Figure 4.

Ibrutinib and acalabrutinib inhibit healthy control platelet activation by bacteria and IV.3 mAb mediated FcγRIIA crosslinking in the presence of plasma. (A) Characterization of the effect of ibrutinib on platelet aggregation. PRP from healthy controls was incubated in vitro with different doses of ibrutinib for 5 minutes before stimulation with the following agonists: crosslinked IV.3 mAb (IV.3-xl; 4 μg/ml IV.3 mAb followed by 30 μg/ml F(ab’)2 rabbit anti-mouse IgG; n=5), S. aureus Newman (n=7), E. coli RS218 (n=7), 3 μg/ml CRP-xl (n=5) and 3 μM TRAP-6 (n=5). Aggregation was measured by light transmission aggregometry. Reactions were run for 20 minutes from onset of aggregation for IV.3-xl and bacteria, and 10 minutes for the rest of agonists, and maximum aggregation was calculated. (B) Characterization of the effect of acalabrutinib on platelet aggregation. PRP from healthy controls was incubated in vitro with different doses of acalabrutinib for 5 min before stimulating with agonists: IV.3-xl (n=5), S. aureus Newman (n=6), E. coli RS218 (n=5), 3 μg/ml CRP-xl (n=5) and 3 μM TRAP-6 (n=5). Aggregation was measured by light transmission aggregometry as above. (C) PRP from healthy controls (n=5) was incubated with vehicle or inhibitors, 9 μM eptifibatide (αIIbβ3 inhibitor), 20 μg/ml IV.3 mAb (to inhibit FcγRIIA), 4 μM dasatinib (Src inhibitor), or iBtks, 5 μM ibrutinib and 15 μM acalabrutinib. Samples were then stimulated with stated agonists. Supernatants from aggregation reactions were collected at 20 minutes of onset of aggregation for IV.3-xl and bacteria, and 10 minutes for TRAP-6. Levels of PF4 as a marker of α-granule secretion were measured by PF4 ELISA. Data is shown as mean ± SD. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison correction (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).