Human Platelet Storage Organelles A Review

SummaryPlatelets contain numerous electron-dense subcellular organelles which have been referred to in the literature by various names such as α-granules, electron-dense and very electrondense granules, lysosomes, dense bodies, etc. Most of the organelles are secretory granules, since induction of secretion by appropriate stimuli causes degranulation of platelets and the appearance of the granule contents in the extracellular medium. Among the substances that are known to be stored and secreted by platelets are: serotonin, ATP, ADP, calcium, pyrophosphate, acid hydrolases, fibrinogen, vascular permeability factor, β-thromboglobulin, platelet factor 4 and growth factor.The recent literature concerning the localization of the secreted substances within specific platelet organelles is reviewed here. Results from electron microscopy and microprobe analysis, selective secretion experiments, subcellular fractionation studies and studies on platelets from patients with storage pool deficiency indicate that there are as many as four types of storage organelles in human platelets.

Download Full-text

Secretion, subcellular localization and metabolic status of inorganic pyrophosphate in human platelets. A major constituent of the amine-storing granules

Biochemical Journal ◽

10.1042/bj1920099 ◽

1980 ◽

Vol 192 (1) ◽

pp. 99-105 ◽

Cited By ~ 29

Author(s):

M H Fukami ◽

C A Dangelmaier ◽

J S Bauer ◽

H Holmsen

Keyword(s):

Time Course ◽

Subcellular Fractionation ◽

Human Platelets ◽

Major Constituent ◽

Inorganic Pyrophosphatase ◽

Acid Hydrolases ◽

Bivalent Cation ◽

Inorganic Pyrophosphate ◽

Dense Granules ◽

Amine Storage

The platelet content of PPi is 1.90 +/- mumol/10(11) platelets (S.E.M., n = 19) or about 10.5 nmol/mg of protein, several hundred times that found for rat liver. Some 80% of this PPi is secreted by platelets treated with thrombin with a time course and dose-response relationship similar to secretion of ATP, ADP and 5-hydroxytryptamine (serotonin) from the platelet dense granules. During platelet aggregation induced by ADP and adrenaline, substantial amounts of PPi were secreted, but no release of acid hydrolases was observed. Subcellular-fractionation studies showed that the PPi is highly enriched in the same fraction that contains the storage organelles which store ATP, ADP, Ca2+ and 5-hydroxytryptamine. Inorganic pyrophosphatase was present mainly in the soluble fraction and in the mitochondria. Secretion studies done with platelets prelabelled with [32P]Pi showed that the sequestered PPi was relatively metabolically inactive, as is the ATP and ADP in the storage organelles. The possible participation of PPi in the formation of a bivalent-cation-nucleotide complex associated with amine storage is discussed.

Download Full-text

Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient

10.1055/s-0038-1652358 ◽

1981 ◽

Cited By ~ 1

Author(s):

F Rendu ◽

M Lebret ◽

J P Caen

Keyword(s):

Plasma Membranes ◽

Blood Platelets ◽

Human Platelets ◽

Release Mechanism ◽

Inherited Disorders ◽

Platelet Factor ◽

Functional Studies ◽

Dense Bodies ◽

Serotonin Uptake And Release

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.

Download Full-text

Differential energy requirements for platelet responses. A simultaneous study of aggregation, three secretory processes, arachidonate liberation, phosphatidylinositol breakdown and phosphatidate production

Biochemical Journal ◽

10.1042/bj2080009 ◽

1982 ◽

Vol 208 (1) ◽

pp. 9-18 ◽

Cited By ~ 48

Author(s):

Holm Holmsen ◽

Karen L. Kaplan ◽

Carol A. Dangelmaier

Keyword(s):

