scholarly journals A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses

1983 ◽  
Vol 210 (1) ◽  
pp. 145-155 ◽  
Author(s):  
J W N Akkerman ◽  
G Gorter ◽  
L Schrama ◽  
H Holmsen
Keyword(s):  
Energy Consumption ◽  
Rapid Determination ◽  
Dense Granule ◽  
Metabolic Energy ◽  
Acid Hydrolase ◽  
Temperature Changes ◽  
Novel Method ◽  
Dense Granule Secretion ◽  
Granule Secretion

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.

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.

scholarly journals Comparative studies on the energetics of platelet responses induced by different agonists

1986 ◽  
Vol 236 (3) ◽  
pp. 879-887 ◽  
Author(s):  
A J M Verhoeven ◽  
M E Mommersteeg ◽  
J W N Akkerman
Keyword(s):  
Energy Consumption ◽  
Energy Cost ◽  
Cytochalasin B ◽  
Dense Granule ◽  
Acid Hydrolase ◽  
Total Consumption ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
The Cost ◽  
Effect Of Inhibitors

The correlation between energy consumption and platelet responses induced by collagen, A23187 and ADP was investigated and compared with the energetics of thrombin-stimulated platelets established in earlier work. Aggregation, measured as single-platelet disappearance, and secretion correlated quantitatively with the increment but not with the total consumption of energy, suggesting that the former reflects the energy cost of these responses. The cost of complete aggregation was 2-3 mumol of ATP equivalents/10(11) platelets with collagen, ADP and thrombin as the stimulus. The cost of complete dense-granule secretion was 0.5-0.8 mumol of ATP equivalents/10(11) platelets with all agonists tested. The cost of combined secretion of alpha-granule and acid hydrolase granule contents was 5-7 mumol of ATP equivalents/10(11) platelets with thrombin and collagen. However, in the presence of A23187 much more energy was consumed during aggregation and secretion. Also ADP triggered more energy consumption during secretion than was seen with the other inducers. The effect of inhibitors of aggregation and secretion was investigated in thrombin-stimulated platelets. Raising the cellular cyclic AMP content sharply decreased the increment in energy consumption as well as aggregation and secretion. The cytoskeleton-disrupting agents cytochalasin B and colchicine left the increment in energy consumption intact, but decreased the basal consumption seen in unstimulated platelets. This was accompanied by normal (cytochalasin B) or diminished (colchicine) aggregation and secretion. Apart from the latter exception, all inhibitors decreased secretion and incremental energy consumption in parallel, thereby preserving the energy-versus-secretion relationship established in earlier work. In contrast, aggregation and energy consumption varied independently, suggesting that the coupling with energy consumption is much weaker for this response.

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.

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.

Energy Control During Platelet Secretion:Predominant Role Of ATP-Turnover

1981 ◽  
Author(s):  
A Deijns ◽  
J W N Akkerman
Keyword(s):  
Control Function ◽  
Energy Charge ◽  
Dense Granule ◽  
Metabolic Energy ◽  
Adenylate Energy Charge ◽  
Glucose Addition ◽  
Atp Level ◽  
Atp Turnover ◽  
Dense Granule Secretion ◽  
Granule Secretion

Platelet aggregation and secretion of granular contents require metabolic energy. This implies the existence of a control mechanism that adjusts the rate of energy producing pathways to the energy need of these functions. Such a control function has been attributed to the level of metabolic ATP, to the adenylate energy charge (AEC =(ATP + 1/2ADP)/(ATP + ADP + AMP) and to the velocity of energy generation and consumption (ATP-turnover). In this study we investigated which factor dominates in control of dense granule (3H-serotonin)-, α-granule (β thromboglobulin)- and lysosomal granule (N acetyl β glucosaminidase) secretion. Human gel- filtered platelets were incubated in glucose-free, CN containing medium. Under these conditions ATP-generation only took place in glycogenolysis, which alone was unable to maintain ATP homeostasis. Consequently, metabolic ATP and AEC fell. Addition of glucose restored glycolytic ATP resynthesis wich restored the AEC but the loss of ATP was for the main part irreversible due to hypoxanthin formation. With this system a broad range of metabolic ATP levels and AEC’s could be obtained both at decreasing ATP turnover (before glucose addition) and at increasing ATP turnover (after glucose addition). Analysis of secretion velocity of dense-, α- and lysosomal granules after initiation with thrombin (5 U/ml) showed that at decreasing turnover the secretion velocity of all types of granules depended on both metabolic ATP level and AEC. In contrast, at increasing ATP turnover the correlation between secretion velocity and metabolic ATP level or AEC was lost. Instead, dense granule secretion was faster and lysosomal granule secretion was slower than expected on the basis of metabolic ATP level or AEC, whereas α-granule secretion showed intermediate levels. The data indicate that the three types of granule secretion differ in their dependence on metabolic energy and that apart from metabolic ATP and AEC, ATP turnover is of crucial importance for secretion of granular contents.

