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Title: VWF-dependent platelet 'priming' potentiates novel leukocyte interactions and mediates NETosis under flow
Author: Constantinescu-Bercu, Adela
ISNI:       0000 0004 9350 1030
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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Platelet-leukocyte interactions are important in diverse pathophysiological settings from infection to DVT. Previously characterised interactions require robust activation of platelets (e.g. via P-selectin) and/or leukocytes (e.g. via certain β2-integrins). However, recent studies reveal that platelets captured by von Willebrand factor (VWF) under flow can acquire the ability to bind leukocytes. I hypothesised that, under flow, VWF ‘primes’ platelets, in turn facilitating an uncharacterised platelet-leukocyte interaction. My aim was to characterise the interaction between VWF-‘primed’ platelets and leukocytes under flow. Using microfluidic assays, I demonstrated that, under flow, binding of platelets to the VWF A1 domain causes intracellular Ca2+-release and αIIbβ3 activation. VWF-‘primed’ platelets captured neutrophils and T-cells (but not monocytes and B-cells) under low shear. Leukocyte binding was independent of P-selectin and β2-integrins, but significantly reduced by αIIbβ3 blockade, and was enhanced in regions of turbulent flow. Bound neutrophils underwent Ca2+-release and formed neutrophil extracellular traps (NETs), in a Ca2+, NADPH-oxidase and shear-dependent manner. The neutrophil receptor was identified as SLC44A2 through differential gene expression analysis using RNA-sequencing data from the Blueprint consortium. Neutrophils and SLC44A2-transfected HEK293T cells bound activated αIIbβ3, in a manner that was inhibited by blocking the first extracellular loop of SLC44A2. A SNP in SLC44A2 (rs2288904-G/A, M.A.F.-0.22) encoding the R154Q substitution was recently shown to be protective against DVT. Neutrophils homozygous for SLC44A2 rs2288904-A and SLC44A2(R154Q)-transfected HEK293T cells exhibited a significant reduction in the ability to bind VWF-‘primed’ platelets. Platelets from a novel transgenic mouse (GpIbasig/sig) exhibit a decreased ability to become ‘primed’ by VWF and recruit neutrophils under flow. Taken together, these data reveal a previously unreported interaction between platelets and neutrophils, while providing novel mechanistic insights into platelet-mediated NET formation and into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.
Supervisor: Crawley, James ; Salles, Isabelle ; Woollard, Kevin Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral