Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and -Granules

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3791-3799 ◽  
Author(s):  
Harry F.G. Heijnen ◽  
Anja E. Schiel ◽  
Rob Fijnheer ◽  
Hans J. Geuze ◽  
Jan J. Sixma
Keyword(s):  
Platelet Activation ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Multivesicular Bodies ◽  
Factor X ◽  
Activated Platelets ◽  
Immuno Electron Microscopy ◽  
Different Types ◽  
Platelet Aggregates

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and -granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and -granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains IIb-β3 and β1, GPIb, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and -Granules

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3791-3799 ◽  
Author(s):  
Harry F.G. Heijnen ◽  
Anja E. Schiel ◽  
Rob Fijnheer ◽  
Hans J. Geuze ◽  
Jan J. Sixma
Keyword(s):  
Platelet Activation ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Multivesicular Bodies ◽  
Factor X ◽  
Activated Platelets ◽  
Immuno Electron Microscopy ◽  
Different Types ◽  
Platelet Aggregates

Abstract Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and -granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and -granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains IIb-β3 and β1, GPIb, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

Movement of Calcium Ions and their Role in the Activation of Platelets

1978 ◽  
Vol 40 (02) ◽  
pp. 212-218 ◽  
Author(s):  
P Massini ◽  
R Käser-Glanzmann ◽  
E F Lüscher
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Calcium Ions ◽  
Binding Sites ◽  
Shape Change ◽  
Release Reaction ◽  
Dense Bodies ◽  
Activated Platelets ◽  
Different Types ◽  
Ca Ions

SummaryThe increase of the cytoplasmic Ca-concentration plays a central role in the initiation of platelet activation. Four kinds of movements of Ca-ions are presumed to occur during this process: a) Ca-ions liberated from membranes induce the rapid shape change, b) Vesicular organelles release Ca-ions into the cytoplasm which initiate the release reaction, c) The storage organelles called dense bodies, secrete their contents including Ca-ions to the outside during the release reaction, d) At the same time a rearrangement of the plasma membrane occurs, resulting in an increase in its permeability for Ca-ions as well as in an increase in the number of Ca-binding sites.Since most processes occurring during platelet activation are reversible, the platelet must be equipped with a mechanism which removes Ca-ions from the cytoplasm. A vesicular fraction obtained from homogenized platelets indeed accumulates Ca actively. This Ca- pump is stimulated by cyclic AMP and protein kinase; it may be involved in the recovery of platelets after activation.It becomes increasingly clear that the various manifestations of platelet activation are triggered by a rise in the cytoplasmic Ca2+-concentration. The evidence for this and possible mechanisms involved are discussed in some detail in the contributions by Detwiler et al. and by Gerrard and White to this symposium. In this article we shall discuss four different types of mobilization of Ca-ions which occur in the course of the activation of platelets. In addition, at least one transport step involved in the removal of Ca2+ must occur during relaxation of activated platelets.

scholarly journals P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 134-142 ◽  
Author(s):  
LG de Bruijne-Admiraal ◽  
PW Modderman ◽  
AE Von dem Borne ◽  
A Sonnenberg
Keyword(s):  
T Cells ◽  
Divalent Cations ◽  
Phosphatidyl Inositol ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Immunofluorescence Assay ◽  
Cd8 Cells ◽  
Activated Platelets ◽  
Selectin Ligands ◽  
Different Types

Abstract Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P- selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P- selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.

The Effect Of Prostacyclin On The Participation Of Platelets In X-Activation And Thrombin Formation

1981 ◽  
Author(s):  
E Bevers ◽  
G V Dieijen ◽  
J Rosing ◽  
G Hornstra ◽  
R F A Zwaal
Keyword(s):  
Platelet Activation ◽  
Vascular Tissue ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Chromogenic Substrates ◽  
Clotting Factors ◽  
Clotting System ◽  
Inhibiting Effect ◽  
Activated Platelets ◽  
Dose Dependent

Damaged vascular tissue triggers the intrinsic and extrinsic clotting system, resulting in the clotting of plasma. Platelets, especially after their activation, promote this clotting response, which is inhibited by prostacyclin (PGI2).The present experiments were devised to characterize this anticoagulant effect of PGI2.Using specific chromogenic substrates, the intrinsic activation of factor X and the conversion of prothrombin into thrombin (IIa) was measured in reaction mixtures containing highly purified clotting factors. Phospholipids were added as vesicles, platelet lysates, or as whole platelets, either or not activated with a mixture of collagen and thrombin. Phospholipid vesicles and lysed platelets greatly promoted Xa and IIa formation, which was not affected byPGI2.The very low formation of Xa and IIa occurring in the presence of non-activated platelets was not inhibited byPGI2 either. Platelets activated with a mixture of collagen and thrombin stimulated Xa and IIa formation considerably. PGI2 inhibited this effect in a dose-dependent way.These results demonstrate that prostacyclin does not interfere with Xa and IIa formation as such, but specifically inhibits the process by which a mixture of collagen and thrombin stimulates the participation of platelets in these reactions.The inhibiting effect of PGI2 is only partial. This is most probably explained by the fact that prostacyclin can only inhibit but not block platelet activation by a mixture of collagen and thrombin.

Role of Active Center and Lysine Binding Sites of Plasmin in Plasmin-Induced Platelet Activation and Disaggregation

1991 ◽  
Vol 65 (01) ◽  
pp. 067-072 ◽  
Author(s):  
He Lu ◽  
Claudine Soria ◽  
Hong Li ◽  
Jeannette Soria ◽  
H Roger Lijnen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Center ◽  
Platelet Activation ◽  
Binding Sites ◽  
Aminocaproic Acid ◽  
Molar Ratio ◽  
Activated Platelets ◽  
Platelet Aggregates ◽  
Epsilon Aminocaproic Acid

SummaryPrevious studies have shown that plasmin can activate platelets and - can also disperse platelet aggregates by degradation of fibrinogen bound to platelets. In this study, the role of the active center and the lysine binding sites (LBS) of human plasmin in activating platelets and in dispersing platelet aggresates is investigated using aprotinin and the tripeptide Val-Phe-Lys-CH2Cl to inhibit the active center and using epsilon-aminocaproic acid (EACA) to specifically block the LBS. Our results show that the catalytic activity of plasmin is indispensable both for activating platelets and for dispersing platelet aggregates. Binding of plasmin to platelets through the LBS enhances its activating potential, since both EACA (1 mM) and Lys-plasminogen (molar ratio of plasminogen:plasmin at 2:l to 4:1) inhibit plasmininduced platelet activation, whereas Glu-plasminogen at a molar ratio of 15: I had no effect. Furthermore, plasmin which lacks the LBS (miniplasmin), is about 3 fold less effective in activating platelets. Flowever, plasmin binding through the LBS is not absolutely required to disperse platelet aggregates, since EACA at 30 mmol/l was unable to prevent disaggregation by plasmin (half disaggregation time: 40 min in the presence of EACA against 27 min in its absence). It also appeared that fibrinogen receptors on activated platelets are resistant to plasmin degradation, and that disaggregation of plasmin-induced platelet aggregates was much slower than the degradation of fibrinogen by plasmin.

scholarly journals P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 134-142 ◽  
Author(s):  
LG de Bruijne-Admiraal ◽  
PW Modderman ◽  
AE Von dem Borne ◽  
A Sonnenberg
Keyword(s):  
T Cells ◽  
Divalent Cations ◽  
Phosphatidyl Inositol ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Immunofluorescence Assay ◽  
Cd8 Cells ◽  
Activated Platelets ◽  
Selectin Ligands ◽  
Different Types

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P- selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P- selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.

Abstract 203: Cardiomyocyte T-tubule Membrane Turns Over, Releasing BIN1 Containing Microparticles into Blood

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Bing Xu ◽  
Ying Fu ◽  
TingTing Hong
Keyword(s):  
Flow Cytometry ◽  
Adult Mouse ◽  
Cardiac Tissue ◽  
Muscle Protein ◽  
Three Dimensional ◽  
Super Resolution ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Fluorescent Imaging

