Page 190 - 2019_11 Resto del Mondo-web

J. Series et al.
with 0.5 mM ibrutinib or 2 mM acalabrutinib for 1 h, was perfused over a collagen matrix and platelet adhesion and thrombus formation were monitored by real-time imaging. Acalabrutinib had no effect on platelet surface coverage in both groups, indicating that platelet adhesion was spared (Figure 4A, B). However, while this drug had no impact on the thrombus volume in the ibrutinib LS group of healthy donors, it significantly decreased thrombus volume in the HS group (Figure 4A, B). As expected, in similar conditions, ibrutinib significantly decreased surface coverage and thrombus volume in the ibrutinib HS group and tended to decrease thrombus volume in the LS group.
Effect of associations of antiplatelet drugs and ibrutinib or acalabrutinib
The management of bleeding risk in patients with cardio- vascular disease under dual antiplatelet therapy for primary or secondary prevention treated with Btk inhibitors is of concern in clinical practice.4,5,28 There is currently little infor- mation to guide clinicians in making decisions about antiplatelet therapy concurrently with Btk inhibitors. We therefore tested the effect of combinations of ibrutinib or acalabrutinib with indomethacin (an aspirin-like drug) or cangrelor (ARC69931MX), an antagonist of the P2Y12 ADP receptor, on platelet aggregation evoked by collagen in PRP from the two groups of healthy donors (Figure 5). In both
A
B
groups, platelet aggregation was significantly inhibited by indomethacin and to a lesser extent by cangrelor. Importantly, the combination of indomethacin or cangrelor with ibrutinib at a clinically relevant dose amplified the inhibition of platelet aggregation in the ibrutinib HS group. In the ibrutinib LS group, ibrutinib at 0.5 mM did not have a significant effect on the maximal platelet aggregation induced by collagen but increased the effect of indomethacin or cangrelor (in accordance with the 6-8% grade 3-4 bleeding events reported in clinical trials). Interestingly, acalabrutinib, at a clinically relevant dose which had no impact on maximal platelet aggregation induced by collagen, also strongly potentiated, in a dose- dependent manner, the effect of indomethacin and can- grelor in both groups (Figure 5A, B). These data indicate that these two Btk inhibitors potentiated the effect of cyclooxygenase inhibition and P2Y12 antagonism, even in the ibrutinib LS group (Figure 5).
Discussion
The first-generation Btk inhibitor ibrutinib has revolu- tionized the therapy of CLL and mantle cell lymphoma but the drug can cause some side effects such as atrial fibrilla- tion and bleeding.1-3,28 The occurrence of side effects is the
Figure 3. Effect of ibrutinib and acalabrutinib on tyrosine phosphorylation events. (A) Washed platelets from healthy donors were treated or not with increasing doses of acalabrutinib (ACP) for 1 h at 37°C and stimulated with collagen 3.3 mg/mL. Platelet aggregation was assessed by turbidimetry during 10 min and results, expressed as percentage of maximal aggregation, are mean ± standard error of mean (SEM). Ten donors with low sensitivity (LS) and eight with high sensitivity (HS) to ibrutinib were analyzed. **P<0.01, according to one-way analysis of variance (ANOVA). Half maximal inhibitory concentrations (IC50) were determined using GraphPad Prism software. (B) In parallel to aggregation, the effect of ibrutinib and acalabrutinib on platelet tyrosine phosphorylation events (PLCγ2 phosphorylation on Tyr-753 and Src phosphorylation on Tyr-418) in response to 1 min stimulation with collagen 3.3 mg/mL was assessed by western blotting. The results of the west- ern blot quantification by densitometric analysis are shown as means ± SEM from ten independent experiments for LS and seven independent experiments for HS. *P<0.05, **P<0.01, according to one-way ANOVA. Representative western blots are shown for each group.
2296
haematologica | 2019; 104(11)