Actin Destabilization Enhances Thermally‐induced Morphological Abnormalities in Erythrocytes

Abstract

Thermal burns are a significant clinical problem with limited treatment options. Burn area progresses radially over several days, blocking oxygenated flow to a progressively increasing region. By 4 hours post‐burn, red blood cell (RBC) aggregates form at the burn margin, without evidence of fibrin clots, indicating a potential for early intervention (Clark, 2013). The present study focuses on understanding the early aggregation process. The RBC’s biconcave shape is due to the cytoskeletal structure that is tethered to the membrane; many pathologies occur when this is disrupted. The RBC cytoskeleton includes spectrin‐actin meshwork that affects membrane curvature, mechanics, and microdomain formation. Our specific hypothesis is that elevated temperatures in arterial oxygen conditions (10%) contributes to the actin cytoskeleton destabilization, thus permitting membrane deformation. Washed human RBCs (BioIVT, Westbury, NY) were suspended in saline (PBS) containing 1% DMSO, 3.7μM Jasplakinolide (JASPA, polymerizes F‐actin), 26.14μM Latrunculin A (LatA, depolymerizes F‐actin), 0.5μM Cytochalasin D (CytoD, disrupts all cytoskeletal integrity), 2μM 4,4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS, binds to band 3, untethering the cytoskeleton from the membrane) (1% DMSO) or control (PBS and platelet poor plasma, PPP). All conditions were exposed to five temperatures (37, 41, 43, 45, or 49°C) and three oxygen levels (0, 5, or 10% O2) for 10 minutes. Suspensions were fixed with 4% paraformaldehyde (PFA) at the test temperature/oxygen and imaged using a 60X water immersion objective. Singlet RBCs were scored by their morphology, in accordance with clinical standards, and identified by degree of echinocyte (I, II, and III), type of non‐echinocyte abnormal shape, or as discocytes. With increasing temperature, the population shifted from the discocyte shape for all control conditions. Increasing oxygen (in plasma) likewise shifted RBC morphology to pathological shapes, but with a less pronounced effect. CytoD, DIDS, JASPA and LatA each eliminated the discocyte populations at all temperatures and oxygen levels. Cells were also prepared for SEM imaging using 1.5% glutaraldehyde and 1.5% PFA and serial dehydration. RBC shapes obtained from brightfield images (current clinical practice) were compared to shapes obtained from SEM images. Population distribution of shapes from these two image pools were not different. Overall, we show that heat and increasing oxygen promotes pathological RBC morphologies. Deliberate disruption of the actin cytoskeleton induces these morphologies, which is exacerbated by increasing temperature. Further, LatA and JASPA cause similar population distributions of shapes, thereby indicating any imbalance of the equilibrium between filamentous and globular actin triggers morphological abnormalities.