The role of stomatin in vesicle formation in red cells

Red blood cells (RBCs) lose membrane by vesiculation at different stages of their lifespan. Nascent reticulocytes must lose 20% of their membrane area as part of their maturation process.1 Mature RBCs shed damaged membrane proteins in vesicles throughout their 120 day lifespan in the circulation, and stored RBCs shed vesicles during the 35 days cold-storage pre-transfusion.2 The vesicles produced are removed from the circulation by splenic macrophages but when produced in excess (from immature cultured RBCs or 35 day stored RBCs) or when the spleen is insufficient (haemoglobinopathy patients) these vesicles have the potential to be pro-coagulatory and pro-inflammatory and have been implicated in causing thrombosis.

Stomatin is a membrane-associated, cytoplasmic, peripheral protein that is found at high levels in RBCs vesicles.3 The purpose of this research project is to understand the role of stomatin in reticulocyte maturation in order to better mature nascent cultured RBCs, to investigate the role of stomatin in prolonging the lifespan of mature RBCs, and to develop a simple test for the level of vesiculation in stored blood which may also inform the development of strategies to delay vesiculation, leading to further improvements in the quality of stored blood.

The plan of investigation consists of three approaches. The first to establish the role of stomatin in reticulocyte maturation. Studies of rare patients with RBCs that lack normal levels of stomatin (Overhydrated stomatocytosis (OHSt)) suggest that stomatin is essential for efficient reticulocyte maturation and for the stability of mature RBCs. We will include the culture of OHSt erythroid progenitor cells to produce a pure reticulocyte population, which will then be monitored to measure the level of reticulocyte maturation and vesicle production in comparison with a control sample. Previous studies have shown that nearly all stomatin is lost from these cells between enucleation and formation of the mature RBCs but the mechanism of this loss is not known. The cultured OHSt and control erythroid cells will also be compared to cultured band 3 null erythroid progenitor cells (grown from an immortalised cell line currently under construction) in order to compare vesicles produced by membrane blebbing with regulated vesiculation.

The second line of investigation will examine the role of stomatin in preserving mature RBCs in the circulation. It is known that RBCs with higher levels of oxidative damage to the membrane, such as sickle cell RBCs, have a higher rate of vesiculation. A greater understanding the involvement of stomatin in recognising the damaged protein and removing it by vesiculation is needed. In podocytes in the kidney glomerulus there is a stomatin-like protein, highly homologous to stomatin, known as podocin. There are no known disease-associated mutations in the STOM gene however mutations in podocin have been associated with ineffective blood filtration (nephrotic syndrome). Using site-directed mutagenesis the equivalent mutations will be made in the stomatin gene and the mutant gene expressed in cultured erythroid progenitor cells to investigate their effect on the ability of the cell to form vesicles.

The third line of investigation will look at the role of stomatin in stored RBCs. RBC units show a large degree of variability in the rate at which they release vesicles, but the cause of this variation is unknown. The NCBI SNP database (http://www.ncbi.nlm.nih.gov/snp) shows that the stomatin gene has 17 coding single nucleotide polymorphisms (SNPs). In order to investigate whether this donor effect is due to variations in the stomatin sequence we will apply to the NIHR-BioResource for permission to screen for these SNPs and recall individuals with known stomatin SNPs in order to determine whether there is any correlation between inheritance of a stomatin SNP and rate of vesiculation during RBC storage. This will potentially lead to the identification of key SNPs to be included on a routine genotyping screen for blood donors in order to better match blood to recipient based on the stored red cells’ propensity to release vesicles.