Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient

1981 ◽  
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.

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

1979 ◽  
Vol 83 (1) ◽  
pp. 126-142 ◽  
Author(s):  
R D Allen ◽  
L R Zacharski ◽  
S T Widirstky ◽  
R Rosenstein ◽  
L M Zaitlin ◽  
...  
Keyword(s):  
Shape Change ◽  
Human Subjects ◽  
Blood Platelets ◽  
Time Lapse ◽  
Human Platelets ◽  
Transformation Process ◽  
Epifluorescence Microscopy ◽  
Scanning Electron Micrographs ◽  
Dense Bodies ◽  
Short Wavelength Light

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.

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

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.

scholarly journals Ultrastructural localization of coagulation factor V in human platelets

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 244-249 ◽  
Author(s):  
JD Wencel-Drake ◽  
B Dahlback ◽  
JG White ◽  
MH Ginsberg
Keyword(s):  
Plasma Membranes ◽  
Coagulation Factor ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Platelets ◽  
Immunofluorescent Staining ◽  
Resting Cells ◽  
Factor V ◽  
Platelet Factor ◽  
Coagulation Factor V

Abstract The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed. Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established. We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V.

scholarly journals cADP-ribose formation by blood platelets is not responsible for intracellular calcium mobilization

1998 ◽  
Vol 331 (2) ◽  
pp. 431-436 ◽  
Author(s):  
Philippe OHLMANN ◽  
Claude LERAY ◽  
Catherine RAVANAT ◽  
Adel HALLIA ◽  
Dominique CASSEL ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Plasma Membranes ◽  
Calcium Release ◽  
Blood Platelets ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Dependent Manner ◽  
Intact Cells ◽  
Adp Ribosyl Cyclase ◽  
Intracellular Calcium Mobilization

Human platelet CD38 is a multifunctional ectoenzyme catalysing the synthesis and hydrolysis of cADP-ribose (cADPR), a recently identified calcium-mobilizing agent that acts independently of d-myo-inositol 1,4,5-trisphosphate and is known to be expressed by human platelets. The present work shows that ADP-ribosyl cyclase activity is exclusively a membrane activity, of which the major part is located in plasma membranes and a small part in internal membranes. In broken cells, cyclase activity was insensitive to the presence of calcium and was not modulated by agonists such as thrombin or ADP, whereas in intact cells thrombin increased cADPR formation by 30%, an effect due to fusion of granules with the plasma membrane. In order to assess the role of cADPR as a calcium-mobilizing agent, vesicles were prepared from internal membranes and loaded with 45CaCl2. These vesicles were efficiently discharged by IP3 in a dose-dependent manner, but were not responsive to cADPR or ryanodine in the presence or absence of calmodulin. Thus cADPR is unlikely to play a role in intracellular calcium release in human blood platelets.

scholarly journals Role of kidney in the catabolic clearance of human platelet antiheparin proteins from rat circulation

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 233-238
Author(s):  
CP Bastl ◽  
J Musial ◽  
M Kloczewiak ◽  
J Guzzo ◽  
I Berman ◽  
...  
Keyword(s):  
Renal Excretion ◽  
Kidney Tissue ◽  
Rat Plasma ◽  
Renal Tissue ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Metabolic Clearance ◽  
Platelet Factor ◽  
Functioning Kidney

Stimulated platelets release at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) from which beta-thromboglobulin (beta TG) is derived. We have found previously marked elevation of LA-PF4/beta TG antigen in platelet poor plasma of patients with chronic renal failure, whereas levels of PF4 remained normal. Therefore, we examined the role of the kidneys in the metabolic clearance of LA-PF4/beta TG and PF4. The supernates of aggregates of thrombin-stimulated human platelets were injected into sham operated control rats, nephrectomized rats, and into rats with acute ureteral ligation. The disappearance of human LA-PF4/beta TG antigen and PF4 in rat plasma determined by specific radioimmunoassays followed biphasic exponential curves. The half-lives (t1/2) for the fast and slow components of LA-PF4 in control rats were 6.4 and 68.4 min. Nephrectomy significantly increased these times to 9.7 and 144 min, while ureteral ligation resulted in no significant change. Comparison of the level of LA-PF4/beta TG antigen and of creatinine in aorta and in renal vein showed 25%-30% extraction of these compounds by the kidney. Less than 0.1% of the total LA-PF4 antigen injected was recovered in the urine of control rats. In contrast to these results, the clearance of PF4 was not affected by nephrectomy. In conclusion: (1) functional renal tissue is necessary for normal clearance of LA- PF4/beta TG, but renal excretion does not play a major role in its elimination suggesting that the protein is catabolized by the kidney; and (2) catabolic clearance of PF4 does not depend on functioning kidney tissue.

scholarly journals Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport

1979 ◽  
Vol 184 (3) ◽  
pp. 651-661 ◽  
Author(s):  
J E B Fox ◽  
A K Say ◽  
R J Haslam
Keyword(s):  
Calcium Ion ◽  
Prostaglandin E1 ◽  
Plasma Membranes ◽  
Human Platelets ◽  
Particulate Fraction ◽  
Ionophore A23187 ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Calcium Ion Transport

Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.

