTH 2014) with modest effects noted on thrombosis and hemostasis. These enzymes do not require increased cytoplasmic calcium for their activity in cleaving the acyl group from the sn2 position of phospholipids to yield a free fatty acid and a lysophospholipid. However, the precise role of iPLA2β in human and mouse platelet activation has not been elucidated. Neither has the contribution of iPLA2β in the response to FcγRIIA-mediated activation been reported. We identified the presence of iPLA2β protein in western blots of human and FcγRIIA transgenic mouse platelets. Of interest, multiple isoforms arising from proteolytic cleavage were detected. We treated washed human and FcγRIIA transgenic mouse platelets with agonists to FcγRIIA (IV.3 + GAM) and to GPVI (collagen or collagen-related peptide) in the absence and presence of pharmacologic inhibitors of iPLA2β. At a range of agonist doses up to 3X threshold, we observed significant inhibition of aggregation, dense granule secretion and alpha granule secretion (p<0.05 vs. vehicle only, n = 3 to 4 each). Inhibition occurred with either S-BEL (bromo-enolactone) or with FKGK18 (a fluoroketone), two chemically distinct iPLA2β inhibitor molecules with different modes of action. The IC50 for S-BEL was found to be 1.02 uM for human FcγRIIA, 2.04 uM for human GPVI, and 2.76 uM for transgenic mouse FcγRIIA activated platelets. FKGK18 was less potent, with IC50s at 7.88 uM for human FcγRIIA. In contrast, iPLA2γ inhibitor R-BEL was able to inhibit FcγRIIA -mediated activation, but at an IC50 of 2.62 uM. Notably, iPLA2β inhibition could eliminate ATP secretion from dense granules downstream of FcγRIIA and GPVI activation. When we added ADP to FcγRIIA stimulation in the presence of inhibitory doses of S-BEL, we overcame the inhibition. We have identified for the first time that iPLA2β contributes to aggregation and secretion of both human and FcγRIIA transgenic mouse platelets. The platelets were slightly more sensitive to FcγRIIA than to GPVI inhibition. In other activatable secretory cells, iPLA2β plays both a homeostatic and signaling role. The mechanisms of iPLA2β action in platelets merit further study. Studies are in progress with genetic knockdown and knockout of the enzyme, to complement the findings with inhibitors. Disclosures No relevant conflicts of interest to declare.

Requirement for Thrombin Receptor Occupancy During Platelet Secretion Under Aggregating and Non-Aggregating Conditions

1987 ◽  
Vol 58 (04) ◽  
pp. 1053-1059 ◽  
Author(s):  
Kazuo Koike ◽  
Holm Holmsen
Keyword(s):  
Receptor Occupancy ◽  
Human Platelets ◽  
Dense Granule ◽  
Secretory Process ◽  
Cell Contact ◽  
Acid Hydrolase ◽  
High Concentration ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Fold Excess

SummaryThe requirement for receptor occupancy in thrombin-induced secretion in human platelets has been studied. When increasing concentrations of thrombin were added to gel-filtered platelets containing a constant, high concentration of hirudin, dense granule secretion was initiated at lower thrombin concentrations than those required for α-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 62-fold excess of hirudin produced abrupt stop of dense granule secretion and a-granule secretion when added to non-aggregating (no stirring) platelets shortly after thrombin; it had no affect after these secretory process had reached about 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, the three secretory processes increased their rates and were abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule and α-granule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum.It is concluded that a-granule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for acid hydrolase secretion. α-Granule secretion might, however, require longer occupancy than dense granule secretion. Aggregation is believed to potentiate secretion through close cell contact and the secretion processes were inhibited by hirudin through hirudin’s effect on aggregation.

scholarly journals Lyn, PKC-δ, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3056-3063 ◽  
Author(s):  
Ramya Chari ◽  
Soochong Kim ◽  
Swaminathan Murugappan ◽  
Archana Sanjay ◽  
James L. Daniel ◽  
...  
Keyword(s):  
Human Platelets ◽  
Sh2 Domain ◽  
Dense Granule ◽  
Protease Activated Receptors ◽  
Inositol Phosphatase ◽  
Glycoprotein Vi ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Granule Release ◽  
Stimulation Of

Protein kinase C-δ (PKC-δ) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-δ positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-δ in platelets. SH2 domain–containing inositol phosphatase-1 (SHIP-1) is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists SFLLRN and AYPGKF or GPVI agonist convulxin. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-δ on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-δ–null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1 and Y311 of PKC-δ were inhibited. In murine platelets lacking Lyn or SHIP-1, GPVI-mediated dense granule secretions are potentiated, whereas PAR-mediated dense granule secretions are inhibited. Therefore, we conclude that Lyn-mediated phosphorylations of PKC-δ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.

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