Simultaneous Assay of Agonist-Induced Endogenous and Non-Radioactive Isotopic Serotonin Secretion from Small Numbers of Human Platelets By Mass Spectrometry

Abstract Background: Light transmission aggregometry (LTA) with simultaneous assay of ATP released from platelet dense granules (lumi-aggregation) provides the ability to assess platelet function in a variety of clinical settings. This methodology, however, requires the ability to obtain adequate sample volume from the patient, if a number of platelet agonists are to be assessed. The platelet count within the patient's blood is also a limiting factor, and LTA interpretations can become difficult when platelet counts in the sample fall significantly below 150,000/µL. Analysis of platelet function by flow cytometry circumvents the limitations of both blood volume and low platelet counts, typically providing information as to glycoprotein IIb/IIIa activation state and release of platelet alpha granules in response to platelet agonists. Flow cytometry, however, is not presently capable of providing quantitative analysis of the release of platelet dense granule constituents. Given the clinical importance of platelet dense granule secretion, we sought to develop a non-radioactive method for quantitation of agonist-induced release of serotonin (5-HT) from blood sample volumes obtainable from small infants and from patients with at least moderate thrombocytopenia. Methods: In the initial studies, total platelet content and released 5-HT following stimulation by a panel of agonists were assessed using 200 µL of freshly drawn, normal citrated blood. In subsequent studies, uptake and agonist-induced secretion of non-radioactive, deuterated serotonin (d-5HT) were additionally measured in parallel with the endogenous 5-HT in these 200 µL samples. In brief, the citrated blood was initially incubated for 15' at 37° with d-5HT, then diluted 10-fold with buffer, and platelet stimulus (collagen, TRAP, ADP, epinephrine, ionophore A23187, thromboxane analogue U46619, ristocetin, or buffer blank) added. Following stimulation, samples were further diluted with buffer, prostaglandin E1 added, and then centrifuged to yield supernatants for analysis. 5-HT and d-5HT total content in unstimulated platelets was additionally assayed. N-methyl-5HT was employed as an internal standard. 5-HT, d-5HT and N-methyl-5HT were subsequently detected and quantified simultaneously by liquid chromatography/tandem mass spectrometry (MS) (Eksigent MicroLC 200/QTrap 6500 Mass Spectrometer, Sciex, Framingham, MA). Results and Conclusions: MS analysis of 5-HT was linear over the range of 50-2000 pg/mL. For whole blood platelet counts of at least 120,000/µL, this sensitivity was sufficient to permit analysis of 5-HT released in response not only to strong stimuli, but even to weaker stimuli such as low concentration (2 µg/mL) collagen. Since the goal of this project, however, was to be able to extend functional analysis to patients with more significant degrees of thrombocytopenia, further efforts were made to improve sensitivity. Assay of agonist-induced release, following platelet uptake, of d-5HT was assessed in parallel with the assay of released endogenous 5-HT. Release of 5-HT and d-5HT in response to increasing concentrations of collagen showed generally similar kinetics. However, not only was the absolute signal increased for the d-5HT, but the analytic assay itself proved significantly more sensitive as compared to that for 5-HT. Combined, these effects increased the overall sensitivity of the assay by at least an order of magnitude. For example, d-5HT released in response to 2 µg/mL collagen in a donor blood having a platelet count of 188,000/µL produced an MS signal approximately 30-fold higher than the lower limit of detection for the assay. Although it will require actual study of thrombocytopenic patients to verify, these results suggest that by use of this MS approach with d-5HT it may be possible to measure platelet dense granule secretion to a panel of agonists from 200 µL of blood in patients with thrombocytopenia potentially as severe as 10,000/µL. Disclosures No relevant conflicts of interest to declare.

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

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqz112.044 ◽

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.

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

10.1055/s-0038-1653271 ◽

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.

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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 ◽

10.1182/blood.v61.1.154.154 ◽

1983 ◽

Vol 61 (1) ◽

pp. 154-162 ◽

Cited By ~ 20

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.

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Misshapen/NIK-related kinase (MINK1) is involved in platelet function, hemostasis, and thrombus formation

Blood ◽

10.1182/blood-2015-07-659185 ◽

2016 ◽

Vol 127 (7) ◽

pp. 927-937 ◽

Cited By ~ 12

Author(s):

Ming Yue ◽

Dongjiao Luo ◽

Shanshan Yu ◽

Pu Liu ◽

Qi Zhou ◽

...

Keyword(s):

Platelet Function ◽

Thrombus Formation ◽

Dense Granule ◽

Key Points ◽

Dense Granule Secretion ◽

Granule Secretion

Key Points MINK1 promotes hemostasis and thrombosis in vivo. MINK1 specifically regulates platelet dense-granule secretion.

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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 ◽

10.1182/blood.v61.1.154.bloodjournal611154 ◽

1983 ◽

Vol 61 (1) ◽

pp. 154-162 ◽

Cited By ~ 1

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.

