Verbatim): The research objective is to develop a detailed understanding of the molecular and structural basis for the abnormal rheological and adherence properties of sickle red blood cells and define the in vivo pathologic sequelae of these cellular changes. To achieve our objective we propose the following series of studies: 1) Using novel experimental strategies, determine the relative contributions of the Gardos channel and the K-Cl cotransporter to the morphologic and rheologic heterogeneity of dense sickle red cells. Using intravital microscopy, document effects of the observed rheological heterogeneity on flow behavior in vivo. Generate sickle mice in which the Gardos channel and/or K-CL cotransporter are inactivated to directly establish the relative contributions of these two transport proteins to cell dehydration and compromised blood flow. 2) To develop a mechanistic understanding of premature sickle red cell destruction, distinct populations of mouse sickle cells will be isolated based on differences in their cellular and membrane characteristics and their in vivo life span will be determined. The surface characteristics and surface area of these different cell sub-populations will be documented. Data from these in vitro and in vivo studies will be used to define the contribution of surface area loss and associated membrane changes to the decreased life span of sickle cells. 3) As it is likely that the dynamic strength of a specific adhesive complex will determine its impact on in vivo flow, we will measure the dynamic strength for various specific adhesive interactions identified in mediating sickle red cell adherence to vascular endothelial cells. These data should enable us to define the physiologically relevant adhesive interactions. We will validate the in vitro studies by performing in situ studies on sickle mice in which genes encoding specific adhesive receptors are inactivated. For the proposed studies, we will use multiple techniques in biophysics, molecular biology, cell biology, mouse genetics and circulatory physiology. The sickle mice that we developed expressing exclusively human sickle hemoglobin and exhibiting many clinical features of human disease are a key component of our experimental strategy. These mice should enable us to perform deliberate and specific perturbations for defining the determinants of adherence and flow, which would not be possible in human studies. Our application of these refined systems offers great promise for elucidating the cause of painful vaso-occlusion and identifying potential targets for future therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL031579-21
Application #
6559434
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1989-10-01
Project End
2006-03-31
Budget Start
2001-12-01
Budget End
2002-03-31
Support Year
21
Fiscal Year
2001
Total Cost
$93,420
Indirect Cost
Name
New York Blood Center
Department
Type
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10065
Koshino, Ichiro; Mohandas, Narla; Takakuwa, Yuichi (2012) Identification of a novel role for dematin in regulating red cell membrane function by modulating spectrin-actin interaction. J Biol Chem 287:35244-50
Safeukui, Innocent; Buffet, Pierre A; Deplaine, Guillaume et al. (2012) Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen. Blood 120:424-30
Mohandas, Narla; An, Xiuli (2012) Malaria and human red blood cells. Med Microbiol Immunol 201:593-8
Liu, Jing; Mohandas, Narla; An, Xiuli (2011) Membrane assembly during erythropoiesis. Curr Opin Hematol 18:133-8
Chen, Lixiang; Hughes, Richard A; Baines, Anthony J et al. (2011) Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of beta1 integrin. J Cell Sci 124:2478-87
An, Xiuli; Guo, Xinhua; Yang, Yang et al. (2011) Inter-subunit interactions in erythroid and non-erythroid spectrins. Biochim Biophys Acta 1814:420-7
Yang, Shaomin; Weng, Haibo; Chen, Lixiang et al. (2011) Lack of protein 4.1G causes altered expression and localization of the cell adhesion molecule nectin-like 4 in testis and can cause male infertility. Mol Cell Biol 31:2276-86
Gauthier, Emilie; Guo, Xinhua; Mohandas, Narla et al. (2011) Phosphorylation-dependent perturbations of the 4.1R-associated multiprotein complex of the erythrocyte membrane. Biochemistry 50:4561-7
Blanc, Lionel; Salomao, Marcela; Guo, Xinhua et al. (2010) Control of erythrocyte membrane-skeletal cohesion by the spectrin-membrane linkage. Biochemistry 49:4516-23
Manno, Sumie; Mohandas, Narla; Takakuwa, Yuichi (2010) ATP-dependent mechanism protects spectrin against glycation in human erythrocytes. J Biol Chem 285:33923-9

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