Stibenz D

[The altered membrane of the erythrocyte. I. Ultrahistochemical and biocellular investigations for the detection of activated acetylcholinesterase (ACHE) and demasking of IgG receptor sites (author's transl)].

Zusammenfassung Es wurde versucht, verschiedene Zustandsformen der Erythrocytenmembran im Hinblick auf den Grad ihrer Desintegration naher zu charakterisieren. Als Kriterien wurden die Bindungsfahigkeit gegenuber autologem bzw. allogenem IgG, der Aktivierungsgrad der am intakten Erythrocytenplasmalemm inhibierten membransessilen Acetylcholinesterase und die Mernbranvesiculation herangezogen. Die Befunde belegen, das offenbar mit dem Schweregrad der Membrangefugestorung die IgG-Bindung anwachst und die Acetylcholinesterase zunehmend aktiviert wird. Das Enzym kann dann spektrophotometrisch und ultrahistochemisch erfast werden. Die Mikrovesiculation wird als Ausdruck tiefgreifender Membrangefugestorungen auigefast. Am konservierten Erythrocyten wird sie als ein lokaler Membran-Remodellierungsprozes angesehen, Starkere Erythrocytenschadigung, wie nach mechanischem Stress, Warmesohock und Inkubation in Harnstoff, fuhrt zu vergleichsweise hoheren Vesiculationsraten, teilweise zu Zellfragmentation. An stark spektrinverarmten Erythrocytenschatten tritt Mikrovesiculation spontan ein und fuhrt zum volligen mikrovesicularen Zerfall der Schattenmembran. Das membranassoziierte autologe IgG wird mit immunologischen und ultrahistochemischen Methoden nachgewiesen. Seine Bedeutung als homoostatisch wirksames Immunsignal fur die Elimination von in vivo oder in vitro gealterten Erythrocyten, Erythrocytenschatten und -vesiceln durch das Reticulo-Histiocytare-System wird anhand von Modellversuchen aufgezeigt. Molekulare Mechanismen zur IgG-Receptordemaskierung und A.cetylcholinesteraseaktivierung in der alterierten Erythrocytenmembran werden diskutiert.

Reversible conformational changes of plasmalemmal glycoproteins.

Repeated incubations of human red blood cells in low ionic isotonic sucrose result in an instantaneous agglutination. In the same medium which had caused the agglutination, erythrocytes completely disagglutinate within 60 to 90 min. Disagglutination is accompanied by the efflux of cellular ions, which causes a 500-fold increase of extracellular K+. Decomposition of agglutinates occurs at once upon addition to the medium of about 3 mM KCL. It will be inhibited for hours, if the medium is renewed twice an hour. Erythrocytes washed successively with phosphate buffered saline and isotonic sucrose are devoid of adhering blood plasma proteins. If these cells were fixed with glutaraldehyde in isotonic sucrose they had lost a) their anisotropic staining with toluidine blue, and b) most of their colloidal iron binding capacity. The staining with ruthenium red and the electrophoretic velocity of these erythrocytes apparently were identical with the controls. The findings are considered evidence of the reversible unfolding of glycocalyx glycoproteins in the low ionic medium.

Topo-optical investigations of the human erythrocyte glycocalyx-age related changes

SummaryThe conformational state of the glycocalyx of the intact and altered erythrocyte membrane was studied by means of the topo-optical toluidine blue reaction, i.e. induced membrane birefringence. High membrane anisotropy represents the normal glycocalyx structure and its decline represents their perturbation. The results show that the glycocalyx structure is changed during ageing of the erythrocytes in vivo as well as in vitro. During fluid preservation, in vitro ageing and vesiculation of cells in vitro, a subpopulation of cells showed a decline of membrane anisotropy, but other cells demonstrated abnormally high values. In the latter cases, there is usually a correlation to spherocytes. From this point of view, it is to be assumed that spherogenesis during cell ageing is induced by cell vesiculation. This leads to a remodelling of an intact plasmalemma. In contrast, the cell fractions which are probably non-vesiculating seem to be more or less damaged by membrane and/or plasmic hydrolases. This can be mimicked by neuraminidase and protease treatment of erythrocytes in vitro. Membrane lesions caused by freeze preservation of red blood cells are rare. The topo-optical results are interpreted according to the assumptions of the theory of membrane anisotropy, i.e. the formation of dye-stuff micelles at distinct, clustered, sialylated carbohydrate chains of the gluycophorin A.

