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Effects of Btk inhibitors on platelet activation
However, at a lower concentration, ibrutinib (70 nM) caused a time-dependent delay in aggregation in response to a high concentration of CRP, which was apparent at incubation times of ≥5 min (Figures 1D and 2Ai). This con- centration of ibrutinib also caused a reduced response to a sub-maximal concentration of CRP (Online Supplementary Figure S2A). The time-dependent delay is consistent with an irreversible action and contrasts with the rapid onset of inhibition observed at the high concentration of ibrutinib. A similar set of observations was seen in washed platelets stimulated by collagen (Online Supplementary Figure S1A). Similar results were also seen in the presence of 0.3% bovine serum albumin, which was used in case the results were influenced by adsorption of ibrutinib to the surface of the aggregometer tube (Online Supplementary Figure S1B).
Platelet secretion and Ca2+ mobilization play key roles in platelet activation. Consistent with the results for aggrega- tion, low (70 nM) and high (700 nM) concentrations of ibrutinib had, respectively, no effect or blocked ATP secre- tion in response to a high concentration of CRP (Figure 2Bi, 2Biii). Similarly, the peak Ca2+ concentration follow- ing administration of a high concentration of CRP was not altered in the presence of a low concentration of ibrutinib (70 nM) but was markedly reduced by a high concentra- tion (700 nM). The dose-response curve for inhibition of aggregation was similar to that for loss of Ca2+ mobiliza- tion (Figure 2Bii, 2Biii) and was not affected by the pres- ence of the cyclooxygenase inhibitor indomethacin (Online Supplementary Figure S1D). There was no statistical difference between the IC50 of ibrutinib for secretion,
AB
C
D
Figure 1. Increasing ibrutinib incubation time has no effect on degree of inhibition of platelet aggregation and this inhibition is reversed by washing. (A) Representative traces of washed platelets at 4x108/mL stimulated with CRP (10 mg/mL for 180 s). Prior to addition of the agonist, platelets were pre-incubated with either ibrutinib or vehicle (DMSO) for 5 min. Alongside, washed platelets at 4x108/mL identically treated with either ibrutinib or vehicle were washed twice in Tyrode buffer and platelets resuspended to 4x108/mL; platelets were then stimulated with CRP (10 mg/mL for 180 s). The data shown are representative of three identical experiments. (B) Mean data for (A) (n=3) analyzed with one-way ANOVA. Results are shown as mean ± SEM. *P<0.05. (C) Washed platelets (4x108/mL) were incubated with ibrutinib or vehicle (DMSO) for 30 s - 60 min before being stimulated with CRP (10 mg/mL). The optical density (OD) of platelet suspensions was measured in a ChronoLog Model 700 aggregometer with stirring at 1200 rpm. Traces representative of three similar experiments are shown. (D) Delay in aggregation seen with ibrutinib-treated washed platelets (n=3) analyzed with one-way ANOVA. Results are shown as mean ± SEM. *P<0.05.
haematologica | 2018; 103(12)
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