Calpain-1 Contributes to Platelet Hyperactivity and Pain Sensitivity in a Humanized Mouse Model of Sickle Cell Disease.
Nwankwo, Jennifer.
2016
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Abstract: Sickle 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 vessel oc... read moreclusion, 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 SCD.
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 - ID:
- qf85np58z
- Component ID:
- tufts:20480
- To Cite:
- DCA Citation Guide EndNote