Bridging integrator 1 (BIN1) is a cardiac muscle protein that folds cardiomyocyte T-tubule membrane. BIN1 is intrinsic to cardiac health, and is reduced in acquired heart failure. Interestingly, we have found that BIN1 is also blood available, and that plasma BIN1 correlates with cardiac function, suggesting cardiac origin of plasma BIN1. We found that low plasma BIN1 correlates with failing muscle and predicts ventricular arrhythmia. However, the paradigm does not exist for an intracellular membrane associate cardiomyocyte protein to be homeostatically turned over into blood. In this study, using a mouse model with cardiac specific deletion of Bin1 gene, we identified with biochemical techniques that plasma BIN1 levels directly correlate with cardiac tissue BIN1 levels, indicating cardiac origin. Furthermore, investigations using both super-resolution fluorescent imaging and flow cytometry analysis revealed that adult ventricular cardiomyocytes constantly release BIN1 into blood via membrane microparticle production. Microparticles are small membrane vesicles shed from plasma membrane of a variety of cell types including platelets, leukocytes, and endothelial cells. Using super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we found similar to the blood cells, isolated adult mouse cardiomyocytes release Annexin V positive microparticles with diameters ranging between 0.1 to 1.0 μm. These microparticles also carry BIN1 protein. Flow cytometry was also used to detect and quantify microparticles <1.0 μm in size from medium bathing a pure population of adult mouse cardiomyocytes. BIN1 microparticle release is proportional to actin stability and amount of T-tubule membrane folds. Compared to wild type cardiomyocytes, microparticle release is significantly reduced from myocytes with heterozygous deletion of Bin1 gene. These data indicate that cardiomyocyte membrane undergoes dynamic turnover, releasing T-tubule folds into blood as microparticles. Furthermore, plasma BIN1 can be used as a direct measure of cardiomyocyte health and reserve.

scholarly journals Platelet Activation by Streptococcus pyogenes Leads to Entrapment in Platelet Aggregates, from Which Bacteria Subsequently Escape

2014 ◽  
Vol 82 (10) ◽  
pp. 4307-4314 ◽  
Author(s):  
Lisbeth Svensson ◽  
Maria Baumgarten ◽  
Matthias Mörgelin ◽  
Oonagh Shannon
Keyword(s):  
Platelet Activation ◽  
Streptococcus Pyogenes ◽  
Dense Granule ◽  
Antibacterial Effects ◽  
Active Protein ◽  
Content Type ◽  
Activated Platelets ◽  
Viable Bacteria ◽  
Platelet Aggregates ◽  
Granule Release

ABSTRACTPlatelet activation and aggregation have been reported to occur in response to a number of Gram-positive pathogens. Here, we show that platelet aggregates induced byStreptococcus pyogeneswere unstable and that viable bacteria escaped from the aggregates over time. This was not due to differential activation in response to the bacteria compared with physiological activators. All the bacterial isolates induced significant platelet activation, including integrin activation and alpha and dense-granule release, at levels equivalent to those induced by potent physiological platelet activators that induced stable aggregates. The ability to escape the aggregates and to resist the antibacterial effects of platelets was dependent on active protein synthesis by the bacteria within the aggregate. We conclude thatS. pyogenesbacteria can temporarily cover themselves with activated platelets, and we propose that this may facilitate survival of the bacteria in the presence of platelets.

Study of the Molecular Architecture of Keratin Filaments by Electron Microscopy

1982 ◽  
Vol 40 ◽  
pp. 118-119
Author(s):  
U. Aebi ◽  
P. Rew ◽  
T.-T. Sun
Keyword(s):  
Electron Microscopy ◽  
Structural Organization ◽  
Cell Types ◽  
Structural Features ◽  
Molecular Architecture ◽  
The Past ◽  
Keratin Filaments ◽  
Different Types ◽  
10 Nm Filaments ◽  
Different Cell Types

Various types of intermediate-sized (10-nm) filaments have been found and described in many different cell types during the past few years. Despite the differences in the chemical composition among the different types of filaments, they all yield common structural features: they are usually up to several microns long and have a diameter of 7 to 10 nm; there is evidence that they are made of several 2 to 3.5 nm wide protofilaments which are helically wound around each other; the secondary structure of the polypeptides constituting the filaments is rich in ∞-helix. However a detailed description of their structural organization is lacking to date.

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
Author(s):  
Dzung The Le ◽  
Samuel I Rapaport ◽  
L Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Damage ◽  
Specific Binding ◽  
Surface Membrane ◽  
Annexin V ◽  
Factor X ◽  
Binding Studies ◽  
Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

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