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P.L.R. Nicolson et al.
a delay in aggregation; and the curves for inhibition of ATP secretion and Ca2+ mobilization lie slightly to the left of that for aggregation (Online Supplementary Figure S3A- E). As with ibrutinib, acalabrutinib blocked tyrosine phos- phorylation of Btk on Y223 and PLCg2 on Y759 and Y1217 at a concentration (2 mM) that caused a delay in onset but no reduction in aggregation (Figure 7Ai,ii). Higher concen- trations of acalabrutinib (up to 200 mM) had no effect on phosphorylation of Src Y418, Syk Y525/6 and LAT Y200 but caused a small reduction in phosphorylation of Btk Y551 and SLP-76 Y145 (Figure 7Ai,iii-iv). Interestingly, acalabrutinib also caused a biphasic inhibition of Tec phosphorylation with partial inhibition observed at approximately 1 mM and full blockade at 200 mM (Figure 7B). The IC50 values for each phosphorylation event are included in Online Supplementary Table S1. Concentrations of acalabrutinib that blocked phosphorylation of Btk in platelets had no effect on NFAT activation by CRP in DT40 cells transfected with WT or KD Btk (Figure 7C).
Allowing for the fact that acalabrutinib has a 5-fold lower potency for Btk, these results are in line with those for ibrutinib.
Glycoprotein VI-mediated platelet aggregation is blocked ex vivo in patients taking ibrutinib, but not acalabrutinib
We investigated the effect of ibrutinib and acalabrutinib in patients with chronic lymphoid leukemia (CLL) taking ibrutinib 420 mg once daily, acalabrutinib 100 mg twice daily or a non-Btk targeting control chemotherapy regi- men. GPVI-induced platelet aggregation was blocked in the platelet-rich plasma of patients taking ibrutinib but was not blocked in patients taking acalabrutinib or in the control group despite complete inhibition of autophos- phorylation of Btk pY223 and its downstream substrate PLCg2 at pY1217 by both inhibitors (Figure 5Bi-iv). Platelet aggregation induced by the GPCR agonists, ADP and PAR1 peptide, was not altered in the patients taking either inhibitor (data not shown).
Discussion
In this study we show that (i) irreversible blockade of Btk by ibrutinib and acalabrutinib delays but does not block the platelet aggregation induced by high concentra- tions of GPVI agonists; (ii) blockade of GPVI-mediated aggregation by ibrutinib and acalabrutinib occurs at a con- centration one to two orders of magnitude higher than is required to block Btk due to an off-target action which is reversible; (iii) the ratio between inhibition of Btk kinase activity and platelet aggregation induced by GPVI is the same for ibrutinib and acalabrutinib; (iv) clinically relevant concentrations of ibrutinib but not acalabrutinib block activation of platelets by GPVI; (v) platelet adhesion and aggregation under flow conditions is maintained follow- ing inhibition of Btk; (vi) Btk supports platelet activation by GPVI by acting as an adapter protein and as a tyrosine kinase; and (vii) ibrutinib blocks platelet aggregation in XLA patients at concentrations that block Btk.
These results show that platelets, in which Btk kinase function and downstream PLCg2 phosphorylation have been blocked, have a slight delay in aggregation in response to high concentrations of GPVI ligands, while platelet adhesion and aggregation under arterial flow con-
ditions are unaltered. These observations, together with reports that patients with XLA or those treated with acal- abrutinib do not experience major bleeding,15,17 provide powerful evidence that inhibition of Btk does not give rise to major bleeding. The major bleeding observed in patients treated with ibrutinib relative to acalabrutinib is due to the differential dosing regimens of the two Btk inhibitors, with the clinical dose of ibrutinib blocking acti- vation of platelets by GPVI due to one or more off-target effects.
The conclusion that inhibition of Btk does not give rise to major bleeding on treatment with ibrutinib contrasts with the conclusion of the studies by Levade et al.11 and Bye et al.13. Levade et al.11 demonstrated a close correlation between inhibition of autophosphorylation of Btk at Y223 and aggregation in GPVI-activated platelets. While we are unable to explain this in the light of the present observa- tions, we note that Levade et al.11 also reported that phos- phorylation of PLCg2 at Y753, which is mediated by Btk, was inhibited at a 10-fold lower concentration of ibrutinib as is seen in the present study. Bye et al.13 used a single, supramaximal concentration of ibrutinib which also blocked Src phosphorylation for their biochemical and flow-based assays. The determination of full concentra- tion-response curves in the present study has highlighted the mismatch between inhibition of Btk and loss of platelet aggregation, and has provided evidence that the bleeding diathesis that is seen in some ibrutinib-treated patients is due to off-target effects.
An unexpected observation in the present study was that platelets are able to aggregate in response to a high dose of CRP despite the absence of detectable PLCg2 phosphorylation. One explanation for this is that Btk also supports activation of PLCg2 as an adapter protein as shown by the observation that transfection of KD Btk restores GPVI signaling in DT40 cells. A similar result has been previously shown for Btk in B-cell receptor signaling.24,26 This is in keeping with previous studies showing that phosphorylation of PLCg2 at Y1217 is not required for its enzymatic activity in Ramos cells.27
We were surprised to find that platelets from XLA patients, who lack Btk protein, have increased susceptibil- ity to ibrutinib relative to platelets from controls. The only known difference between the XLA patients and controls in the presence of ibrutinib is the absence of Btk protein, although this could also change the balance of activatory and inhibitory phosphorylation within the GPVI signaling cascade. Furthermore, the absence of Btk renders aggrega- tion of these platelets critically dependent on PLCg2 phos- phorylation in contrast to controls. The target for ibrutinib which gives rise to inhibition of aggregation in the XLA patients is not known. There are several kinases that are inhibited by ibrutinib over a similar range of concentra- tions to that for inhibition of Btk.18 Within this group only Csk is known to be expressed in platelets.28
We have shown that blockade of GPVI-mediated platelet aggregation by ibrutinib is reversible, which con- trasts with the irreversible blockade of Btk and Tec.18 The reversibility provides evidence that blockade is not medi- ated by inhibition of Tec family kinases, as was previously postulated,9,11,17 because Tec also has a cysteine residue in its ATP binding domain analogous to C481 on Btk. This is further supported by the observation that ibrutinib-medi- ated blockade of NFAT signaling in DT40 cells, which lack Tec, follows a similar pattern as that for platelet aggrega-
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haematologica | 2018; 103(12)