Release of spectrin-free vesicles from human erythrocytes during ATP depletion: 1. characterization of spectrin-free vesicles

Abstract

Human erythrocytes incubated without glucose at 37~ (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h.93% of all the membrane particles released constitute a uniform population of spheres with a diameter of 185 _ 23 nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipid phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities per surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipid composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes.These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.Human blood contains a mixed population of old and young erythrocytes that can be separated into different age groups due to subtle changes in their properties (11,41). Old cells are denser (33) but slightly smaller, have a decreased sialic acid content (21) and thereby a reduced surface charge (10, 48), have lost their deformability (46), and have become osmotically fragile (12, 44). One of the mechanisms responsible for the above changes in the senescent cell is a so-called "fragmentation" 548

Uncoupling of the membrane skeleton from the lipid bilayer. The cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickled cells.

“…Spicules, used at the same total phospholipid concentration of 1 MM, induced a thrombin formation at a rate of 66 nM/min (Table II). This procedure produces a homogeneous preparation within a relatively short period of time compared with other methods used to isolate microvesicles (42,43). In agreement with the observations of others (19,38,39), the spicules thus isolated as microvesicles were largely deficient in spectrin (Fig.…”

Iron release, membrane protein oxidation and erythrocyte ageing

“…Therefore we set up a model of rapid in vitro ageing of erythrocytes to investigate whether a similar iron release could be detected during ageing and whether such a release could be related to the alterations of membrane proteins leading to the formation of senescent antigen. Since it has been reported [26][27][28] that the aerobic incubation in buffer markedly accelerates the ageing of erythrocytes as measured by vesciculation, we incubated calf erythrocytes in saline phosphate buffer for 24, 48 and 60 h under aerobic conditions and compared the results with similar incubates carried out under anaerobic conditions. As shown in Table 2, the aerobic incubation induced a marked and progressive release of iron, which was accompanied by an extensive methemoglobin formation and severe GSH depletion.…”

Vesiculation of healthy and defective red blood cells

“…Subsequently, these protrusions grow further, as membrane particles flow towards them to relax the compression stress on the lipid bilayer, and finally detach from the membrane [15]. This could explain why the composition of the microvesicles is more heterogeneous than that of nanovesicles and similar to that of the parent RBC membrane [16][17][18][19].…”