Functional Assessment of Platelet Dense Granule ATP Release

2021 ◽  
Author(s):  
Joseph H Cho ◽  
Geoffrey D Wool ◽  
Melissa Y Tjota ◽  
Jocelyn Gutierrez ◽  
Krzysztof Mikrut ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Atp Release ◽  
Dense Granule ◽  
Assay Optimization ◽  
Specimen Processing ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Optimized Conditions ◽  
Formalin Fixed ◽  
Granule Release

Abstract Objectives This study was undertaken to explore the feasibility of assessing platelet dense granule release in response to platelet stimuli, using less than 1 mL of whole blood (WB). Methods Optimization of the luciferin-luciferase (LL) assay for ATP release, together with additional modifications, was applied to 1:10 diluted WB. Results LL assay optimization using nonstirred 1:10 diluted WB resulted in dense granule ATP release in response to thrombin receptor-activating peptide (TRAP) of similar magnitude to that observed using stirred platelet-rich plasma. Stirring of the 1:10 diluted WB restored collagen-induced dense granule secretion. Addition of lyophilized, formalin-fixed platelets, together with stirring, restored dense granule secretion responsiveness to ADP. TRAP, ADP, and collagen all stimulated ATP release in 1:10 diluted WB under the optimized conditions of this study at levels close to those observed using platelet-rich plasma. Blood sample reconstitution experiments offer hope that this assay may prove robust down to WB platelet counts as low as 50 × 103/μL. Conclusions Platelet dense granule release in response to a number of classic stimuli, including ADP, was accomplished from less than 1 mL WB with minimal specimen processing, using widely available reagents and instrumentation.

An Optimized Assay for Assessing Platelet Dense Granule Secretion Using Diluted Whole Blood

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S23-S23
Author(s):  
Joseph Cho ◽  
Krzysztof Mikrut ◽  
Jonathan Miller
Keyword(s):  
Whole Blood ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Dense Granule ◽  
Platelet Counts ◽  
Agonist Stimulation ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Assessment of dense granule secretion following agonist stimulation is an integral part of platelet function testing and can be performed in the clinical laboratory using lumi-aggregometry. A significant limitation of traditional lumi-aggregometry is the relatively large volume of blood required and the normal platelet counts needed to perform the assay, which often precludes usage of this test in pediatric and thrombocytopenic patients. By optimizing the luciferase-luciferin reaction with commercially available reagents and using a conventional lumi-aggregometer, we have developed a method that measures platelet dense granule secretion following agonist stimulation in diluted whole blood, at up to 10-fold dilutions. With the goal of developing a testing method independent of platelet count, the assay intentionally does not include specimen stirring. Direct comparison of the optimized reagents with the standard Chrono-lume reagent in 10-fold diluted whole blood showed an improved lower limit of detection for exogenously added ATP by at least one order of magnitude. We reproducibly observed dose-dependent responses in platelet ATP release using this assay upon stimulation with platelet agonists such as the thrombin receptor-activating peptide (TRAP) and the collagen receptor agonist convulxin. ATP release in 10-fold diluted whole blood, for example, was 168.6 ± 24.7 pmoles/107 platelets in response to 40 μM TRAP and 17.1 ± 2.9 pmoles/107 platelets in response to 1 nM convulxin (mean ± SEM, N = 6). Method comparisons of this assay were performed using 10-fold diluted whole blood without stirring, compared to standard methodology using undiluted platelet-rich plasma stirred at 1000 rpm. On concurrently run, matched specimens, ATP release in response to a series of agonists of varying stimulus intensity showed overall moderate concordance, with an r2 = 0.722. Based on ATP standard curves and the observed agonist responses to date, we are hopeful that this assay may prove capable of assessing platelet dense granule release in patient blood with platelet counts as low as 20,000/μL. Additionally, the assay requires less than 60 μL of whole blood per agonist with testing performed at two different agonist concentrations. The extended analytical range and robustness of the assay, with no need for centrifugation, offer promise that it may be useful for the assessment of platelet dense granule secretion in pediatric or thrombocytopenic patients who require assays amenable to limited blood volumes and low platelet counts.

