The following proposal will serve to further characterize red blood cell (RBC) membrane cytoskeleton interactions. Alterations in these components have been shown to play a role in many hereditary hemolytic anemias, aging changes in stored blood and have provided a model for other cellular cytoskeletal networks with isoforms of the erythrocyte proteins. There is strong evidence that the 4.1-spectrin-actin ternary complex, an essential component of the RBC cytoskeleton, is partially linked to the membrane via an attachment of 4.1 to the integral membrane protein, glycophorin C (GPC). Recent evidence suggests that this linkage is not essential to the structural integrity of the RBC as measured by ektacytometry. We hypothesize that the function of the 4.1-GPC linkage, although invisible to the ektacytometer, will be revealed by careful morphologic and in vivo survivability studies. In this project, specific 4.1 interactions will be quantitatively modulated using osmotic lysis and resealing techniques whereby exogenous proteins in the form of recombinant canine and human peptides are loaded into canine RBCs. Using fragments of 4.1 and GPC, we will selectively sever the band 4.1-GPC linkage without disrupting the band 4.1-spectrin-actin complex. The perturbed cells will then be biotinylated and transfused into a canine model for post-transfusional survivability studies. We hypothesize that quantitative addition of the canine 3Ok 4.1 peptide (the GPC binding domain) in the canine RBC is inversely correlated with in vivo survivability of the RBC. Separate experiments will also test that quantitative addition of the cytoplasmic domain of GPC, or antibodies to the 3Ok 4.1 fragment peptide and tail of GPC, will result in decreased survivability of the RBC. These survivability studies conducted in the dog will evaluate our hypothesis as well as introduce a new in vivo model for evaluating membrane cytoskeletal interactions.
| Andrews, Dina A; Yang, Lu; Low, Philip S (2002) Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells. Blood 100:3392-9 |
| Yang, L; Andrews, D A; Low, P S (2000) Lysophosphatidic acid opens a Ca(++) channel in human erythrocytes. Blood 95:2420-5 |
| Andrews, D A; Low, P S (1999) Role of red blood cells in thrombosis. Curr Opin Hematol 6:76-82 |
