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Platelet Biology & its Disorders
Inhibition of Btk by Btk-specific concentrations of ibrutinib and acalabrutinib delays but does not block platelet aggregation mediated by glycoprotein VI
Phillip L.R. Nicolson,1 Craig E. Hughes,2 Stephanie Watson,1 Sophie H. Nock,2 Alexander T. Hardy,1 Callum N. Watson,1 Samantha J. Montague,3
Hayley Clifford,4 Aarnoud P. Huissoon,4 Jean-Daniel Malcor,5 Mark R. Thomas,1 Alice Y. Pollitt,2 Michael G. Tomlinson,6 Guy Pratt7 and Steve P. Watson1,8
1Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, UK; 2Institute for Cardiovascular and Metabolic Research, Harborne Building, University of Reading, UK; 3ACRF Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, Australian National University, Canberra, ACT, 2601, Australia; 4Department of Immunology, Heartlands Hospital, Birmingham, UK; 5Department of Biochemistry, University of Cambridge, UK; 6Department of Biosciences, College of Life and Environmental Sciences, University of Birmingham, UK; 7Department of Haematology, Queen Elizabeth Hospital, Birmingham, UK and 8Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, UK
ABSTRACT
Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton tyro- sine kinase used in the treatment of B-cell malignancies. They bind irreversibly to cysteine 481 of Bruton tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of down- stream substrates including phospholipase C-g2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton tyrosine kinase activity, as shown by loss of phosphorylation at tyrosine 223 and phospholipase C-g2, delay but do not block aggregation in response to a maximally-effective concentration of collagen-related peptide or collagen. In contrast, 10- to 20-fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation in response to glyco- protein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, showed a reduction of platelet aggregation in response to collagen-related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton tyrosine kinase block- ade. Unexpectedly, low concentrations of ibrutinib inhibited aggregation in response to collagen-related peptide in patients deficient in Bruton tyrosine kinase. The increased bleeding seen with ibrutinib over acal- abrutinib is due to off-target actions of ibrutinib that occur because of unfavorable pharmacodynamics.
Introduction
The major physiological ligands that activate platelets in hemostasis and throm- bosis signal through G protein-coupled and tyrosine kinase-linked receptors. The former includes receptors for thrombin (PAR1, PAR4), thromboxane A2 (TP) and ADP (P2Y1, P2Y12), and the latter receptors for collagen/fibrin (glycoprotein VI: GPVI), podoplanin (CLEC-2), von Willebrand factor (GPIb-IX-V) and fibrinogen (integrin αIIbβ3).1,2
GPVI is a receptor for collagen and fibrin which forms a complex with the Fc receptor g-chain (FcRg).2-4 GPVI triggers powerful platelet activation through Src, Syk and Tec family tyrosine kinases leading to activation of phospholipase C-g2 (PLCg2).5 GPVI is expressed exclusively on platelets and the platelet precursor cell, the megakaryocyte.6 Mice deficient in GPVI have a minor increase in tail bleeding times but fail to form occlusive thrombi in a FeCl3 injury arterial thrombosis assay.7
Ferrata Storti Foundation
Haematologica 2018 Volume 103(12):2097-2108
Correspondence:
p.nicolson@bham.ac.uk or s.p.watson@bham.ac.uk
Received: March 16 2018. Accepted: July 18, 2018. Pre-published: July 19 2018.
doi:10.3324/haematol.2018.193391
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/12/2097
©2018 Ferrata Storti Foundation
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