Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy.

Blood platelets from 10 normal human subjects have been examined with a sensitive differential interference contrast (DIC) microscope. The entire transformation process during adhesion to glass is clearly visible and has been recorded cinematographically, including the disk to sphere change of shape, the formation of sessile protuberances, the extension and retraction of pseudopodia, and the spreading, ruffling, and occasional regression of the hyalomere. The exocytosis of intact dense bodies can be observed either by DIC microscopy, or by epifluorescence microscopy in platelets stained with mepacrine. Details of fluorescent flashes indicate that the dense bodies usually release their contents extracellularly, may do so intracytoplasmically under the influence of strong, short wavelength light on some preparations of mepacrine-stained platelets. The release of one or more dense bodies leaves a crater of variable size on the upper surface of the granulomere. Such craters represent the surface component of the open canalicular system and their formation and disappearance can be directly observed. Because these techniques permit quantitation of several parameters of motility which are not readily observable by other techniques, it is suggested that high extinction DIC microscope examination may become a rapid and useful method of studying congenital and acquired platelet disorders. Many features of platelet transformation have been confirmed and extended by scanning electron micrographs. These can in turn be interpreted by reference to time-lapse films of living platelets.

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Cinemicroscopic Visualization of Shape Change and Motility in Normal and Abnormal Living Human Platelets

10.1055/s-0039-1687279 ◽

1979 ◽

Author(s):

L. R. Zacharski ◽

R. D. Allen ◽

R. Rosenstein ◽

S. Widirtsky

Keyword(s):

Shape Change ◽

Blood Platelets ◽

Rapid Test ◽

Human Platelets ◽

Epifluorescence Microscopy ◽

Optical Sectioning ◽

Dense Bodies ◽

Cine Film ◽

20 Nm ◽

Human Blood Platelets

Living human blood platelets (P) have been examined with a high extinction differential interference contrast (DIC) microscope capable of detecting structures as small as 20 nm in diameter. During the quarter hour required for complete transformation, the entire shape change sequence is clearly visible, including disk to sphere transformation, extension and retraction of pseudopodia, and spreading and ruffling of the hyalomere. The exocytosis of intact 5-hydroxy tryptamine (serotonin)-containing dense bodies has been observed both by DIC microscopy and by epifluorescence microscopy in P stained with mepacrine. The release of dense bodies is associated with the formation of one or more “craters” in the upper surface of the granulomere. With optical sectioning, it is evident that certain “craters” represent internal chambers of the open canalicular system. Using these techniques, abnormalities in P motility have been observed in hereditary P disorders. In summary, the ability to observe and record permanently on cine film the motility of living P provides a rapid test of P function which allows quantitation of normal vs. abnormal motile behavior.

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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.

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Effects of Lectins on Blood Platelets from Various Species

10.1055/s-0039-1682742 ◽

1977 ◽

Author(s):

O. Tangen ◽

B. Karlstam ◽

S. Bygdeman

Keyword(s):

Shape Change ◽

Blood Platelets ◽

Human Platelets ◽

Serotonin Release ◽

Variable Degree ◽

Con A ◽

Platelet Release ◽

Induced Aggregation ◽

Aggregation Of Platelets ◽

Platelet Shape

Earlier it has been shown that different lectins induce a variable degree of aggregation of platelets. The present study confirmed previous data and demonstrated that wheat germ agglutinin (WGA) was very active, 1eucoagglutinin had about a tenth of the activity of WGA on a concentration basis, and Con A had a weak aggregating effect on human gel filtered platelets (GFP). Soy bean lectin did not aggregate human GFP.The fact that adenosine inhibited WGA- and leucoagglutinin-induced aggregation that WGA and Con A caused serotonin release, and that the aggregation- curves indicated platelet shape change are indications that the lectins influenced glycosyl moieties involving one or more molecules relevant to release and aggregation reaction.GFP were markedly more responsive to the lectins than platelets in plasma, probably due to interfering glycosyl groups amongst the plasma constituents.Platelets from man, rabbit, rat, cow and pig reacted differently towards the lectins, human platelets being the most reactive and bovine and porcine platelets being almost unreactive. These results pose intriguing questions regarding the glycosyl content of platelet membranes in different species and their relation to platelet release and aggregation.

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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.

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Conditions Affecting the Responses of Human Platelets to Epinephrine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647031 ◽

1988 ◽

Vol 60 (02) ◽

pp. 209-216 ◽

Cited By ~ 23

Author(s):

Chantal Lalau Keraly ◽

Raelene L Kinlough-Rathbone ◽

Marian A Packham ◽

Hidenori Suzuki ◽

J Fraser Mustard

Keyword(s):

