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.

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.

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.

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.

REQUIREMENT FOR THROMBIN RECEPTOR OCCUPANY DURING PLATELET SECRETION UNDER AGGREGATING CONDITIONS

1987 ◽  
Author(s):  
Kazuo Koike ◽  
Holm Holmsen
Keyword(s):  
Receptor Occupancy ◽  
Dense Granule ◽  
Secretory Process ◽  
Thrombin Receptor ◽  
Cell Contact ◽  
Acid Hydrolase ◽  
High Concentration ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Secretion

We have previously showed that hirudin abruptly arrests thrombin-induced secretion of acid hydrolase at any stage of its progress, whereas it only affects dense granule secretion only at its initial stages; these results have been interpreted to show that acid hydrolase secretion requires sustained while dense granule secreion ony requires a brief receptor occupancy (Holmsen et al. JBC 256(1981)9393). The requirement for receptor occupancy in thrombin-induced α-granule secretion and secretion during aggregation have been studied. Increasing concentrations of thrombin were added to gel-fitered platelets containing a constant, high concentration of hirudin. Dense granule secretion was initiated at lower thrombin concentration than those required for α-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 14-fold exess of hirudin produced abrupt stop of dense granule secretion and α-granule secretion when added to platelets shortly after thrombin; it had no affect after these secretory process had reached 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, all three secretory processes increased their rates and could now be abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule andoαgranule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum. It is concluded thatαgranule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for hydrolase secretion.α-granule secretion might, however, require longer occupancy than dense granule secretion. It is possible that aggregation potentiates all secretory responses through close cell contact and that the abrupt inhibition by hirudin of all secretions may have been caused by its effect on the slower aggregation.

scholarly journals Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 154-162 ◽  
Author(s):  
B Lages ◽  
H Holmsen ◽  
HJ Weiss ◽  
C Dangelmaier
Keyword(s):  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Magnesium Content ◽  
Calcium Pyrophosphate ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Strongly Correlated ◽  
Storage Pool ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.

scholarly journals Platelet functions and energy metabolism in a patient with hexokinase deficiency

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 147-153 ◽  
Author(s):  
JW Akkerman ◽  
G Rijksen ◽  
G Gorter ◽  
GE Staal
Keyword(s):  
Adenine Nucleotides ◽  
Energy Charge ◽  
Energy Generation ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Glycolytic Flux ◽  
Adenylate Energy Charge ◽  
Severe Reduction ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK- deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N- acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.

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 Platelet functions and energy metabolism in a patient with hexokinase deficiency

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 147-153
Author(s):  
JW Akkerman ◽  
G Rijksen ◽  
G Gorter ◽  
GE Staal
Keyword(s):  
Adenine Nucleotides ◽  
Energy Charge ◽  
Energy Generation ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Glycolytic Flux ◽  
Adenylate Energy Charge ◽  
Severe Reduction ◽  
Dense Granule Secretion ◽  
Granule Secretion

We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK- deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N- acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.

Assessment Of Energy Costs Of Secretory Responses In Platelets By Abrupt Arrest Of ATP Regeneration

1981 ◽  
Author(s):  
J W N Akkerman ◽  
G Gorter ◽  
H Holmsen
Keyword(s):  
Energy Consumption ◽  
Dense Granule ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Energy Costs ◽  
Dense Granules ◽  
Metabolic Conditions ◽  
Energy Consuming ◽  
Dense Granule Secretion ◽  
Granule Secretion

A new method has been developed for the quantitative assessment of energy consuming processes in platelets. Under carefully controlled metabolic conditions ATP resynthesis is abruptly blocked by a cocktail of metabolic inhibitors. This leads to a fall in metabolic ATP, which is linear with time between 0 and 30 sec after addition of the inhibitors. Evidence is presented that this fall reflects the velocity by which the platelets consume metabolic energy prior to addition of the inhibitors. Resting platelets consume 4 μmol ATP equivalents/min/1011 cells at 37° and 0.5 μmol (same units) at 15°C. When thrombin (5 U/ml) is included in the inhibitor-mixture, aggregation and secretion of dense granules (3H-serotonin), α-granules (β-thromboglobulin) and lysosomal granules (N acetyl β glucosaminidase) follow despite the arrest in ATP resynthesis. The fall in metabolic ATP is now much steeper, reflecting an increase in energy consumption during these functions. Using changes in temperature as a means to affect secretion and energy metabolism, secretion velocity (measured between 0 and 10 sec after thrombin addition) can be compared with simultaneous energy consumption (measured between 0 and 30 sec after thrombin addition). At a consumption of 12 ymol ATP/min/1011 cells secretion velocity of dense-, α- and lysosomal granules is 100, 95 and 50% of uninhibited suspensions, respectively. At 6 μmol (same units) these percentages are 70, 35 and 25%.If thrombin is added after addition of the inhibitors thereby initiating secretion at lowered metabolic ATP levels, secretion is slower as metabolic ATP is lower. Again lysosomal granule secretion is more inhibited than α-granule secretioiy. which is slower than dense granule secretion. These data reflect an increasing need for metabolic energy in the order: dense-, α- and lysosomal granule secretion.

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