Transformation of membrane nanosurface of red blood cells under hemin action

Abstract

Hemin is the product of hemoglobin oxidation. Some diseases may lead to a formation of hemin. The accumulation of hemin causes destruction of red blood cells (RBC) membranes. In this study the process of development of topological defects of RBC membranes within the size range from nanoscale to microscale levels is shown. The formation of the grain-like structures in the membrane ("grains") with typical sizes of 120-200 nm was experimentally shown. The process of formation of "grains" was dependent on the hemin concentration and incubation time. The possible mechanism of membrane nanostructure alterations is proposed. The kinetic equations of formation and transformation of small and medium topological defects were analyzed. This research can be used to study the cell intoxication and analyze the action of various agents on RBC membranes.

Figure 1. Stages of transformation of RBC shape and its membrane under the hemin action for different concentrations C.

(a) C = 0 (control), AFM 3D- images 100 × 100 μm and 30 × 30 μm of cells in control smear, 95 ± 2% discocytes. (b) C = 0.8 mM, AFM 3D-images 30 × 30 μm, 85 ± 5% deformed discocytes and stomatocytes. (c) C = 1.2 mM, AFM 3D-images 30 × 30 μm, 78 ± 6% planocytes and rising of topological “grain” defects. (d) C = 1.5 mM, AFM 3D-images 30 × 30 μm, 84 ± 10% cells with “grains”-containing domains on the membrane surface. (e) C = 2.5 mM, AFM 3D-images 30 × 30 μm, 79 ± 8% cells with merging domains and spheroechinocytes. Time of incubation was 60 min for (a–e). (f) The dependence of domains arising on incubation time t_i for the same hemin concentration C = 1.5 mM (t₁ = 3 min, t₂ = 60 min and t₃ = 180 min). Statistical processing performed by 100 × 100 μm AFM 3D-images of cells in monolayers in 45 smears. (60–100 cells in each image) for each concentration and each time.

Figure 2. The process of domains rising on the membrane under the hemin action. Comparison with the control.

(a), (b),(c) For hemin concentration C = 0 (control). (d), (e),(f) For hemin concentration C = 1.1–1.2 mM. (a) and (d) The AFM 3D- images of RBCs 8 × 8 μm. (b) and (e) AFM 3D-images of membrane surfaces 1300 × 1300 nm (control) and 2400 × 2400 nm (surface with incipient domais). (c) and (f) Typical profiles of the surface in control membrane and in membrane with incipient domains. Interaction time of hemin with RBCs was 1 hour. The typical control cells were chosen out of the ensemble of 2820 cells, under the hemin action – out of 3010 cells. Membrane nanoscructure and profiles are represented as typical for given concentration and incubation time among 108 areas.

Figure 3. Domains with “grain” structures on the membrane surface under the hemin action C = 1.5 mM. AFM images.

(a) RBC 3D-image with “grains”-containing domains. White circles indicate the domains with “grains”. (b) 3D- image 1500×1500 nm of “grains” in the domain, indicated by yellow arrow. (c) Profile of the domain structure. Interaction time of hemin with RBCs was 1 hour. Typical cells were chosen out of the ensemble of 3250 cells. Membrane nanoscructure and profiles are represented as the typical for given concentration and incubation time among 108 areas.

Figure 4. Cells with “grain”-containing domains.

(a) AFM 3D-image 18 × 18 μm of 5 cells with topologic defects in membrane in form of “grain”-containing domains. (b) Fragment of membrane with “grain”-containing domains, AFM 2D-image 4×4 μm. (c) “Grains” in a domain, AFM 3D-image 1500 × 1500 nm. Interaction time of hemin with RBCs was 1 hour, C = 1.5 mM. These cells were typical among 45 smears. Membrane nanoscructure is represented as typical for given concentration and incubation time among 108 areas of membrane surface.

Figure 5. Profiles and histograms of spatial periods of membrane nanostructures for the control cells and cells after the hemin action.

(a) Profile of the control membrane. (b) Profile of the membrane surface in the domain. (s) Histogram of the spatial periods of structures for the control membrane. (d) Histogram of the spatial periods in the field of domains. Interaction time of hemin with RBCs was 1 hour, C = 1.5 mM. Profiles are represented as typical for given concentration and incubation time among 108 areas. Sample volume for control histograms was 310, after hemin action – 215.

Figure 6. Merging of “grains” in the domain.

(a) RBC 3D- image – in its membrane partial merging of “grains” in a domain occurred (arrows). (b) 3D-image of the fragment of membrane surface with merged “grains” in domain (arrows). (c) Profile of this surface. Interaction time of hemin with RBCs was 1 hour, C = 2.5 mM. Cells, membrane nanoscructure and profiles are represented as representative for given concentration and incubation time among 3120 cells.

Figure 7. Fragment of nanosurface of control cell, C = 0.

(a) AFM 3D- image of membrane nanosurface fragment 900 × 900 nm. (b) Model of profile. (c) AFM- profile in the adjusted section on the surface. Arrows show images of protein complexes in a model. Membrane nanoscructure and profiles are represented as typical for given concentration S = 0 among 108 areas.

Figure 8. Fragment of membrane surface of planocyte with “grain” structures after hemin action, C = 1.5 mM.

(a) AFM 3D- image of membrane nanosurface fragment 900 × 900 nm. (b) Model of profile. (c) AFM profile in the adjusted section on the surface. The region of rupture of connection band 4.1R–spectrin is shown by the arrow 1, rupture in spectrin is shown by arrow 2. Incubation time was 1 hour. Membrane nanostructure and profiles are represented as typical among 108 areas.

Figure 9. Fragment of membrane surface with merged “grains” in the domain after hemin action, C = 2.5 mM.

(a) AFM 3D- image of the nanosurface fragment 900 × 900 nm. (b) Model of profile. (s) AFM profile in the adjusted section on the surface. Arrow 3 points on the process of crosslinking of spectrin filament. Incubation time was 1 hour. Membrane nanoscructure and profiles are represented as typical among 108 areas.

Figure 10. Kinetics of formation and development of topologic defects.

(a) Theoretical curves of dependences of the number of small defects (“grains”) on time n(t) and of the number of medium defects (merged “grains”) on time N (t). (b–d) - AFM 2D- image of membrane surface: small – separate “grains” in domains (b), medium – merged “grains” in domains (c), large – merging of domains. (d). Values t, n(t) and N(t) are in relative units.