Energy Charge ◽

Human Platelets ◽

Dense Granule ◽

Metabolic Inhibitors ◽

Energy Requirements ◽

Adenylate Energy Charge ◽

Acid Hydrolases ◽

Platelet Factor ◽

Dense Granules ◽

Collagen Secretion

Previous studies have indicated different energy requirements for some platelet responses; these differences could, however, be due to inadequate methodology and differences in platelet preparation. The present study describes the effect of decreasing ATP availability on seven platelet responses measured in gel-filtered human platelets. The cells, prelabelled with 5-hydroxy[3H]tryptamine, [3H]- or [14C]adenine, [32P]Pi or [3H]arachidonate, were incubated with antimycin A and 2-deoxy-d-glucose. Platelet responses induced by thrombin and collagen (secretion only), level of metabolic ATP and the adenylate energy charge (AEC) were determined at various times during incubation. Platelet aggregation was rapidly inhibited after a lag of 5–15 min and with 50% inhibition at AEC = 0.55–0.60. Secretion of 5-hydroxy[14C]tryptamine and ATP + ADP from dense granules and of fibrinogen and β-thromboglobin from α-granules were inhibited in parallel, without a lag and with 50% inhibition at AEC = 0.65–0.70. The inhibition of secretion of platelet factor 4 from the α-granules followed another pattern with 50% inhibition at AEC = 0.70–0.80. Breakdown of [3H]-phosphatidylinositol, formation of [3H]- and [32P]-phosphatidate, liberation of [3H]arachidonate and secretion of acid hydrolases were inhibited in parallel and inhibition was present at the start of incubation with 50% inhibition at AEC = 0.80–0.87. These results suggest that the responses have different energy requirements, increasing in the order: aggregation < dense granule and α-granule secretion < acid hydrolase secretion, phosphatidylinositol breakdown, phosphatidate formation and arachidonate liberation. The powerful inhibition of phosphatidylinositol breakdown by metabolic inhibitors suggests that energy-requiring steps are involved in the activation of phospholipase C.

Download Full-text

Platelet Heparin-Neutralizing Factor (Platelet Factor 4)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647812 ◽

1975 ◽

Vol 33 (01) ◽

pp. 066-072 ◽

Cited By ~ 11

Author(s):

E. F Lüscher ◽

R Käser-Glanzmann

Keyword(s):

Molecular Weight ◽

Basic Protein ◽

Adenine Nucleotide ◽

Human Platelets ◽

Clotting System ◽

Platelet Factor ◽

High Molecular Weight Complex ◽

Dense Bodies ◽

Peptide Moiety ◽

Platelet Aggregates

SummaryThe heparin-neutralizing platelet factor (PF4) is released from platelets under the influence of inducers of aggregation and nucleotide-release in the form of a high-molecular weight complex. The heparin-neutralizing activity is carried by a basic protein of molecular weight 29,700, four of which combine with a proteoglycan carrier, which in turn consists of 4 chondroitin sulfate A residues and a peptide moiety.The carrier-PF4-complex dissociates at higher ionic strength into proteoglycan and PF4 proper ; both forms (bound and free) are active in the inactivation of heparin, which displaces PF4 from its natural mucopolysaccharide carrier.PF4 is localized in human platelets in storage organelles, most likely in the α-granules and not in the adenine nucleotide and serotonin containing dense bodies. It is released simultaneously with the contents of these latter organelles.The physiological significance of PF4 is mainly seen in the interference with the inhibition of the clotting system by heparin within and in the vicinity of platelet aggregates. Released in circulation in the course of intravascular platelet damage, it may contribute to a facilitation of thrombin formation and action, by removing heparin.Adequately purified PF4 does not interact with fibrinogen and its derivatives and therefore is not directly involved in the induction of intravascular coagulation or platelet aggregation.

Download Full-text

Distribution of phospholipids, fatty acids, and platelet factor 3 activity among subcellular fractions of human platelets

Blood ◽

10.1182/blood.v47.6.963.963 ◽

1976 ◽

Vol 47 (6) ◽

pp. 963-971 ◽

Cited By ~ 37

Author(s):

MJ Broekman ◽

RI Handin ◽

A Derksen ◽

P Cohen

Keyword(s):