Effect Of Prostacyclin (PGI2) On Human Platelet Aggregation And Secretion

1981 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Factor V ◽  
Platelet Factor ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Data Representative

PGI2,which increases platelet cAMP(Prostaglandins 13: 389,1977),is a potent inhibitor of aggregation and secretion .We stidued the time course of the same return of platelet function after exposure of platelets to PGI2.Sepharose 2B columns were equilibrated with Tyrode’s albumin buffer, pH7.5 (no Ca2+) containing PGI2 (534nM). Platelet rich plasma was applied and eluted with the same buffer. The filtered platelets(GFP) were then subsampled hourly after elution from the column. Fibrinogen was added to finel concentration of 1.7mg/ml. Platelet aggregation(PA) and release of 14C serotonin (5HT),platelet factor 4(PF4), and factor V (FV) were assayed after stimulation of the platelet by collagen(C), ADP,epinephrine(E), arachidonic acid(AA) and ionophore A23187(I). Data representative of 5 separate studies follow.I(20μg/ml) induced PA was 76%(Ohr),52%(1hr) and 61%(2hr and beyond). Release of 5HT, FV,and PF4 were 60%,1.89u,and 7.97 yg/10 pit, respectively, at time 0 and increased progressively, reaching a plateau at 2 hr. AA(500μg/ml) was 10%(0hr),30%(2hr),68%(3hr) and 8%(4hr). Release of 5HT paralleled PA but release of FV and PF4 remained suppressed for 4 hrs. In contrast α-granule (PF4 and FV)release by C(μg/ml)increased as PA increased while dense granule secretion remained suppressed. PA as well as a and dense granule secretion by ADP (10μM) were minimal during 4 hrs. PA and FV secretion by E (55μM) also remain inhibited for 4 hrs. In spite of this normal dense granule release occurred initially and declined progressively over 4 hours.

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.

Differential Regulation of α-Granule and Dense Granule Secretion by an Actin Cytoskeletal Barrier.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3528-3528
Author(s):  
Robert Flaumenhaft ◽  
James R. Dilks ◽  
Nataliya Rozenvayn ◽  
Rita A. Monahan-Earley ◽  
Dian Feng ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Actin Polymerization ◽  
Dense Granule ◽  
Snare Protein ◽  
Latrunculin A ◽  
Cytochalasin E ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Cytoskeletal Disruption ◽  
Granule Release

Abstract Platelet granule secretion is an essential component of normal arterial thrombus formation. Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and loss of granules. These observations have lead to the supposition that cytoskeletal contraction facilitates granule secretion. Yet, the influence of the actin cytoskeleton in controlling membrane fusion events required for granule secretion remains largely unknown. Initial studies using electron microscopy revealed that the actin disrupting agents latrunculin A (4 μM) or cytochalasin E (4 μM) prevented pseudopod formation and granule centralization in platelets exposed to SFLLRN or PMA, but did not prevent degranulation. We next determined the effects of disruption of the actin cytoskeleton on α-granule secretion by monitoring P-selectin expression and β-thromboglobulin release. Incubation of platelets with either latrunculin A or cytochalasin E failed to stimulate α-granule secretion, but increased the rate of SFLLRN-induced α-granule secretion by 3.5-fold. Cytoskeletal disruption also augmented the degree of SFLLRN-induced α-granule secretion by 41±18% and reduced the amount of SFLLRN required to cause half-maximal stimulation by 2-fold. Incubation with latrunculin A stimulated α-granule secretion by the weak secretogues epinephrine or ADP by 7.6-fold and 5.4-fold, respectively. Cytoskeletal disruption also facilitated β-thromboglobulin release in response to SFLLRN, epinephrine, or ADP. In platelets permeabilized in the absence of ATP, exposure to 2 μM latrunculin A resulted in a 6.5- and 3.5-fold increase in α-granule release induced by Ca2+- or GTP-γ-S, respectively. Antibodies directed at a SNARE protein termed vesicle-associated fusion protein (VAMP) inhibited latrunculin A-dependent α-granule secretion. Thus, disruption of the actin cytoskeletal barrier by latrunculin A supports SNARE protein-dependent membrane fusion. Since actin acts as a barrier to α-granule secretion, we evaluated α-granules purified by subcellular fractionation for the presence of F-actin. Purified α-granules, but not phospholipid micelles, bound the F-actin probe FITC-phalloidin as determined by flow cytometry. FITC-phalloidin binding was inhibited in a dose-dependent manner by latrunculin A. These data indicated that α-granules are coated with F-actin that could serve a barrier function. We next evaluated the effects of cytoskeletal disruption on dense granule secretion by monitoring ADP/ATP release using a luciferin-luciferase based assay and by quantifying [3H]serotonin release. Cytoskeletal disruption by 4 μM latrunculin A failed to affect the degree of dense granule secretion from platelets stimulated by either SFLLRN, epinephrine, or ADP. Yet, 200 μM latrunculin A stimulated substantial dense granule release in the absence of agonist exposure and augmented SFLLRN-induced dense granule release by 2-fold. In contrast, 200 μM latrunculin A abolished SFLLRN-induced α-granule secretion. These observations indicate that the cytoskeleton differentially regulates α-granule and dense granule secretion. Our results also suggest that while some degree of actin polymerization is required for α-granule secretion, dense granule secretion is not dependent on actin polymerization.

