Red Blood Cell (RBC) Echinocyte SEM Structure Directly Affected by Oxygen, Heat and F‐actin Destabilization

Abstract

The RBC cytoskeleton is a spectrin-actin meshwork tethered to the integral membrane protein, band 3, through several proteins, including ankyrin. Their interactions affect membrane curvature, mechanics, microdomain formation, and may participate in oxygenation of hemoglobin (Hb) as deoxyHb displaces ankyrin to bind band 3. Our focus is to understand how hypoxia and elevated temperature cause RBC shape changes that may impact microvascular rouleaux formation and vessel blockage with thermal burn injury. We hypothesized that both oxygen and F-actin destabilization are required for shape change. Washed human RBCs (BioIVT) suspended in saline with 1% DMSO, 3.7μM Jasplakinolide (JASPA, polymerizes F-actin), 26.14μM Latrunculin A (LatA, depolymerizes F-actin), 0.5μM Cytochalasin D (CytoD, disrupts cytoskeleton), 2μM 4,4ʹ-diisothiocyano-2,2ʹ-stilbenedisulfonic acid (DIDS, binds band 3, untethers cytoskeleton from membrane) or control (platelet poor plasma, PPP). Each was tested at 2 temperatures (T: 37, 45°C) and 3 oxygen levels (O2: 0, 5, 10%). At T/O2, suspensions were fixed with 1.5% paraformaldehyde 1.5% glutaraldehyde, then with OsO4. Samples were serially dehydrated in ethanol, and coated with 12-15nm Au-Pd in Ar (6mA, Polaron E5100). SEM images (FEI Nova NanoSEM 450, high vacuum mode 4-5 mm working distance, beam 2kV, Everhart-Thornley Detector) were obtained and analyzed using ImageJ (NIH) for 44 samples and 656 RBC. RBC long and short diameters were obtained. For echinocytes (ECH), ‘edge’ spicule (seen on periphery), height, H, width at base (BW) and mid-height (MW) were measured; ‘face’ spicule (seen on presenting face) topology was measured (inter-node distances, nearest neighbors, angles). RBC were classified per the clinical standard as biconcave (discocyte), ECH-I (short broad spicules, flat cell), ECH-II (formed spicules, elliptical cell), ECH-III (well formed spicules, spherical). Independent of treatment, T or O2, we first confirmed that spicule shape progressed to taller with a narrower base, with closer packed conformation as ECH progressed from I to III. Independent of treatment or O2, spicules at 45°C for ECH-I were smaller in H and BW/MW, and more closely packed than at 37°C; ECH-II/III only displayed closer packing. In PPP ECH-I spicules at 0%/37°C were broader and further spaced, while spicules at 10%/45°C were significantly smaller in BW/MW and closer packed; ECH-II/III were not affected by oxygen. Concentrating on ECH-I, DIDS removed the effects on W and spacing at all oxygen/temps. Actin destabilization (CytoD) removed the effect of O2 and T on W, but not spacing; while depolymerization (LatA) had little effect on W or spacing. Actin polymerization (JASPA) had no effect on W, but removed the effect of O2 on spacing. Thus, decoupling the cytoskeleton from band 3 (0% oxygen or DIDS) resulted in fewer broad spicules; depolymerization only (LatA) had little effect, while destabilization (CytoD) reduced the W but not spacing of spicules, and polymerization (JASPA) affected spacing but not W.