Fatty Acids ◽

Phosphatidyl Ethanolamine ◽

Total Content ◽

Subcellular Fractionation ◽

Human Platelets ◽

Phospholipid Content ◽

Platelet Factor ◽

New Information ◽

Phospholipase A2 Activity ◽

Relative Purity

Abstract As compared with other methods, our recently reported method for subcellular fractionation of human platelets improves the separation of mitochondria, alpha granules, and lysosomal enzyme activities. The relative purity of these fractions has led us to undertake the present study to compare the subcellular distribution of phospholipids, fatty acids, and platelet factor 3 (clot-promoting) activity. Two findings pertaining to distribution of phospholipids were entirely new. (1) In the alpha granule zone, plasmalogen phosphatidyl ethanolamine peaked at the expense of diacyl phosphatidyl ethanolamine. (2) The fatty acid composition of the membrane lysophosphatidyl choline suggested that it may have been formed by the action of platelet phospholipase A2 activity. The fatty acids of the membranes showed a markedly asymmetrical distribution in noncholine versus choline phospholipids. The latter held 94%, 72%, and 85%, respectively, of the total content of 16:0, 18:1, and 18:2 fatty acids, whereas 55% of the 18:0, 72% of 20:4, and 67% of higher polyenoic acids other than 20:4 were esterified to the noncholine group. The most important new information related to clot-promoting activity, which, on the basis of protein content, was highest in the membrane fractions, but on the basis of phospholipid content in the nonmembranous fractions. The discussion centers on possible explanations for this novel finding.

Download Full-text

Distribution of phospholipids, fatty acids, and platelet factor 3 activity among subcellular fractions of human platelets

Blood ◽

10.1182/blood.v47.6.963.bloodjournal476963 ◽

1976 ◽

Vol 47 (6) ◽

pp. 963-971 ◽

Cited By ~ 1

Author(s):

MJ Broekman ◽

RI Handin ◽

A Derksen ◽

P Cohen

Keyword(s):

Fatty Acids ◽

Phosphatidyl Ethanolamine ◽

Total Content ◽

Subcellular Fractionation ◽

Human Platelets ◽

Phospholipid Content ◽

Platelet Factor ◽

New Information ◽

Phospholipase A2 Activity ◽

Relative Purity

As compared with other methods, our recently reported method for subcellular fractionation of human platelets improves the separation of mitochondria, alpha granules, and lysosomal enzyme activities. The relative purity of these fractions has led us to undertake the present study to compare the subcellular distribution of phospholipids, fatty acids, and platelet factor 3 (clot-promoting) activity. Two findings pertaining to distribution of phospholipids were entirely new. (1) In the alpha granule zone, plasmalogen phosphatidyl ethanolamine peaked at the expense of diacyl phosphatidyl ethanolamine. (2) The fatty acid composition of the membrane lysophosphatidyl choline suggested that it may have been formed by the action of platelet phospholipase A2 activity. The fatty acids of the membranes showed a markedly asymmetrical distribution in noncholine versus choline phospholipids. The latter held 94%, 72%, and 85%, respectively, of the total content of 16:0, 18:1, and 18:2 fatty acids, whereas 55% of the 18:0, 72% of 20:4, and 67% of higher polyenoic acids other than 20:4 were esterified to the noncholine group. The most important new information related to clot-promoting activity, which, on the basis of protein content, was highest in the membrane fractions, but on the basis of phospholipid content in the nonmembranous fractions. The discussion centers on possible explanations for this novel finding.

Download Full-text

Loss of Thrombin-Induced Ca2+ Mobilization in a Subpopulation of Platelets during Storage

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646419 ◽

1991 ◽

Vol 66 (03) ◽

pp. 350-354 ◽

Cited By ~ 5

Author(s):

Rob Fijnheer ◽

Christa H E Homburg ◽

Berend Hooibrink ◽

Martine N Boomgaard ◽

Dirk de Korte ◽

...

Keyword(s):

Human Platelets ◽

Flow Cytometer ◽

Maximal Concentration ◽

Gradual Decline ◽

Platelet Storage ◽

Functional Defect ◽

Total Fluorescence ◽

Induced Changes

SummaryThrombin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were studied in human platelets that had been stored for up to 6 days. Changes in [Ca2+]i were measured with Indo-1-loaded platelets and quantitated with two different methods: (i) measurement of the changes in total fluorescence; (ii) measurement of the [Ca2+]i changes in individual platelets in a flow cytometer, allowing the detection of non-responding platelets. The maximal concentration of [Ca2+]i after stimulation with 0.5 U of thrombin/ml decreased from 544 ± 58 nM (mean ± SEM, n = 6) on day 0, to 276 ± 9 nM on day 3 and to 203 ± 23 nM on day 6. The percentage of platelets responding to 0.5 U of thrombin/ml declined from 90 ± 2% on day 0 to 72 ± 4% on day 3, and to 47 ± 8% on day 6. Nevertheless, also the responding platelets showed a decreased rise in [Ca2+]i.The study shows that during platelet storage a decrease in the rise in [Ca2+]i upon thrombin stimulation occurs. This decrease is partly due to the formation of a subpopulation of platelets that is completely unresponsive and partly due to a decreased responsiveness in the remainder of the platelets; it is not due to a gradual decline in [Ca2+]i rise in all platelets. This phenomenon provides new insight in the functional defect of stored platelets.

