The isolation and characterization of 60 nm vesicles (‘nanovesicles’) produced during ionophore A23187-induced budding of human erythrocytes

Abstract

In addition to the microvesicles released during the treatment of human erythrocytes with Ca2+ and ionophore A23187, a new subpopulation of still smaller dense vesicles ('nanovesicles') has now been identified. Nanovesicles are about 60 nm in diameter, have an acetylcholinesterase activity higher than that of microvesicles and appear to be relatively enriched in sphingomyelin and correspondingly depleted of phosphatidylethanolamine. They have a polypeptide composition quite different from those of erythrocyte membranes or microvesicles, consisting largely of components of mol.wts. 60 000 and 26 000 in addition to haemoglobin. These two major polypeptides do not appear to represent contaminating cytoplasmic proteins or proteolytic subfragments of a larger protein.

Cell‐derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII–dependent mechanism

“…In vitro, platelets are known to release microparticles that expose PS and bind coagulation factors V, VIII, IX, and XI and/or their activated forms (5)(6)(7)(8). Monocytes, endothelial cells, and erythrocytes also release PS-exposing microparticles upon appropriate activation, which supports coagulation in vitro (9)(10)(11). In vivo, microparticles can be found in the systemic circulation; their numbers and cellular origin are dependent on the state of health of the individual.…”

Ca++-dependent vesicle release from erythrocytes involves stomatin-specific lipid rafts, synexin (annexin VII), and sorcin

“…AFM determination of the sizes of quasi-native microvesicles and nanovesicles ( Figure 1A,B) is in good agreement with the data obtained by electron microscopy. 4 The patchy, fine structure of the microvesicular membrane revealed at higher magnification ( Figure 1C) may reflect various lipid domains and/or lipid-protein complexes.Whereas the size distribution of nanovesicles is rather narrow, the microvesicles reveal a higher size variation ( Figure 1D). …”

“…Two sorts of vesicles, differing in size, have been described; these have been named microvesicles (150 nm diameter) and nanovesicles (60 nm diameter). 4 Elevated cytosolic Ca ϩϩ levels cause alterations in the membrane protein pattern, notably the aggregation of proteins catalyzed by transaminases and cytoskeletal rearrangements owing to the proteolysis of protein 4.1. 5 Another Ca ϩϩ -mediated effect is the breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a concomitant increase in 1,2-diacylglycerol and phosphatidic acid.…”

“…These data are in good agreement with earlier results obtained by electron microscopy. 4 Additionally, the size distribution indicates the cross-contamination of nanovesicles with small microvesicles (about 16%) and of microvesicles with large nanovesicles owing to the limited separation efficiency of method A.As the GPI-linked protein AChE is abundant in the microvesicles and nanovesicles, 4 we were specifically interested whether other lipid raft proteins, like stomatin and the flotillins, are similarly enriched within these vesicles. Figure 2A shows the protein pattern of washed erythrocyte membranes (lane 1) compared with that of whole microvesicles and nanovesicles (lanes 2 and 3), normalized to AChE activity.…”

HDL and apolipoprotein A-I protect erythrocytes against the generation of procoagulant activity.

“…After centrifugation, 600 fiL of the supernatant was diluted to 1.2 mL of pH 8.0 phosphate buffer, and the solution was assayed for acetylcholinesterase activity 30 as a measure of microvesicle formation. 21 Replacement of NaCl with KC1 in the buffer suppresses microvesiculation." This was confirmed in the present study.…”