*Rha and Rhag are MGI nomenclature. SummaryMouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rh null haemolytic anaemia nor any clinical or cellular abnormalities. Rhd)/) and Rhag)/) erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag)/) but only slightly in Rhd)/) animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.

BackgroundAbnormal adhesiveness of red blood cells to endothelium has been implicated in vaso-occlusive crisis of sickle cell disease. The present study examined whether the SAD mouse model exhibits the same abnormalities of red blood cell adhesion as those found in human sickle cell disease. Design and MethodsThe repertoire of adhesive molecules on murine erythrocytes and bEnd.3 microvascular endothelial cells was determined by flow cytometry using monoclonal antibodies or by western blotting. Adhesion was investigated in dynamic conditions and measured at different shear stresses. ResultsCD36, CD47 and intercellular adhesion molecular-4, but not Lutheran blood group antigen/basal cell adhesion molecule, are present on mouse mature erythrocytes. a4β1 are not expressed on SAD and wild type reticulocytes. Endothelial bEnd.3 cells express aVβ3, a4β1, CD47, vascular cell adhesion molecule-1, and Lutheran blood group antigen/basal cell adhesion molecule, but not CD36. Adhesion of SAD red cells is: (i) 2-to 3-fold higher than that of wild type red cells; (ii) further increased on platelet activating factor-activated endothelium; (iii) not stimulated by epinephrine; (iv) inhibited after treating the endothelium with a peptide reproducing one of the binding sequences of mouse intercellular adhesion molecular-4, or with monoclonal antibody against murine av integrin; and (v) inhibited after pretreatment of red blood cells with anti-mouse CD36 monoclonal antibodies. The combination of treatments with intercellular adhesion molecular-4 peptide and anti-CD36 monoclonal antibodies eliminates excess adhesion of SAD red cells. The phosphorylation state of intercellular adhesion molecular-4 and CD36 is probably not involved in the over-adhesiveness of SAD erythrocytes. ConclusionsIntercellular adhesion molecular-4/avβ3 and CD36/thrombospondin interactions might contribute to the abnormally high adhesiveness of SAD red cells. The SAD mouse is a valuable animal model for investigating adhesion processes of sickle cell disease. Haematologica 2010;95:730-737. doi:10.3324/haematol.2009 Intercellular adhesion molecule-4 and CD36 are implicated in the abnormal adhesiveness of sickle cell SAD mouse erythrocytes to endothelium © F e r r a t a S t o r t i F o u n d a t i o n