Differences and similarities in the effects of ibrutinib and acalabrutinib on platelet functions

Abstract

While efficient at treating B-cell malignancies, Bruton tyrosine kinase (BTK) inhibitors are consistently reported to increase the risk of bleeding. Analyzing platelet aggregation response to collagen in platelet-rich plasma allowed us to identify two groups in the healthy population characterized by low or high sensitivity to ibrutinib in vitro Inhibition of drug efflux pumps induced a shift from ibrutinib low-sensitive platelets to high-sensitive ones. At a clinically relevant dose, acalabrutinib, a second-generation BTK inhibitor, did not affect maximal collagen-induced platelet aggregation in the ibrutinib low-sensitive group but did inhibit aggregation in a small fraction of the ibrutinib high-sensitive group. Consistently, acalabrutinib delayed aggregation, particularly in the ibrutinib high-sensitive group. In chronic lymphocytic leukemia patients, acalabrutinib inhibited maximal platelet aggregation only in the ibrutinib high-sensitive group. Acalabrutinib inhibited collagen-induced tyrosine-753 phosphorylation of phospholipase Cγ2 in both groups, but, in contrast to ibrutinib, did not affect Src-family kinases. Acalabrutinib affected thrombus growth under flow only in the ibrutinib high-sensitive group and potentiated the effect of cyclooxygenase and P2Y₁₂ receptor blockers in both groups. Since the better profile of acalabrutinib was observed mainly in the ibrutinib low-sensitive group, replacement therapy in patients may not systematically reduce the risk of bleeding.

Figure 1.

Effect of ibrutinib and acalabrutinib on collagen-induced platelet aggregation in vitro, in healthy donors. Platelet-rich plasma (PRP) from healthy volunteers was treated or not with ibrutinib (A-D) or acalabrutinib (ACP) (C-E) at the indicated concentrations for 1 h at 37°C and stimulated with collagen 3.3 μg/mL. Platelet aggregation was assessed by turbidimetry during 10 min and results, expressed as percentage of maximal platelet aggregation and area under the curve, are mean ± standard error of mean. A maximal platelet aggregation response below 50% indicated ibrutinib high-sensitive donors (HS, in red, n=39) while a maximal aggregation response above 50% indicated ibrutinib low-sensitive donors (LS, in blue, n=31). The same analysis was performed 6 months later (After 6 months) in 29 out of the 70 healthy donors (A). Platelet aggregation curves showing representative platelet responses to ACP and ibrutinib on PRP from ibrutinib HS healthy donors are shown (D). The numbers of donors analyzed in each experiment were: (A) n=70, after 6 months: n=29; (B) LS: n=15, HS: n=7; (C) n=52; (E) LS: n=10, HS: n= 12. *P<0.05, **P<0.01, ***P<0.001, ^#P<0.05, ^##P<0.01, ^###P<0.001 according to one-way analysis of variance.

Figure 2.

Effect of ibrutinib and acalabrutinib on collagen-induced platelet aggregation in vitro in patients with chronic lymphocytic leukemia. Platelet-rich plasma from 16 Bruton kinase inhibitor-naïve patients with chronic lymphocytic leukemia was treated or not with ibrutinib or acalabrutinib (ACP) for 1 h at 37°C and stimulated with collagen 3.3 μg/mL. Platelet aggregation was assessed by turbidimetry during 10 min. (A) Four patients were identified as having low sensitivity (LS) to ibrutinib (reduction of maximal platelet aggregation <50%, blue) and 12 as having high sensitivity (HS) to ibrutinib (reduction of maximal platelet aggregation >50%, red). (B) Results, expressed as percentage of maximal aggregation in the two groups, are mean ± standard error of mean. **P<0.01, ***P<0.001 according to one-way analysis of variance.

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 μg/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 μg/mL was assessed by western blotting. The results of the western 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.

Figure 4.

Effect of acalabrutinib on platelet thrombus formation ex vivo on collagen under arterial flow. (A, B) DIOC₆-labeled platelets from healthy donors with (A) low sensitivity (LS) or (B) high sensitivity (HS) to ibrutinib (determined on the basis of their aggregation response as in Figure 1) pre-incubated with acalabrutinib (ACP) 2 μM, ibrutinib 0.5 μM or dimethylsulfoxide (control) for 1 h at 37°C were perfused through a collagen-coated microcapillary at a physiological arterial shear rate of 1500 s⁻¹ for 180 or 360 s. Surface coverage (%) and thrombi volume (% of vehicle response) were analyzed using ImageJ software. Results are presented as mean ± standard error of mean of three or four independent experiments. *P<0.05, **P<0.01, ***P<0.001 according to two-way analysis of variance (ANOVA) for surface coverage, one-way ANOVA for thrombi volume at 360 s and a one sample t-test for thrombi volume at 180 s.

Figure 5.

Effect of ibrutinib and acalabrutinib in association with anti-platelet drugs. (A, B) Platelet-rich plasma from healthy donors with (A) high sensitivity (HS) or (B) low sensitivity (LS) to ibrutinib was pre-treated or not with the indicated doses of acalabrutinib (ACP) or ibrutinib for 1 h at 37°C and anti-platelets drugs (10 μM indomethacin and/or 10 μM ARC69931MX) were added 10 min before stimulation with collagen 3.3 μg/mL. Platelet aggregation was assessed by turbidimetry during 10 min and results, expressed as percentage of maximal aggregation, are the mean ± standard error of mean of three to five independent experiments. *P<0.05, **P<0.01, ***P<0.001 according to one-way and two-way analyses of variance.