scholarly journals Quantification of energy consumption in platelets during thrombin-induced aggregation and secretion. Tight coupling between platelet responses and the increment in energy consumption

1984 ◽  
Vol 221 (3) ◽  
pp. 777-787 ◽  
Author(s):  
A J Verhoeven ◽  
M E Mommersteeg ◽  
J W N Akkerman
Keyword(s):  
Energy Consumption ◽  
Energy Requirement ◽  
Metabolic Energy ◽  
Resting Cells ◽  
Strong Positive Correlation ◽  
Acid Hydrolase ◽  
Tight Coupling ◽  
Total Energy Consumption ◽  
Granule Secretion ◽  
Induced Aggregation

The involvement of metabolic energy in platelet responses was investigated by measuring the energy consumption during aggregation and secretion from dense, alpha- and acid-hydrolase-containing granules. Gel-filtered human platelets were stimulated with different amounts of thrombin (0.05-5.0 units X ml-1). At various stages during aggregation and secretion the energy consumption was measured from the changes in metabolic ATP and ADP following abrupt arrest of ATP resynthesis. Stimulation with 5 units of thrombin X ml-1 increased the energy consumption from 6.2 +/- 0.9 to 17.8 +/- 0.4 mumol of ATPeq. X min-1 X (10(11) platelets)-1 during the first 15 s. It decreased thereafter and returned to values found in resting cells after about 30 s. With 0.05 unit of thrombin X ml-1, the energy consumption accelerated more slowly and took at least 3 min before it normalized. A strong positive correlation was found between the velocities of the three secretion responses and the concurrent energy consumption (a) at different stages of the responses induced by a given dose of thrombin, and (b) at different secretion velocities initiated by different amounts of thrombin. When, at different stages of the responses, the extent of secretion was compared with the amount of energy that had been consumed, a strong linear correlation was found with the increment in energy consumption but not with the total energy consumption. This correlation was independent of the concentration of thrombin and indicated that complete secretion from dense, alpha- and acid-hydrolase-containing granules was paralleled by an increment of 4.0, 6.5 and 6.7 mumol of ATPeq. X (10(11) platelets)-1, respectively. An energy cost of 0.7 mumol of ATPeq. X (10(11) platelets)-1 was calculated for separate dense-granule secretion, whereas the combined alpha- and acid-hydrolase granule secretion required 5.3 mumol of ATPeq. X (10(11) platelets)-1. There was no correlation between energy consumption and optical aggregation. In contrast, the rate of single platelet disappearance, which is a measure for the early formation of small aggregates, correlated closely with the rate of energy consumption. Compared with secretion, however, the energy requirement of single platelet disappearance was minor, since 2mM-EDTA completely prevented this response but decreased the energy consumption only slightly. An increase of 0.5-1.0 mumol of ATPeq. X (10(11) platelets)-1 was seen before single platelet disappearance and the three secretion responses were initiated, indicating an increase in energy consuming processes that preceded these responses.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

scholarly journals Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability

2014 ◽  
Vol 290 (3) ◽  
pp. 1536-1545 ◽  
Author(s):  
Ewelina M. Golebiewska ◽  
Matthew T. Harper ◽  
Christopher M. Williams ◽  
Joshua S. Savage ◽  
Robert Goggs ◽  
...  
Keyword(s):  
Dense Granule ◽  
Dense Granule Secretion ◽  
Granule Secretion

scholarly journals Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis

2021 ◽  
Vol 5 (3) ◽  
pp. 674-686
Author(s):  
Tony G. Walsh ◽  
Yong Li ◽  
Christopher M. Williams ◽  
Elizabeth W. Aitken ◽  
Robert K. Andrews ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Surface Expression ◽  
Dense Granule ◽  
Conditional Knockout ◽  
Integrin Activation ◽  
Platelet Factor ◽  
Glycoprotein Vi ◽  
Exocyst Complex ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract The exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5′-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.

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