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Effects of Btk inhibitors on platelet activation
Ai Aii
Aiii
Aiv
Bi Bii
C
Figure 7. Acalabrutinib dose-dependently inhibits glycoprotein VI-mediated signaling. (A) Eptifibatide (9 mM)-treated washed human platelets (4×108/mL) were stimu- lated with CRP (10 mg/mL for 180 s) followed by lysis with 5X SDS reducing sample buffer. Prior to addition of agonist, platelets were pre-incubated with either acalabru- tinib or vehicle (DMSO). (i) Whole cell lysates were then separated by SDS-PAGE and western blot with the stated antibodies for whole cell phosphorylation, kinases and proteins downstream of GPVI. Blots are representative of three experiments. (ii - iv) The percentage of tyrosine phosphorylation as compared to that of vehicle-treated platelets was measured and is represented as the mean ± SEM of three identical experiments. The dose-response curve for inhibition of aggregation from Online Supplementary Figure S3D is shown as a dotted line to enable comparison. (B) Eptifibatide (9 mM)-treated washed human platelets (8×108/mL) were stimulated with CRP (10 mg/mL for 180 s) followed by lysis with 2X ice cold lysis sample buffer. Lysates were precleared and Tec was immunoprecipitated before addition of SDS reducing sample buffer and separation by SDS-PAGE and western blot with the anti-pY antibody 4G10. Membranes were stripped and reprobed with the pan-Tec antibody. The trace is representative of three identical experiments. (C) Btk-deficient DT40 cells were transfected with either wild-type (WT) or kinase-dead (KD) Btk with or without GPVI/FcRg. All cells were transfected with a NFAT-luciferase reporter plasmid. Cells were stimulated with collagen (10 mg/mL) in the presence or absence of acalabrutinib (0.5-10 mM). Serum was excluded during stimulation to avoid plasma binding of the drugs. Luciferase activity between vehicle and drug-treated samples was measured and is shown as the mean ± SEM of three independent experiments. ns = non-significant.
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