Platelet Rich Plasma ◽

Shape Change ◽

Human Platelets ◽

Dense Granule ◽

Primary Phase ◽

Lag Phase ◽

Acid Citrate Dextrose ◽

Two Phases ◽

Citrate Dextrose ◽

Thromboxane Formation

SummaryConditions affecting the responses of human platelets to epinephrine were examined. In platelet-rich plasma prepared from blood anticoagulated with hirudin or PPACK (D-pheny- lalanyl-L-prolyl-L-arginine chloromethyl ketone), epinephrine did not cause shape change or aggregation. In a Tyrode-albumin- apyrase solution containing a concentration of Ca2+ in the physiological range, and fibrinogen, epinephrine in concentrations as high as 40 μM did not induce platelet shape change, caused either no primary aggregation or very slight primary aggregation, and did not induce thromboxane formation, release of dense granule contents, or secondary aggregation. In contrast, in citrated platelet-rich plasma, epinephrine induced two phases of aggregation. This is not attributable to the generation of traces of thrombin since the same effects were evident when blood was taken into a combined citrate-hirudin anticoagulant or a combined citrate-PPACK anticoagulant. In a modified Tyrode-albu- min-apyrase solution containing approximately 20 μM Ca2+, 1 mM Mg2+, and fibrinogen, epinephrine induced extensive aggregation after a lag phase, but no primary phase was evident; thromboxane formation and release of dense granule contents accompanied the aggregation response. These responses were also observed when PPACK was included with the acid-citrate- dextrose anticoagulant, and in the washing and resuspending fluids. In the presence of aspirin or the thromboxane receptor blocker BM 13.177 a few small aggregates were detected by particle counting and by scanning electron microscopy; with the latter inhibitor, the platelets in the aggregates retained their disc shape; secondary aggregation and the responses associated with it did not occur. Thus thromboxane A2 formation is not necessary for the formation of these small aggregates, but is required for extensive aggregation and release. As with other weak agonists, the close platelet-to-platelet contact in the low Ca2+ medium appears to be necessary for full secondary aggregation. Omission of fibrinogen from the low Ca2+ medium prevented both primary and secondary aggregation in response to epinephrine. An antibody (10E5) to the glycoprotein Ilb/IIIa complex was completely inhibitory in the presence of fibrinogen. Thus the response of human platelets to epinephrine is influenced by the concentration of Ca2+ and the presence of fibrinogen in the medium in which they are suspended.

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Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645981 ◽

1987 ◽

Vol 58 (02) ◽

pp. 737-743 ◽

Cited By ~ 12

Author(s):

Frarnçois Lanza ◽

Alain Beretz ◽

Martial Kubina ◽

Jean-Pierre Cazenave

Keyword(s):

Intracellular Calcium ◽

Shape Change ◽

Calcium Ionophore ◽

Human Platelets ◽

Arachidonic Acid Metabolism ◽

Free Calcium ◽

Membrane Bilayer ◽

Fluorescent Indicators ◽

Fura 2 ◽

Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

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Reaction of Acetaldehyde with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648393 ◽

1992 ◽

Vol 67 (01) ◽

pp. 126-130 ◽

Cited By ~ 8

Author(s):

Olivier Spertini ◽

Jacques Hauert ◽

Fedor Bachmann

Keyword(s):

Platelet Aggregation ◽

Inhibitory Action ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Shape Change ◽

Reaction Medium ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

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Physiological Effects of Nonimmune Platelet Associated Immunoglobulin G

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650123 ◽

1981 ◽

Vol 45 (01) ◽

pp. 027-033 ◽

Cited By ~ 10

Author(s):

K Sugiura ◽

M Steiner ◽

M Baldini

Keyword(s):

Shape Change ◽

Fluorescein Isothiocyanate ◽

Recovery Phase ◽

Human Platelets ◽

Serotonin Release ◽

Platelet Volume ◽

Igg Antibody ◽

Heterologous Antiserum ◽

Igg Binding ◽

Series Of Experiments

SummaryThe function of nonimmune IgG associated with platelets is unknown. In a series of experiments we have investigated this problem, relating amount of platelet-associated IgG (PAIgG) to platelet volume, serotonin release, adherence of platelets to monocytes and platelet senescence. Most of these studies were performed with human platelets. Platelets freed of preexisting PAIgG by incubation at 22° C were incubated with IgG in a series of concentrations ranging from 0.4 — 27.0 X10-6 M. The IgG preparations used were demonstrably free of aggregated forms of the protein. The amount of PAIgG bound to platelets was determined by the use of fluorescein isothiocyanate-conjugated anti-IgG antibody (F-anti-IgG antibody) which was quantified in a fluorospectrophotometer. Newly bound IgG was assayed similarly by the use of F-IgG. A dose-dependent increase in platelet volume was associated with the binding of nonimmune IgG by platelets. The process which leveled off at an IgG concentration of 1.2 —1.5 X10-5 M was almost fully reversible and was not due to platelet shape change or aggregation. Release of serotonin from IgG-treated platelets was relatively small but to the extent that it occurred was positively related to the IgG concentration to which platelets were exposed. Adherence to autologous monocytes studied quantitatively by the use of formaldehyde-fixed cells was also positively related to the amount of IgG on the platelets. Normal or IgG-defident serum had a potent inhibitory (noncompetitive) action on the binding of F-IgG and F-anti-human IgG antibody to human platelets. Cohorts of platelets prepared in rabbits during the recovery phase of immunological thrombocytopenia induced by injection of heterologous antiserum, showed an age-dependent increase of PAIgG and of IgG binding. These results suggest that PAIgG plays a role in the clearance of senescent platelets.

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Platelet Aggregation Caused by Dithiothreitol

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661036 ◽

1984 ◽

Vol 51 (01) ◽

pp. 119-124 ◽

Cited By ~ 59

Author(s):

M B Zucker ◽

N C Masiello

Keyword(s):

Shape Change ◽

Blood Platelets ◽

Dense Granule ◽

Metabolic Inhibitors ◽

Electrophoretic Patterns ◽

Discernible Effect ◽

Variable Extent ◽

Triton X ◽

Induced Aggregation ◽

Platelet Shape

SummaryMacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DTT- induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0° C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to “expose” the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I- labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

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Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657048 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1473-1482 ◽

Cited By ~ 17

Author(s):

A Dup Heyns ◽

P N Badenhorst ◽

H Pieters ◽

M G Lötter ◽

P C Minnaar ◽

...

Keyword(s):

Platelet Rich Plasma ◽

Blood Platelets ◽

Kinetic Studies ◽

Physiological Saline ◽

Human Platelets ◽

Autologous Plasma ◽

Platelet Suspension ◽

Viable Population ◽

Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

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