The roles of αIIbβ3-mediated outside-in signal transduction, thromboxane A2, and adenosine diphosphate in collagen-induced platelet aggregation

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2646-2651 ◽  
Author(s):  
Moon J. Cho ◽  
Junling Liu ◽  
Tamara I. Pestina ◽  
Shirley A. Steward ◽  
Dennis W. Thomas ◽  
...  
Keyword(s):  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Signaling Proteins ◽  
Platelet Signaling ◽  
Protein Kinase C Inhibitor ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
High Level ◽  
Granule Release

Collagen-induced activation of platelets in suspension leads to αIIbβ3-mediated outside-in signaling, granule release, thromboxane A2 (TxA2) production, and aggregation. Although much is known about collagen-induced platelet signaling, the roles of TxA2 production, adenosine diphosphate (ADP) and dense-granule secretion, and αIIbβ3-mediated outside-in signaling in this process are unclear. Here, we demonstrate that TxA2 and ADP are required for collagen-induced platelet activation in response to a low, but not a high, level of collagen and that αIIbβ3-mediated outside-in signaling is required, at least in part, for this TxA2 production and ADP secretion. A high level of collagen can activate platelets deficient in PLCγ2, Gαq, or TxA2 receptors, as well as platelets treated with a protein kinase C inhibitor, Ro31-8220. Thus, activation of αIIbβ3 in response to a high level of collagen does not require these signaling proteins. Furthermore, a high level of collagen can cause weak TxA2 and ADP-independent aggregation, but maximal aggregation induced by a high level of collagen requires TxA2 or secretion.

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

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 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.

SHIP-1 Differentially Regulates Dense Granule Secretion in Platelets through Its Association with Protein Kinase C δ.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3630-3630
Author(s):  
Ramya Chari ◽  
Soochong Kim ◽  
Swaminathan Murugappan ◽  
James L. Daniel ◽  
Satya P. Kunapuli
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dense Granule ◽  
Protease Activated Receptors ◽  
Kinase C ◽  
Inositol Phosphatases ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Granule Release ◽  
Stimulation Of

Abstract Collagen-induced glycoprotein (GP) VI-mediated and thrombin-induced protease activated receptors (PAR)-mediated activation are important signaling pathways regulating dense granule secretion in platelets. Protein kinase C (PKC) isoforms play a crucial role in platelet secretion and we have previously shown that PKCδ plays a ying-yang role in dense granule release by different agonists (Murugappan et al, J. Biol. Chem. 2004). PKCδ isoform positively regulates PAR-mediated platelet dense granule release, whereas it negatively regulates GPVI-mediated dense granule release. In this study, we investigated the mechanism of such differential regulation by PKCδ downstream of PAR and GPVI pathways. We hypothesize that the differential association of PKCδ with phosphatases downstream of GPVI and PAR receptors differentially regulate dense granule secretion. More specifically, we explored the functional relevance of the interaction of PKCδ with Src homology 2-domain containing Inositol Phosphatases (SHIP), 5′-inositol phosphatases in platelets. In our studies, SHIP-1 was tyrosine phosphorylated by both PARs and GPVI receptors and its phosphorylation followed different activation kinetics. Whereas PAR-mediated SHIP-1 phosphorylation (Y1020) was delayed and occurred as late as 120 seconds, the GPVI-mediated SHIP-1 phosphorylation was rapid, starting as early as 15 seconds and peaked at 60 seconds. Co-immunoprecipitation experiments revealed that SHIP-1, and not SHIP-2, associated with PKCδ upon stimulation of platelets with GPVI agonist, convulxin. However, such association did not occur with the PAR agonists. GPVI-mediated SHIP-1 phosphorylation failed to occur in platelets from mice lacking Lyn kinase suggesting a role for Lyn in regulating SHIP-1 phosphorylation. In murine platelets lacking either Lyn or SHIP-1, dense granule secretion was potentiated by convulxin and not by thrombin. We attribute the phosphorylation and association of SHIP-1 with PKCδ to be critical for the regulation of agonist-induced dense granule secretion in platelets. Based on the above results, we conclude that the preferential association of SHIP-1 with PKCδ upon stimulation of GPVI receptor results in the negative regulation of collagen-induced dense granule release in platelets.

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.

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