Genetic deletion of calpain-1 and pharmacological inhibition of calpain activity improve red blood cell morphology and reduce sickling in humanized mouse model of sickle cell disease
Butabayeva, Aliya.
2019
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Sickle cell disease
(SCD) is a genetic hematological disorder caused by inheriting two copies of mutated
hemoglobin S gene (HbS) located in codon 6 of globin gene causing the insertion of
valine in place of glutamic acid. The mutated hemoglobin undergoes increased
polymerization in deoxygenated state, and changes erythrocyte properties generating
sickle shape. It has significantly short life ... read morespan leading to anemia, vaso-occlusive
events, and organ impairment. In this study, we investigated the remodeling effects of
calpain-1 (CAPN1) on red blood cell (RBC) membrane using calpain-1 null mouse model of
sickle cell disease (SSCKO). CAPN1 associates with RBCs membrane and cleaves membrane
proteins at high calcium concentration. Patients with SCD have a high level of calcium
in erythrocytes that activates calpain-1 and changes its morphology to sickle shape. The
genotype of AA, SS and SSCKO was confirmed by PCR. Blood samples collected from the
facial vein of AA, SS and SSCKO mice in Heparin coated tube were washed with RPMI and
exposed under normoxia (20%-O2, T-37oC) and hypoxia (3%-O2, 5%-CO2, T-37oC) for
different time intervals and visualized under 60X oil objective by microscopy. No
significant difference between the morphology of SS and SSCKO erythrocytes was observed
under normoxia condition; however, SSCKO erythrocytes showed significantly higher
resistance to hypoxia-induced sickling as compared to SS upon 3-hour hypoxia. The
calpain activity measurements showed that SS erythrocytes contain higher activity then
SSCKO mice. However, treatment with a pan-calpain inhibitor significantly reduced
calpain activity in SS compared to vehicle treated control cells. Moreover, treatment of
cells with MDL28170 showed significant reduction in the number of sickle erythrocytes
and reticulocytes upon 3-hour exposure to hypoxia. Together, these findings suggest that
both calpain-1 and calpain-2 serve as potential therapeutic targets for exerting
anti-sickling effects on erythrocytes and reticulocytes under hypoxic
conditions.
Thesis (M.S.)--Tufts University, 2019.
Submitted to the Dept. of Pharmacology and Drug Development.
Advisor: Athar Chishti.
Keywords: Pharmaceutical sciences, and Pharmacology.read less - ID:
- xw42nn29k
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