Platelets of Patients Who Lack Bruton Tyrosine Kinase (BTK) Do Not Aggregate in Response to Fcγriia Activation: Implications for Developing Safe Treatments for Patients with Thrombotic Disorders Related to Fcγriia Activation

Background: Bruton's Tyrosine Kinase inhibitors (BTKi) have transformed the treatment of chronic lymphocytic leukemia (CLL). Paradoxically, while increased bleeding has been reported with BTKi therapy, patients who lack or have a defective BTK (X-linked agammaglobulinemia; XLA) have not been reported to have a bleeding diathesis. Therefore, the increased risk of bleeding with BTKi may reflect effects beyond inhibition of BTK itself. To test this, we examined platelet function in patients with XLA, in the absence and presence of BTKi of varying specificity.

Methods: Blood from healthy volunteers (n=5; 3 males, ages 33 to 61) and patients with XLA (n=2 males, ages 37 and 57) was obtained with informed consent under an IRB-approved protocol. Washed platelets (WP) and platelet-rich plasma (PRP) were prepared and treated with vehicle control (DMSO 0.02%), ibrutinib, or fenebrutinib (DMSO 0.02%) for 20 min at room temperature. Light transmission aggregometry was performed with: 1. collagen-related peptide (CRP; 5-10 µg/ml) to assess platelet activation via the GPVI pathway, 2. the anti-human FcγRIIa receptor (CD32) monoclonal antibody IV.3 (3 µg/ml) crosslinked with goat anti-mouse antibody (12 µg/ml) to assess activation via FcγRIIa, and 3. the platelet PAR-1 thrombin receptor activating peptide SFLLRN (T6). Aggregation of WP platelets was stopped after five min with a perchloric acid lysis buffer and samples were analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation at Tyr223. The patients' bleeding histories were obtained with the International Society of Hemostasis and Thrombosis bleeding assessment tool (ISTH-BAT) and their bleeding scores calculated.

Results: In both WP and PRP, T6 at 25 μM produced rapid and complete aggregation of platelets from the healthy controls and both patients with XLA, and neither BTKi inhibited the aggregation. Platelet activation via FcγRIIa also produced rapid and complete aggregation in control WP and PRP, but the aggregation in both WP and PRP was completely inhibited by both BTKi at 2 µM. In sharp contrast, WP and PRP from patients with XLA failed to aggregate in response to FcγRIIa activation. WP from controls aggregated in response to 5 μg/ml of CRP, whereas the WP of one XLA patient required 10 μg/ml for full aggregation and the WP of the other had only a partial aggregation in response to 10 μg/ml. Both ibrutinib and fenebrutinib at 2 µM completely inhibited CRP-induced aggregation in the controls (using 5 µg/ml CRP) and in the one XLA patient who had a full aggregation response to higher concentration of CRP (using 10 µg/ml CRP). Immunoblot analysis of these samples confirmed the presence of BTK protein in the control platelets and its absolute absence in the platelets from the XLA patients. CRP activation of control platelets induced phosphorylation of BTK at Y223 and both BTKi greatly diminished the phosphorylation as judged by immunoblot.

The XLA patients had ISTH-BAT scores of 0 and 1, which are within the normal range (0-3), despite undergoing an arthroplasty (patient A) and tooth extractions (patient B).

Conclusions: We confirmed that patients with XLA do not have a clinical bleeding diathesis. Queck et al. previously showed that platelets from XLA patients have reduced, but not absent aggregation in response to collagen and CRP (Current Biology 1998, 8:1137) and our data are in accord with their findings. Since both BTKi drugs dramatically inhibited the aggregation response to CRP of platelets from one of our patients with XLA, we conclude that the major effect of BTKi on collagen and CRP-induced aggregation is due to off-target effects. In sharp contrast, since both patients with XLA have a major defect in FcγRIIa-mediated platelet aggregation, it appears that BTK is absolutely required for activation through this pathway. This is consistent with the inhibition of FcγRIIa-mediated platelet aggregation observed with patients treated with BTKi (Goldmann et al. Blood Adv 2019; 3:4021). Collectively, these data suggest that it may be possible to target just BTK itself for pathological conditions dependent on FcγRIIa-mediated activation, including heparin-induced thrombocytopenia and thrombosis and related thrombotic disorders, without compromising hemostasis.

Coller:Chugai Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CeleCor Therapeutics: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Scholar Rock: Current equity holder in publicly-traded company; Accumetrics/Instrumentation Laboratories: Patents & Royalties.