Examination of the erythrocyte membrane during senescence and coagulation

Michael Paul Rettig, Purdue University

Abstract

One hypothesis to explain the age-dependent clearance of RBCs from circulation proposes that denatured/oxidized hemoglobin arising late during an RBC's lifespan induces clustering and immunological recognition of the integral membrane protein, band 3. Because dog RBCs have survival characteristics that closely resemble those of human RBCs, we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. Consistent with the above hypothesis, senescent dog RBCs exhibited: (i) a ∼50-fold increase in membrane-bound globin, (ii) a ∼70% decrease in intracellular reduced glutathione, (iii) a 7-fold enhancement of surface-associated autologous IgG, and (iv) a ∼25% reduction in the number of band 3 molecules attached to the underlying membrane skeleton. In an unrelated study, we found that the conformation of the cytoplasmic domain of band 3 (cdb3) influences the association state of the protein in situ. The compact form of cdb3 that occurs under slightly acidic conditions favors dimer formation, whereas the elongated state of cdb3, induced by alkaline treatment, allows band 3 aggregation. Phosphatidylserine (PS) exposure onto the surface of vascular or peripheral blood cells is a crucial event in the coagulation process. Using prothrombinase and FITC-annexin V binding assays, we demonstrated that: (i) PGE 2 and hypertonically treated RBCs do not expose PS, (ii) RBCs isolated from whole clotted blood do not expose PS, and (iii) patients with overhydrated hereditary stomatocytosis (OHSt) have subpopulations of circulating RBCs that expose PS on their outer surface. Although these data argue that normal RBCs do not promote coagulation through the exposure of PS, the vaso-occlusive crises and increased thrombosis observed in patients with OHSt could be due to the subpopulation of PS-exposing RBCs. Finally, the current studies also indicate that the PLC signaling cascade is not activated in human RBCs treated with either PGE2 or thrombin.

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Biochemistry

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