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Pharmacological Characterization of Calcium-Activated Potassium Channel Function in Human Platelets

  • Author / Creator
    Back, Valentina
  • Background: Platelets and endothelial cells play an important role in maintaining vascular tone and hemostasis, critical to maintaining the health of the cardiovascular system. In resistance arteries, stimulation of endothelial cell small (SKCa) and intermediate (IKCa) conductance calcium-activated potassium channels provides a negative-feedback mechanism to limit agonist-induced vasoconstriction. Additionally, endothelial cell KCa channels in conjunction with nitric oxide (NO) mediate vasodilation in response to agonists and physical stimuli. Platelets, like endothelial cells, possess KCa channels and generate NO via endothelial nitric oxide synthase (eNOS). NO is known to limit platelet aggregation but the role of KCa channels in platelet function and NO-generation has not yet been explored. It was hypothesized that activation of KCa channels inhibits platelet aggregation and enhances platelet NO production. The objective was to pharmacologically characterize SKCa and IKCa channel function in platelets, and investigate their role in platelet NO production.Methods: Blood was collected from healthy human volunteers following ethics approval. The platelets were then isolated from the collected blood, according to standard protocol. Immunofluorescence microscopy was used to determine KCa channel subtypes present within platelets, and their localization within the platelet. Aggregometry was performed in the presence of IKCa (SKA-31) and SKCa (CyPPA) channel activators, and IKCa (TRAM-34) and SKCa (apamin) channel blockers. A novel flow chamber model, the Q-Sense Quartz Crystal Microbalance (QCM), along with confocal microscopy was used to investigate platelet adhesion and aggregation under flow conditions in response to KCa channel activators. Flow cytometry of DAF-FM stained platelets was used to measure changes in NO generation. Calcium signaling was measured using flow cytometry of Fluo-4 AM stained platelets at different time points of aggregation in response to KCa channel activators. ATP and P-selectin secretion were measured as markers of dense and alpha granule secretion, respectively. Results: Confocal microscopy of platelets demonstrated the presence of IKCa channels in platelets, with the presence of SKCa channels in some individuals. IKCa channels were localized to the cytosolic portion of the platelet plasma membrane. Activation of IKCa channel with SKA-31, or SKCa channel activation with CyPPA demonstrated concentration-dependent inhibition of collagen-induced aggregation. IKCa selective channel blocker, TRAM-34, reversed the anti-aggregatory effects of SKA-31 but not CyPPA. SKCa channel-selective blocker, apamin, did not reverse the effect of either SKA-31 or CyPPA, but weakly inhibited platelet aggregation alone. The QCM demonstrated that SKA-31 inhibits platelet aggregation under laminar flow conditions, but CyPPA does not. Surprisingly, SKA-31 and CyPPA treatment inhibited platelet-NO generation. Calcium signaling flow cytometry demonstrated a significant inhibition of calcium flow during aggregation with SKA-31 treatment, but not CyPPA. Further investigation demonstrated SKA-31 inhibiting dense and alpha granule secretion, whereas CyPPA only inhibited dense granule secretion. Conclusions: These results suggest that IKCa may be the dominant KCa channel within platelets, which upon pharmacological activation inhibits platelet aggregation. IKCa channel activation also inhibits platelet NO generation, calcium signaling, as well as dense and alpha granule secretion within platelets. Thus IKCa channels may provide a novel therapeutic target to inhibit platelet aggregation.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/r3-4xvx-5f06
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.