The goal of this research is to test the following hypothesis: adducin plays a critical role in the assembly, stability, and function of the membrane skeleton in red blood cells (RBCs), platelets and the kidney. Genetic defects in adducin result in anemia, platelet dysfunction, and aberrant control of systemic blood pressure. The membrane skeleton is a critical component of most vertebrate cells. In vitro studies in mammalian cells and studies in other organisms such as D. melanogaster suggest that adducin plays a critical role in the developmental assembly of the membrane skeleton in the red blood cell (RBC), while data have also emerged showing that adducin is a component of the platelet membrane skeleton and is important in platelet function (e. g., aggregation, formation of filopodia and lamellipodia). Moreover, adducin polymorphisms have been associated with altered RBC and kidney cation transport activity and a hypertensive phenotype in the Milan hypertensive (MHS) rat strain. Notably, the cation transport differences between MHS and normotensive (MNS) control rats are abolished upon removal of the membrane skeleton. In humans, adducin polymorphisms have been associated with hypertension in some but not all populations. Here, we will determine the role of adducin in RBC membrane skeleton assembly and stabilization of the lipid bilayer, platelet function, and RBC cation transport activity and the regulation of systemic blood pressure in vivo using genetically defined mice.
The specific aims are to: (1) Determine the role of adducin in RBC membrane skeleton assembly and stabilization. We will generate conditional knockouts of the adducin genes in mice using the loxP system and an interferon-inducible Cre-expressing promoter strain (Mxl-Cre) and perform a complete analysis of the RBC phenotype of each mutant strain (singly and in combination). (2) Determine the role of adducin in platelet physiology and pathophysiology. We will perform a complete battery of platelet function tests in adducin null strains to assess shape change, adhesion and spreading, clot retraction, secretion, aggregation, phosphatidylserine exposure and the incidence of thrombi and infarcts. (3) Determine the role of adducin in RBC cation transport and systemic blood pressure regulation. We will determine RBC cation transporter rates (which mirror transport activities in the kidney), systolic blood pressure and heart rates in adducin null strains. Follow up studies will include electrocardiology and echocardiology.
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