Adsorbed proteins mask negatively charged sites of the erythrocyte glycocalyx

Protein masking of charged sites of the erythrocyte glycocalyx was studied by means of the colloidal iron affinity. Washed red cells were fixed with glutaraldehyde such as to stabilize their glycocalyx and to inhibit conformational changes during posttreatment. Following the incubation in an ionic protein solution, proteins adsorbed to the cell surface were insolubilized by repeated treatment with low ionic isotonic surcose. Erythrocytes coated with precipitated proteins exhibited a rough surface. Their iron binding capacity was reduced considerably. In comparison with serum albumin, masking by gamma globulin was more efficient, apparently, because of its insolubility in low ionic media. High ionic incubation of coated erythrocytes resolubilized adsorbed proteins and unmasked negatively charged groups of the glycocalyx.

Metabolic Depletion of Erythrocytes is Accompanied by a Decrease of Sialic Acid during Blood Bank Storage: A Reply

Erythrocytes lose about 10% of their sialic acid during banking in ACD-AG medium. In contrast to these findings, Pessina et al. could not detect a significant banking-related sialic acid decrease. The present paper explains this discrepancy. The vesiculation of erythrocytes is mainly responsible for the observed sialic acid loss during blood preservation.

Alteration of the enucleating erythroblast glycocalyx

Plasmalemmal differentiation of the enucleating normoblast of rabbit and rat was studied by means of cytochemical methods and freeze-etching. Staining with colloidal iron revealed about identical amounts of iron particles bound to various areas of the normoblast membrane. Cationized ferritin and ruthenium red, likewise, failed in the demonstration of significant changes of the enucleating normoblast glycocalyx. Despite these findings the topo-optical staining with toluidine blue showed the plasmalemmal envelope of the protruding normoblast nucleus moderately birefringent, clearly discriminated from the intense anisotropic staining of the future reticulocyte membrane. The ferritin-labeled snail lectin anti AHP localized a great number of binding sites at the plasmalemmal envelope of the nucleus under extrusion. That is in sharp contrast with rather low lectin binding to the future reticulocyte membrane which amounts to about 30 to 50% of the nuclear envelope label. The findings provide evidence of unmasking of bindings sites of the normoblast membrane. Apparently, the effect is due to conformational changes of the cell membrane, rather than it could be attributed to degradation of glycoproteins. Moreover, enucleation kinetics may also be related to supramolecular changes of membrane structure albeit missing evidence for the rearrangement of membrane particles.

Acceleration of Coagulation by Spectrin‐Free Erythrocytic Vesicles: Evidence for Lipid Flip‐Flop during Vesicle Formation

Abstract. Spectrin‐free erythrocytic vesicles isolated from outdated liquid‐preserved blood samples show a distinct increase of the clot‐promoting activity compared with membrane phospholipid equivalents of intact erythrocytes. We suppose that, among other remodelling processes, local spectrin detachment from the inner membrane surface may trigger a flip‐flop‐mediated disturbance of the membrane lipid asymmetry. The interactions of spectrin with the cytoplasmic membrane surface seem to be essential for the structural membrane integrity including the lipid asymmetry.

Acceleration of Coagulation by Spectrin-Free Erythrocytic Vesicles

Spectrin-free erythrocytic vesicles isolated from outdated liquid-preserved blood samples show a distinct increase of the clot-promoting activity compared with membrane phospholipid equivalents of intact erythrocytes. We suppose that, among other remodelling processes, local spectrin detachment from the inner membrane surface may trigger a flip-flop-mediated disturbance of the membrane lipid asymmetry. The interactions of spectrin with the cytoplasmic membrane surface seem to be essential for the structural membrane integrity including the lipid asymmetry.

Phosphorylated spectrins are likely constituents of major Ca2+ affinity sites

Fixation with Ca2+ -glutaraldehyde of ghosts results in opaque membrane associated deposits similar to Ca2+ binding sites of native human erythrocytes. Following brief incubation in an ATP medium the number and size of major Ca2+ affinity sites is considerably enhanced. In addition to major Ca2+ affinity sites multiple minor sites are lining either aspect of the ghost membrane. Ghosts fixed with EDTA-glutaraldehyde are devoid of major Ca2+ affinity sites and they exhibit extreme low overall opacity. Ghosts previously partially despectrinated by incubation in 0.5 mM EDTA have lost major Ca2+ affinity sites, although minor binding sites appear unimpaired. The findings provide evidence of the demonstration of phosphorylated spectrins in major Ca2+ affinity sites.