Blood Platelets Organize Pro- and Anti-Angiogenic Factors into Separate, Distinct Alpha Granules: Implications for the Regulation of Angiogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 393-393 ◽  
Author(s):  
Joseph Italiano ◽  
Jennifer L. Richardson ◽  
Judah Folkman ◽  
Giannoula Klement
Keyword(s):  
Confocal Microscopy ◽  
Blood Platelets ◽  
Local Environment ◽  
Human Platelets ◽  
Angiogenic Factors ◽  
Regulatory Proteins ◽  
Platelet Factor ◽  
Proteinase Activated Receptors ◽  
The Many ◽  
Granule Release

Abstract In addition to contributing to hemostasis, platelets are increasingly being viewed as major contributors to wound healing and tumor growth. This can be attributed to the many angiogenesis regulatory proteins selectively taken up and stored within the alpha granules of platelets and deposited in the local environment of a tumor or wound upon platelet aggregation and adhesion. Platelets contain both pro and anti-angiogenesis regulators, and the mechanisms by which they modulate angiogenesis are unclear. To address this question, we examined the localization of both pro-and anti-angiogenic factors in platelets and megakaryocytes using immunostaining and confocal microscopy. Double immunofluorescence microscopy revealed that VEGF (an angiogenesis stimulator) and endostatin (an inhibitor), are localized in separate and distinct alpha granules. Immunofluorescence for thrombospondin 1, basic FGF, platelet factor 4, and placental growth factor subsequently confirmed the separate and distinct alpha granules. These observations, in combination with a recent report that proteinase-activated receptors (PARs) selectively modulate the release of angiogenesis regulators from platelets (Ma et al., PNAS 102:216), motivated the hypothesis that these distinct populations of alpha granules could undergo selective release as well. To investigate whether PARs facilitate differential release of alpha granules, we treated human platelets with PAR agonists or antagonists and assayed for differential alpha granule release. The selective PAR1 agonist stimulated the release of endostatin-containing alpha granules and suppressed the release of VEGF-containing alpha granules. Furthermore, confocal microscopy studies revealed that in the presence of the PAR1 antagonist (SCH79797), thrombin stimulated the release of endostatin-containing alpha granules, but not VEGF-containing granules. These observations suggest that platelets may be organizing angiogenesis regulatory proteins into pharmacologically and morphologically distinct populations of alpha granules, which are then susceptible to differential regulation upon platelet activation.

Platelet Heparin-Neutralizing Factor (Platelet Factor 4)

1975 ◽  
Vol 33 (01) ◽  
pp. 066-072 ◽  
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.

The Effect of Phospholipase C on Platelet Factor 3 in Human Blood Platelets

1977 ◽  
Vol 37 (01) ◽  
pp. 029-035 ◽  
Author(s):  
A-B Otnaess ◽  
H Prydz
Keyword(s):  
Phospholipase C ◽  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Human Platelets ◽  
Small Reduction ◽  
Platelet Factor ◽  
Human Blood Platelets ◽  
External Attack

SummaryIntact human platelets isolated by gel filtration have been treated with purified phospholipase C. The effect of the enzyme on available and total platelet factor 3 has been tested.The available procoagulant platelet factor 3 was very low. A further small reduction was observed after incubation with phospholipase C when the enzyme was washed away before testing.External attack on platelets by phospholipase C led to a marked inactivation of total platelet factor 3.

Human Platelet Storage Organelles A Review

1977 ◽  
Vol 38 (04) ◽  
pp. 0963-0970 ◽  
Author(s):  
Miriam H. Fukami ◽  
Leon Salganicoff
Keyword(s):  
Secretory Granules ◽  
Subcellular Fractionation ◽  
Calcium Pyrophosphate ◽  
Human Platelets ◽  
Acid Hydrolases ◽  
Vascular Permeability Factor ◽  
Extracellular Medium ◽  
Platelet Factor ◽  
Platelet Storage ◽  
Dense Bodies

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.

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