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Ibrutinib and acalabrutinib: effects on platelet functions
Like ibrutinib, the second-generation Btk inhibitor, acal- abrutinib, inhibits Btk by covalent modification of the cys- teine residue C481 in the ATP binding domain. While ibrutinib is known to irreversibly inhibit both Btk and Tec, acalabrutinib exhibits a higher specificity towards Btk and less activity on Tec,9,19,20 although a recent study suggests that its selectivity for Btk over Tec is not substantial.21 At the highest clinically achievable doses, ibrutinib has also been shown to inhibit Src-kinases in washed human platelets.10-13 Src-kinases are essential for platelet activation and act upstream of Btk and Tec in the GPVI signaling pathway.22 Their inhibition has been shown to induce bleeding in vivo.23 Of note, acalabrutinib seems to have less inhibitory potential than ibrutinib on Src-kinases.19,24 A clinical trial that enrolled 61 relapsed CLL patients demon- strated the efficacy of acalabrutinib and did not document any major bleeding although a significant incidence of low-grade bleeding including petechiae (16%) and contu- sions (18%) was observed.9 Using an elegant experimental mouse model of arterial thrombosis and platelets from acalabrutinib-treated patients, it was also shown in this study that this second-generation Btk inhibitor has fewer anti-platelet effects than ibrutinib. Another recent study in a small cohort of patients also documented significant low-grade bleeding in acalabrutinib-treated patients and less platelet dysfunction in these patients than in ibrutinib- treated patients.24 Of note, a very recent report suggests that ibrutinib and the second generation Btk inhibitors, acalabrutinib and tirabrutinib (ONO/GS-4059), have the capacity to prevent platelet thrombus formation on human atherosclerotic plaque homogenate.25
There are only limited available data on the effects of sec- ond-generation Btk inhibitors to guide physicians who switch from ibrutinib to another kinase inhibitor (e.g. in the case of bleeding, or co-medication with antithrombotic drugs). Therefore, we evaluated in this study whether acal- abrutinib could represent a safer option. We first identified two groups of healthy donors based on their sensitivity to collagen-induced platelet aggregation inhibition by ibruti- nib in vitro. We then characterized the differences and simi- larities in the effects of acalabrutinib and ibrutinib in the two groups and analyzed the impact of an association of acalabrutinib with antiplatelet drugs. Our data suggest that switching Btk therapy may not be a systematically good option for patients who bleed under ibrutinib treatment, and that the association of any of these Btk inhibitors with antiplatelet drugs significantly potentiates the inhibition of collagen-induced platelet aggregation.
Methods
Reagents
The sources of the reagents used in this study are provid- ed in the Online Supplement.
Preparation of human platelets
Human platelets from adult healthy volunteers who had not taken aspirin or any anti-platelet or anti-inflammatory drugs in the preceding 10 days or CLL patients were isolated from blood col- lected under citrate. All experiments were performed within 1 h after blood sample collection for healthy donors and within 2 h after blood sample collection for CLL patients. Platelet-rich plasma (PRP) and washed platelets were prepared as indicated in the Online Supplement and elsewhere.23
Light transmission aggregometry
Platelet aggregation was monitored in siliconized glass cuvettes under continuous stirring (1000 rpm) at 37°C using a turbidimetric method in a multi-channel aggregometer (SD Medical, France). Platelet aggregation was monitored for 10 min and the extent of platelet aggregation and area under curve (AUC) were analyzed using Thrombosoft 1.6 software (SD Medical, France).
Ex vivo model of thrombosis under flow conditions
Glass microcapillaries (Cellix System, New York, NY, USA) were coated with 50 mg/mL type I collagen from equine tendon overnight at 4°C and saturated with a solution of 1% bovine serum albumin (fatty acid-free) in phosphate-buffered saline for 30 min. Heparin-anticoagulated whole blood from healthy human donors was pre-treated with ibrutinib, acalabrutinib or vehicle (dimethylsulfoxide) for 60 min at 37°C and platelets were labeled with DiOC6 (2 mM, 10 min at 37°C). Blood was then perfused through the microfluidic system for the indicated times at an arte- rial shear rate of 1500 s-1 as previously described.26 Platelet adhesion and thrombus formation were measured in real time with an epi- fluorescence microscope (Axiovert 200; Zeiss) with a 40X oil immersion objective (Plan-Apo 40x/1.3 Oil DIC UVVIS-IR) and a Colibri LED System light source (Zeiss, Jena, Germany). The results were recorded in real time (acquisition rate: 1 frame every 30 s) using a high resolution CCD cooled camera (Orca-R2, Hamamatsu, Hamamatsu City, Japan). Image sequences of the time-lapse recording and surface coverage were analyzed using
Image J software.
Statisticalanalysis
Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) with the Bonferroni post-test (GraphPad PRISM software, San Diego, CA, USA). P-values <0.05 were con- sidered statistically significant. Two-way ANOVA with the Bonferroni post-test was used for statistical analysis of the surface coverage in the thrombus formation assay and a one sample t-test was applied to analyze thrombus volume at 180 s.
Ethical approval
Ethical approval for collecting blood from patients and healthy volunteers was granted by the Hémopathies Inserm Midi-Pyrénées (HIMIP) collection declared to the French Ministry of Higher Education and Research (DC 2008-307 collection 1) and a transfer agreement (AC 2008-129) was obtained after approval from the ethical committee Comité de Protection des Personnes Sud-Ouest et Outremer II and the Toulouse Hospital Bio-Resources biobank, declared to the Ministry of Higher Education and Research (DC 2016-2804). In accordance with French law, clinical and biological annotations of patients’ samples were declared to the Comité National Informatique et Libertés (CNIL), the French data protection authority.
Results
Effect of ibrutinib and acalabrutinib on collagen-induced platelet aggregation in healthy donors
It is important to note that only a subset of ibrutinib- treated patients develop spontaneous bleeding and have a defect in collagen-induced platelet aggregation.3,10,11 Moreover, we consistently observed great heterogeneity in the intensity of the in vitro effect of ibrutinib on collagen- induced platelet aggregation in PRP from healthy donors (unpublished observation). We therefore first tested the effect
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