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P.L.R. Nicolson et al.
Patients homozygous for an insertion that introduces a stop codon and prevents expression of the immunoglobu- lin receptor on the platelet surface have a relatively mild bleeding diathesis,8 although there are too few of such individuals to determine whether they are protected from thrombosis.
Bruton tyrosine kinase (Btk) is a member of the Tec fam- ily of tyrosine kinases and mediates phosphorylation and activation of PLCg2 downstream of GPVI and the B-cell antigen receptor. The irreversible Btk inhibitor ibrutinib has been introduced into the clinic for treatment of B-cell malignancies but has been reported to increase rates of major hemorrhage in a subgroup of patients.9,10 The increase in bleeding has been attributed to a loss of platelet activation by GPVI11-13 and GPIb,11 with the inhibi- tion of the two receptors having been shown to corre- late.14
In contrast to ibrutinib-treated subjects, patients with X- linked agammaglobulinemia (XLA) do not bleed exces- sively.15 XLA is caused by mutations in the BTK gene which result in a loss or reduction of Btk expression, or expression of a non-functional protein. A potential expla- nation for this difference in bleeding propensity is that ibrutinib blocks activation of platelets by both Btk and the closely related kinase Tec. Tec is expressed in human and mouse platelets, and has been shown to support PLCg2 activation in mouse platelets.16 Interestingly, major hemor- rhage is not seen in patients treated with the structurally related Btk inhibitor, acalabrutinib, despite this also inhibiting Btk by covalent modification of C481.9,17 It has been postulated that this is due to its greater selectivity for Btk over Tec in comparison to ibrutinib.17,18
In the present study we compared the inhibitory effects of ibrutinib and acalabrutinib on platelet activation and protein phosphorylation by GPVI alongside ex vivo studies on patients prescribed the two inhibitors, as well as on XLA patients.
Methods
Reagents
Details on the source of reagents and chemical analyses can be found in the Online Supplementary Information.
Light transmission aggregometry
Aggregation was measured in siliconized glass vials at 37°C in a Model 700 aggregometer (ChronoLog, Havertown, PA, USA) with stirring at 1200 rpm. Platelets were warmed to 37°C for 5 min before the experiments. Platelets were pre-incubated with ibruti- nib, acalabrutinib or dimethyl sulfoxide (DMSO) vehicle for 5 min prior to agonist addition unless otherwise stated. Results were averaged and the half maximal inhibitory concentration (IC50) val- ues were calculated from these data.
the protease inhibitors sodium orthovanadate (5 mM), leupeptin (10 mg/mL), AEBSF (200 mg/mL), aprotinin (10 mg/mL) and pep- statin (1 mg/mL). Platelet lysates were precleared, and detergent- insoluble debris was discarded. An aliquot was dissolved with SDS sample buffer for detection of total tyrosine phosphorylation. Lysates were incubated with either the indicated antibodies and protein A- or protein G-Sepharose. Lysates were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS- PAGE), electro-transferred, and western blotted. Western blots were imaged using ECL autoradiography film. In order to analyze levels of phosphorylation, western blot films were scanned and band intensity measured using ImageJ 1.5 with values normalized to basal levels. Results were averaged and IC50 values were calcu- lated from these data.
Other
Details on the methods for blood sampling, platelet preparation, granule release, [Ca2+]i mobilization, measurement of platelet adhesion under flow, cell lines, plasmids, transfections and the luciferase assay can be found in the Online Supplementary Information.
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM) with statistical significance taken as P<0.05 unless other- wise stated. Statistical analyses, unless otherwise specified, were performed using one-way analysis of variance (ANOVA) with a Bonferroni post-test. Ex vivo platelet aggregation was determined by optical densities, which were compared using a one-way ANOVA with the Tukey multiple comparison test. Correlations of aggregation with tyrosine phosphorylation were assessed using the Pearson correlation coefficient. IC50 values were analyzed using the Welch t-test. All statistical analyses were performed using GraphPad Prism 7.
Ethical approval
Ethical approval for collecting blood from patients and healthy volunteers was granted by the National Research Ethics Service (10/H1206/58) and Birmingham University Internal Ethical Review (ERN_11-0175), respectively. Work on HLA patients has ethical approval via the University of Birmingham HBRC 16-251 Amendment 1.
Results
Inhibition of GPVI-induced platelet aggregation by high concentrations of ibrutinib is reversible
Ibrutinib is 97% bound to plasma proteins and unbound levels reach approximately 0.5 mM in patients.11 At this concentration, ibrutinib has been shown to block GPVI- induced platelet aggregation.12,19 If this is due to inhibition of Btk and other Tec kinases then the inhibition should be irreversible and time-dependent (i.e. inhibition should increase with time). To test this, platelets were treated with a concentration of ibrutinib that causes complete inhibition of GPVI-mediated aggregation in washed platelets before washout of ibrutinib and stimulation with the GPVI-specific agonist collagen-related peptide (CRP). Platelets showed almost full recovery on washout demon- strating that the inhibitory effect is not due solely to cova- lent modification of Btk or Tec (Figure 1A,B). In support of this, incubation of washed platelets with a high concentra- tion of ibrutinib (700 nM) for ≥30 s was sufficient to block aggregation in response to a high dose of CRP (Figure 1C).
Protein phosphorylation
Washed platelets were pre-treated with 9 mM eptifibatide to block integrin αIIbβ3 activation. Agonists were added while stir- ring at 1200 rpm in an aggregometer at 37°C for 180 s unless oth- erwise stated. The platelets were stimulated in the presence of ibrutinib (17 nM - 7 mM), acalabrutinib (50 nM – 200 mM) or vehi- cle (DMSO). For whole cell lysate experiments, activation was ter- minated with 5X SDS reducing sample buffer. For immunoprecip- itation, 8x108/mL platelets were used and reactions were terminat- ed by addition of 2X ice-cold Nonidet P-40 lysis buffer containing
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haematologica | 2018; 103(12)