4.1R-deficient human red blood cells have altered phosphatidylserine exposure pathways and are deficient in CD44 and CD47 glycoproteins.

BACKGROUND: Protein 4.1R is an important component of the red cell membrane skeleton. It imparts structural integrity and has transmembrane signaling roles by direct interactions with transmembrane proteins and other membrane skeletal components, notably p55 and calmodulin. DESIGN AND METHODS: Spontaneous and ligation-induced phosphatidylserine exposure on erythrocytes from two patients with 4.1R deficiency were studied, using CD47 glycoprotein and glycophorin C as ligands. We also looked for protein abnormalities in the 4.1R-based multiprotein complex. RESULTS: Phosphatidylserine exposure was significantly increased in 4.1R-deficient erythrocytes obtained from the two different individuals when ligands to CD47 glycoprotein were bound. Spontaneous phosphatidylserine exposure was normal. 4.1R, glycophorin C and p55 were missing or sharply reduced. Furthermore there was an alteration or deficiency of CD47 glycoprotein and a lack of CD44 glycoprotein. Based on a recent study in 4.1R-deficient mice, we found that there are clear functional differences between interactions of human red cell 4.1R and its murine counterpart. CONCLUSIONS: Glycophorin C is known to bind 4.1R, and we have defined previously that it also binds CD47. From our evidence, we suggest that 4.1R plays a role in the phosphatidylserine exposure signaling pathway that is of fundamental importance in red cell turnover. The linkage of CD44 to 4.1R may be relevant to this process.

Molecular biology of Rh proteins and relevance to molecular medicine.

The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility.

Expression of phosphatidylserine (PS) on wild-type and Gerbich variant erythrocytes following glycophorin-C (GPC) ligation.

Glycophorin-C (GPC) is a 40 kDa glycoprotein expressed on erythrocytes and is a receptor for the malarial parasite Plasmodium falciparum to invade these cells. A link between GPC binding (ligation) and phosphatidylserine (PS) expression on erythrocytes has been suggested by its appearance on P. falciparum-infected erythrocytes. Phosphatidylserine expression has also been shown to be a marker of cellular death in a number of biological pathways including some in erythrocytes. Using Annexin V binding, we demonstrated that ligation of GPC with mouse mAb (BRIC-10) induced PS expression on normal erythrocytes. Phosphatidylserine exposure was prevented following tryptic digestion of intact erythrocytes. In addition, GPC variant phenotypes Yus (Delta exon 2) and Gerbich (Delta exon 3), which express a truncated extracellular domain, did not express PS following BRIC-10 binding, whereas PS was exposed on Ls(a) erythrocytes (duplication of exon 3). GPC ligation was also shown to result in a concomitant loss of erythrocyte viability in wild-type erythrocytes after 24 h in vitro. These results identify a potential pathway linking GPC to PS exposure on erythrocytes that may have a role in regulating red cell turnover. Further characterization of this pathway may also identify new targets for the treatment of P. falciparum malaria.

Ligation of CD47 mediates phosphatidylserine expression on erythrocytes and a concomitant loss of viability in vitro.

CD47 ligation has been shown to induce phosphatidylserine (PS) expression as part of a death pathway in nucleated blood cells. Using Annexin V binding assays we showed that ligation of CD47 with the specific CD47-binding peptide 41NK, anti-CD47 monoclonal antibody, and its natural ligand thrombospondin-1 also induced PS expression on enucleated erythrocytes. PS expression was associated with a concomitant loss of erythrocyte viability in vitro. Further characterisation of the CD47-PS signalling pathway on erythrocytes may help develop clinical strategies to further preserve the life of blood donations and improve our understanding of certain types of haemolytic anaemias.