MS supervised experiments and contributed to discussions. platelet effects of two novel BTKi that exhibit a high (remibrutinib) or low (rilzabrutinib) selectivity for Btk over Tec. Methods and Results: Remibrutinib and rilzabrutinib were pre-incubated with anticoagulated blood. Platelet aggregation and bleeding time (closure time) were analyzed by Gastrofensin AN 5 free base multiple electrode aggregometry (MEA) and platelet-function analyzer-200 (PFA-200), respectively. Both BTKi inhibited atherosclerotic plaque-stimulated GPVI-mediated platelet aggregation, remibrutinib being more potent (IC50 = 0.03 M) than rilzabrutinib (IC50 = 0.16 M). Rabbit Polyclonal to Keratin 20 Concentrations of remibrutinib (0.1 M) and rilzabrutinib (0.5 M), 80% inhibitory for plaque-induced aggregation, also significantly suppressed ( 90%) Gastrofensin AN 5 free base the Btk-dependent pathways of platelet aggregation upon GPVI, von Willebrand factor/GPIb and FcRIIA activation stimulated by low collagen concentrations, ristocetin and antibody cross-linking, respectively. Both BTKi did not inhibit aggregation stimulated by ADP, TRAP-6 or arachidonic acid. Remibrutinib (0.1 M) only slightly continuous closure time and significantly less than rilzabrutinib (0.5 M). Conclusion: Remibrutinib and rilzabrutinib inhibit Btk-dependent pathways of platelet aggregation upon GPVI, VWF/GPIb, and FcRIIA activation. Remibrutinib being more potent and showing a better profile of inhibition of Btk-dependent platelet activation vs. hemostatic impairment than rilzabrutinib may be considered for further development as an antiplatelet drug. (5), and at higher concentrations prolong bleeding time (19), it is assumed that therapeutic concentrations of these BTKi inhibit in platelets irreversibly Tec in addition to Btk thereby abrogating GPVI signaling. This might contribute to the observed bleeding side effects. Therefore, we hypothesized that off target effects of BTKi with low Btk selectivity over Tec might explain bleeding of BTKi, and investigated in the present study the effects of two novel BTKi on Btk-mediated pathways of platelet aggregation and bleeding time (IC50 values, 1.3 and 0.8 nM, respectively) (22). It forms a reversible covalent bond with Cys481 of Btk, and shows a fast association and a very slow dissociation rate (23). Rilzabrutinib is in clinical trials of pemphigus (24) and idiopathic thrombocytopenic purpura (ITP), a disease exhibiting very low platelet counts ( 50.000/l) and bleeding events. Here, it inhibits platelet destruction mainly via the inhibition of autoantibody/FcR signaling in splenic macrophages (25). Unexpectedly, Gastrofensin AN 5 free base in a previous report clinically relevant concentrations of rilzabrutinib showed no inhibition of platelet activation (26). Materials and Methods Reagents Remibrutinib (LOU064), rilzabrutinib (PRN1008) and fenebrutinib (GDC-0853) were purchased from MedChem Express (New Jersey, USA). Dimethyl sulfoxide (DMSO) was from Sigma-Aldrich (Taufkirchen, Germany). Collagen was from Takeda (Linz, Austria). ADP, ristocetin, arachidonic acid (AA) and TRAP-6 (Thrombin Receptor Activator Peptide 6) were obtained from Roche Diagnostics (Mannheim, Germany). The anti-CD32 antibody AT10 (monoclonal mouse IgG1), cross-adsorbed F(ab’)2-goat anti-mouse IgG (H + L) and the anti-CD9 antibody Ts9 (monoclonal mouse IgG1) were from ThermoFisher Scientific (Waltham, MA, USA). Declaration of Helsinki Informed consent was obtained from healthy volunteers, as approved by the Ethics Committee of the Faculty of Medicine of the University or college of Munich, and in accordance with the ethical principles for medical research involving human subjects, as set out in the Declaration of Helsinki. Human Atherosclerotic Plaque Homogenates Atherosclerotic tissue specimens were obtained from patients who underwent endarterectomy for high-grade carotid artery stenosis. Specimen made up of lipid-rich soft plaques were collected. The atheromatous plaques were cautiously dissected under sterile conditions from other regions of the atherosclerotic tissue. The plaques were weighed, homogenized with a glass pestle and potter, then stored at ?80C (27, 28). Plaque homogenates from 5 patients were pooled. Blood Collection Whole blood from healthy donors who had not taken any antiplatelet drug within 2 weeks was collected by cubital venipuncture into blood tubes (double wall) from Verum Diagnostica GmbH (Munich, Germany) made up of hirudin as anticoagulant (final hirudin concentration in blood: 200 U/ml corresponding to 15 g/ml) for platelet aggregation measurements (29).