Date of Completion

12-19-2013

Embargo Period

12-18-2015

Keywords

sickle cell disease, atomic force microscopy, elasticity, red blood cell, erythrocyte, cytoadhesion

Major Advisor

George Lykotrafitis

Associate Advisor

Anastasios Tzingounis

Associate Advisor

Biree Andemariam

Associate Advisor

Wei Sun

Associate Advisor

Tai-Hsi Fan

Field of Study

Biomedical Engineering

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The biomechanical properties of red blood cells (RBCs), including increased stiffness and abnormal cytoadherence, are integral components in the cascade of events resulting to vasoocclusive episodes (VOEs) in sickle cell disease (SCD). VOEs are the main cause of morbidity in SCD and sickle cell trait (SCT). Using experimental techniques based on atomic force microscopy (AFM), we studied the stiffness and adhesion of RBCs from SCD patients and from subjects with SCT. We found that SCD and SCT RBCs are three-fold stiffer than normal RBCs. Further, a ten-fold increase in the stiffness of sickled RBCs was measured upon deoxygenation. In an effort to rectify the increased stiffness of sickle RBCs, mice were fed a diet supplemented with docosahexanoic acid (DHA), an omega-3 fatty acid. A decrease in RBC stiffness was measured suggesting therapeutic benefits of DHA. Cytoadherence of RBCs to subendothelial laminin via the basal cell adhesion molecule/Lutheran (BCAM/Lu) is implicated in vasculopathy, a common condition in SCD patients. We established the in vitro technique of single-molecule force spectroscopy (SMFS) which enables detection of single BCAM/Lu proteins on the RBC surface via measurement of the unbinding force with laminin. It was shown that epinephrine, acting through the cyclic adenosine monophosphate (cAMP) signaling pathway, increases the population of active BCAM/Lu receptors on SCT RBCs, suggesting a role in exercise-induced VOEs. The sensitivity of the SMFS system was validated in a neuronal system to quantitatively map SK channels and then employed to investigate the effects of cAMP pathway targeting on BCAM/Lu receptor expression on normal and SCD RBCs. We illustrated

that A-kinase anchoring proteins are crucial for BCAM/Lu receptor activation. To examine the relevance of results based on SMFS in the cytoadhesion of entire RBCs, single-cell force spectroscopy (SCFS) was established to measure the adhesion of whole cells with a functionalized substrate. We established a correspondence between the SMFS and SCFS results. Both techniques were able to detect significant changes in the adhesive response of RBCs to cAMP pathway modulation and variability was measured amongst human subjects, suggesting that RBCs maintain diverse intracellular levels of tonic protein kinase A.

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