Page 176 - 2018_12-Haematologica-web

P.L.R. Nicolson et al.
aggregation or Ca2+ mobilization.
Taken together these results show that ibrutinib has
two distinct effects on platelet activation by CRP. At a low concentration (70 nM), ibrutinib delays but does not inhibit activation, whereas at a 10-fold higher concentra- tion of ibrutinib (700 nM) activation is blocked. The latter action is reversible indicating that it is not mediated by covalent modification of Btk or other Tec kinases.
Low-dose ibrutinib blocks Btk but not Tec
The concentration-response curve to ibrutinib on tyro- sine phosphorylation was investigated in washed platelets in the same conditions as for the platelet function studies above. CRP induced robust tyrosine phosphorylation in whole cell lysates which was dose-dependently inhibited by ibrutinib. Correspondingly with the results for aggrega- tion, this inhibitory effect of ibrutinib on global tyrosine phosphorylation was also reversible on washout (Figure
3Ai). Using phosphospecific antibodies, we were able to see that the inhibition of Src pY418 (which lies upstream of Btk) was also reversible but that autophosphorylation of Btk at pY223 and Btk substrates PLCg2 pY753 and pY121711,20 was irreversible (Figure 3Ai-ii).
A detailed analysis of the dose response to ibrutinib on a wider range of proteins in the GPVI signaling cascade was investigated using further phosphospecific antibodies (Figure 3Bi). Autophosphorylation of Btk and downstream PLCg2 was reduced to basal levels by a low dose of ibru- tinib (70 nM) (Figure 3Bii). In contrast, phosphorylation of Btk on Y551, which is mediated by Src family kinases,21 and proteins that lie upstream of Btk, namely Src Y418, Syk Y525/6, SLP-76 Y145 and LAT Y200, was not altered (Figure 3Biii-iv). Inhibition of phosphorylation of Src on its activation site, Y418, was observed at a 10-fold higher concentration of ibrutinib (Figure 3Biv) and was shown to correlate with inhibition of aggregation (Pearson correla-
Ai
Aii
Bi
Bii
Biii
Figure 2. Ibrutinib dose-dependently inhibits glycoprotein VI-mediated platelet aggregation, ATP secretion and Ca2+ mobilization. (A) (i) Representative traces showing the effect of increas- ing doses of in vitro ibrutinib incubated for 5 min with washed platelets at 4x108/mL. (ii) Ibrutinib dose-response curves in washed platelets (n=7). (B) Representative traces showing the effect of increasing doses of in vitro ibrutinib incubated with washed platelets at 4x108/mL for 5 min on (i) ATP secretion and (ii) Ca2+ mobilization in response to stimulation with CRP (10 mg/mL) for 180 s. (iii) Ibrutinib dose-response curves in washed platelets on ATP secretion (n=3) and Ca2+ mobilization (n=3). The dose-response curve for inhibition of washed platelet aggrega- tion from (Aiii) is shown as a dotted line to enable comparison. Results are shown as mean ± SEM. All experiments were stimulated with CRP (10 mg/mL). For comparison of IC50: ns = non- significant.
2100
haematologica | 2018; 103(12)