Calpain-1 Contributes to Platelet Hyperactivity and Pain Sensitivity in a Humanized Mouse Model of Sickle Cell Disease.
cell disease (SCD) was the first genetic disorder for which a molecular mechanism was
identified. SCD is caused by a glutamic acid to valine substitution in the sixth codon
of the beta-globin gene, resulting in sickle hemoglobin, which polymerizes under low
oxygen conditions to form sickle red blood cells (sRBCs). Reduced deformability of
sickle RBCs contributes to blood vess... read moreel occlusion, leading to organ damage,
vaso-occlusive crises, and pain, the hallmark features of SCD. Today, SCD affects ~13
million patients worldwide, and 100,000 in the United States, where the annual economic
burden is estimated to be >$1 billion. Currently, management of SCD is limited to
hydroxyurea, which has limited efficacy, and is not FDA-approved for use in children.
Hence, there is an urgent need for new disease-modifying therapies for SCD. One of the
major contributors to SCD pathobiology is the hemolysis of sRBCs, which release free
plasma hemoglobin and the platelet agonist adenosine 5'-diphosphate (ADP). While
platelet activation/aggregation promotes tissue ischemia, the link between sickle
platelet dysfunction and pain remains poorly understood. Calpain-1, a calcium-activated
cysteine protease, is ubiquitously expressed in hematopoietic cells, and is known to
mediate aggregation of washed platelets in healthy mice. In the SAD mouse model of mild
SCD, pharmacological inhibition of calpain-1 by BDA-410 improved sickle RBC morphology
and density. Thus, the hypothesis of this doctoral dissertation is that genetic
inactivation of calpain-1 in the Townes mouse model of severe SCD would ameliorate one
or more of the three main clinical features of SCD - hemolytic anemia, vaso-occlusive
pain crises, and organ dysfunction. We generated calpain-1 knockout Townes sickle
(SSCKO) mice to investigate the role of calpain-1 in sickle platelet
activation/aggregation and pain. The platelet surface expression of activated GPIIb-IIIa
was elevated in sickle (SS) and SSCKO platelets as compared to control (AA) mice. In
PAR4-TRAP-stimulated whole blood aggregometry, steady state SSCKO mice exhibited
significantly impaired platelet aggregation compared to SS and AA mice. Interestingly,
hypoxia/reoxygenation treatment induced platelet hyperactivity in SS and SSCKO but not
AA mice, and partially restored the attenuated platelet aggregation in SSCKO mice.
Furthermore, PAR4-TRAP-stimulated GPIIb-IIIa activation was normal in SSCKO platelets
suggesting that a mechanism downstream of integrin-fibrinogen binding mediates the
impaired platelet aggregation in steady state SSCKO mice. Notably, knockout of calpain-1
in Townes sickle mice ameliorated chronic hyperalgesia, including deep/musculoskeletal
pain, mechanical hyperalgesia, and thermal sensitivity to heat and cold. Similar to the
findings in the platelet aggregation study, hypoxia/reoxygenation treatment reversed
calpain-1-mediated attenuation of deep/musculoskeletal pain, mechanical hyperalgesia,
and thermal sensitivity to heat and cold, indicating that calpain-1 contributes to
chronic pain but not acute painful crises in sickle mice. In summary, this doctoral
dissertation provides the first evidence that calpain-1 regulates platelet hyperactivity
and pain in Townes sickle mice, and may present a viable pharmacological target to
reduce platelet hyperactivity and pain in
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Pharmacology & Experimental Therapeutics.
Advisor: Athar Chishti.
Committee: John Castellot, David Greenblatt, Henry Wortis, and Andrew Frelinger III.
Keywords: Pharmacology, Genetics, and Biochemistry.read less