Download Full-text

Correlation Between Noradrenaline Fluxes, Metabolism and Compartmentalisation in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657217 ◽

1982 ◽

Vol 48 (01) ◽

pp. 062-066 ◽

Cited By ~ 5

Author(s):

Chantal Legrand ◽

Véronique Dubernard ◽

Philippe Meyer

Keyword(s):

Noradrenaline Release ◽

Human Platelets ◽

Release Of Noradrenaline ◽

Storage Pool ◽

Platelet Storage ◽

Efflux Of Noradrenaline ◽

Preferential Localization

Summary(3H) noradrenaline was taken up by human platelets and partially converted into sulfoconjugated noradrenaline. This uptake was inhibited by drugs which have been previously shown to impair the uptake of 5-HT (ouabain, chlorimipramine) or the storage of 5-HT (tyramine, reserpine) by platelets. In addition, tyramine and reserpine stimulated the formation of sulfoconjugated noradrenaline. The efflux of noradrenaline from platelets was measured in parallel and was found to be directly related to the proportion of non metabolized to metabolized noradrenaline in the cells. Unlike tyramine, which induced a similar release of noradrenaline and 5-HT, reserpine was less effective at inducing noradrenaline release than 5-HT release. This study indicates a preferential localization of noradrenaline in the granular pool of human platelets with the existence of an extragranular sulfoconjugated pool which is increased when the granular storage of noradrenaline is impaired. Studies of noradrenaline fluxes and metabolism may be useful in the understanding of both acquired and inherited platelet storage pool defects.

Download Full-text

Relationship Between Mepacrine-Labelled Dense Body Number, Platelet Capacity to Accumulate 14C-5-HT and Platelet Density in the Bernard-Soulier and Hermansky-Pudlak Syndromes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666908 ◽

1979 ◽

Vol 42 (02) ◽

pp. 694-704 ◽

Cited By ~ 31

Author(s):

F Rendu ◽

A T Nurden ◽

M Lebret ◽

J P Caen

Keyword(s):

Dense Body ◽

Human Subjects ◽

Human Platelets ◽

Dense Bodies ◽

Storage Organelle ◽

Hereditary Disorders ◽

Labelling Procedure ◽

Normal Human ◽

Hermansky Pudlak Syndrome ◽

Bernard Soulier Syndrome

SummaryWe have used the mepacrine-labelling procedure to measure the dense body (serotonin storage organelle) content of the platelets of 2 hereditary disorders where abnormalities in dense body number were suspected. The platelets were incubated with mepacrine and examined by fluorescence microscopy. A mean number of 5.4 ± 0.8 (SD) dense bodies per platelet was calculated from the data obtained using platelets isolated from 40 normal human subjects. In contrast the platelets of 2 patients with the Bernard-Soulier syndrome contained an average of 14 and 17 labelled granules. This increase was associated with a much greater capacity of the platelets to accumulate 14C-5-HT. The opposite result was obtained using the platelets from 2 patients with the Hermansky-Pudlak syndrome which contained few granules labelled by mepacrine and took up less 14C-5-HT than normal human platelets. Centrifugation of the patients’ platelets on discontinuous sucrose gradients showed that the platelets of the 2 Bemard-Soulier patients were much denser than normal whereas a high proportion of low density platelets was observed in the Hermansky-Pudlak syndrome. These results further define the platelet abnormalities in the two syndromes and suggest that dense body number may be one of the factors governing platelet density.

Download Full-text

Loss of 111Indium as indicator of platelet injury

Blood ◽

10.1182/blood.v58.2.350.350 ◽

1981 ◽

Vol 58 (2) ◽

pp. 350-353 ◽

Cited By ~ 18

Author(s):

JH Joist ◽

RK Baker

Keyword(s):

Small Molecules ◽

Adenine Nucleotides ◽

Human Platelets ◽

Metabolic Energy ◽

Platelet Factor ◽

Platelet Protein ◽

Threshold Rate ◽

Immune Injury ◽

Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

Download Full-text