Phase Separation and Crystallization of Hemoglobin C in Transgenic Mouse and Human Erythrocytes

Abstract

Individuals expressing hemoglobin C (beta6 Glu-->Lys) present red blood cells (RBC) with intraerythrocytic crystals that form when hemoglobin (Hb) is oxygenated. Our earlier in vitro liquid-liquid (L-L) phase separation studies demonstrated that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS or HbA, and that L-L phase separation preceded and enhanced crystallization. We now present evidence for the role of phase separation in HbC crystallization in the RBC, and the role of the RBC membrane as a nucleation center. RBC obtained from both human homozygous HbC patients and transgenic mice expressing only human HbC were studied by bright-field and differential interference contrast video-enhanced microscopy. RBC were exposed to hypertonic NaCl solution (1.5-3%) to induce crystallization within an appropriate experimental time frame. L-L phase separation occurred inside the RBC, which in turn enhanced the formation of intraerythrocytic crystals. RBC L-L phase separation and crystallization comply with the thermodynamic and kinetics laws established through in vitro studies of phase transformations. This is the first report, to the best of our knowledge, to capture a temporal view of intraerythrocytic HbC phase separation, crystal formation, and dissolution.

Overexpression of cryoglobulin‐like single‐chain antibody induces morular cell phenotype via liquid–liquid phase separation in the secretory pathway organelles

“…Morular cell phenotype induction and cryoprecipitation by the LLPS‐prone scFv–Fc–stp protein served as another telltale example, demonstrating close relationships between the intrinsic physicochemical properties of an overexpressed secretory cargo and the phenotype of the cells that overexpress it. With regards to LLPS inside cells, it has only been reported for cytosolic proteins such as the disease alleles of erythrocyte hemoglobin and eye lens proteins . This study presented the first example of an LLPS event that took place in the secretory pathway organelles.…”

Pronounced in vivo hemoglobin polymerization in red blood cells of Gulf toadfish: a general role for hemoglobin aggregation in vertebrate hemoparasite defense?

“…In the case of Hb C (␤6 Glu¡Lys), homozygous carriers (with Hb CC RBCs) are prone to intracellular Hb crystallization, predominantly in oxygenated RBCs and at elevated MCHC (13,14,24). Patients show mild chronic hemolytic anemia but are even more strongly protected from clinical P. falciparum malaria than Hb AS heterozygotes.…”

Printed microfluidic filter for heparinized blood

“…The crystals are originated due to the aggregation of fresh whole blood with heparin. 48 The size and amount of crystals have a strong influence on the filtration process/efficiency. Therefore, it is important to emphasize that a minimum of 24 h of blood storage at 4 C guaranteed uniformity and reproducibility of crystal size and distribution, reaching a reproducible filtering process.…”

“…When fresh blood is stored with heparin to prevent the coagulation, small crystals are formed. 48 The accumulation of crystals near the inlet of microchannels can generate a natural micro-filter, with pores smaller than the red blood cells dimension, a fact that helps separating the red cells from plasma. Hence, only plasma can flow through while the RBCs are retained by the crystals.…”