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Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqz094 ◽

2019 ◽

Vol 152 (6) ◽

pp. 718-724 ◽

Cited By ~ 1

Author(s):

Siaw Li Chan ◽

Xin Yi ◽

Emily Wysocki ◽

Rachael Bridgman ◽

Jocelyn Gutierrez ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Dense Granule ◽

Serotonin Uptake ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

Dense Granule Secretion ◽

Granule Secretion

Abstract Objectives Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC–MS/MS. Results This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Conclusions Quantification of d45-HT by micro-LC–MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

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Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel

Blood ◽

10.1182/blood-2012-07-444281 ◽

2012 ◽

Vol 120 (25) ◽

pp. 5041-5049 ◽

Cited By ~ 68

Author(s):

Ban B. Dawood ◽

Gillian C. Lowe ◽

Marie Lordkipanidzé ◽

Danai Bem ◽

Martina E. Daly ◽

...

Keyword(s):

Healthy Volunteers ◽

Platelet Function ◽

Light Transmission ◽

Dense Granule ◽

Phenotypic Analysis ◽

Atp Secretion ◽

Platelet Function Disorders ◽

Platelet Signaling ◽

Dense Granule Secretion ◽

Granule Secretion

Abstract Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A2 (TxA2) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA2 pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y12 ADP and TxA2 receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

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Novel RUNX1 Mutation in a Family with an Uncharacterized Secretion Defect

Blood ◽

10.1182/blood.v126.23.3458.3458 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3458-3458

Author(s):

M. Badin ◽

C. P.M. Hayward ◽

S. Tasneem ◽

G. Pare ◽

A. D. Paterson ◽

...

Keyword(s):

Exome Sequencing ◽

Platelet Function ◽

Index Case ◽

Family Members ◽

Dense Granule ◽

Platelet Function Disorders ◽

Single Base Pair ◽

Dense Granule Secretion ◽

Granule Secretion ◽

Runx1 Mutation

Abstract Background: Platelet function disorders are a common cause of a bleeding problem. While many rare and severe forms of platelet function disorders are well studied, many common platelet function defects are uncharacterized. In a family with uncharacterized platelet secretion defects, we identified a single base pair insertion in the gene RUNX1 by exome sequencing. We report on the clinical and laboratory phenotype of the defect in this family. Methods: Bleeding histories of affected and unaffected family members, obtained using a standardized questionnaire, were scored using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT). Laboratory data evaluated included blood counts, platelet aggregation responses, dense granule ATP release findings (lumiaggregometry) and platelet dense granule counts, evauated by whole mount preparations and electron microscopy. Affection status was based on the recorded opinion of the subject's hematologist, which concurred with diagnostic laboratory findings. Exome sequencing was performed on the index case, followed by evaluation of all family members for the candidate mutation by polymerase chain reaction (PCR) amplification and Sanger sequencing. Results: Family members investigated (median age: 25.5, range 1-69] included 5 males (affected: n=4, unaffected: n=1) and 3 females (affected: n=2, unaffected: n=1). ISTH BAT bleeding scores for affected members were elevated (median: 10.5, range 4-20), unlike unaffected family members (n=2) and healthy controls (n=40) (ranges: 0-1 and 0-2). Platelet counts were low in 2/6 affected individuals (109 platelets/L : affecteds: median: 164, range: 125-169). While unaffected family members had unremarkable platelet function findings, the affected individuals had absent or reduced dense granule secretion with all agonists tested (including thrombin, collagen, epinephrine, U46619 and arachidonic acid), and in aggregation tests, they had absent secondary aggregation with epinephrine, reduced maximal aggregation with collagen (1.25 and 5.0 μg/mL) and thromboxane analogue U46619, variable responses to arachidonic acid (reduced in 4/5 affecteds), and normal responses to ADP and ristocetin. Only some (3/6) affected family members had dense granule deficiency (lower reference interval limit: 4.9/platelet; affecteds: median: 5.2, range: 4-6). A single base pair insertion (A) in exon 6 of the RUNX1 gene (NM_001754.4:c.583dup) was identified in the index case using exome sequencing. The mutation results in a reading frame shift at codon Ile195, that is expected to end in a premature termination codon 17 positions downstream. Sanger sequencing further confirmed this mutation was present in 6/6 affected family members and it was absent in both unaffected family members. Further evaluation of the family history indicated that only one family member (deceased aunt of the index case) had developed leukemia or myelodysplasia although her mutation status is unknown. Conclusion: We identified a novel, frameshift RUNX1 mutation in a family with an inherited platelet function disorder associated with increased bleeding symptoms. This variant has not been previously reported in exome sequencing of over 60 000 individuals as documented by the ExAC consortium. The variable platelet count and dense granule numbers among affected individuals in this family emphasize the importance of phenotyping multiple family members. RUNX1 defects should be considered as a potential cause of inherited abnormalities in dense granule secretion, even in individuals without dense granule deficiency or a striking family history of leukemia/myelodysplasia. Disclosures No relevant conflicts of interest to declare.

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

10.1055/s-0038-1644475 ◽

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.

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

Biochemical Journal ◽

10.1042/bj1820413 ◽

1979 ◽

Vol 182 (2) ◽

pp. 413-419 ◽

Cited By ~ 14

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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