We demonstrate here that fenebrutinib also inhibited platelet aggregation upon low but not high collagen concentrations and inhibited GPIb/VWF-mediated platelet aggregation upon stimulation with ristocetin. after FcRIIA stimulation by antibody-mediated cross-linking (inducing platelet aggregation and secretion) or anti-CD9 antibody (inducing platelet aggregation only). The concentrations that inhibit 50% (IC50) of FcRIIA cross-linkingCinduced platelet aggregation were for the irreversible BTKi’s ibrutinib 0.08 M, zanubrutinib 0.11 M, acalabrutinib 0.38 M, tirabrutinib 0.42 M, evobrutinib 1.13 M, and for the reversible BTKi fenebrutinib 0.011 M. IC50 values for ibrutinib and acalabrutinib were four- to fivefold lower than the drug plasma concentrations in patients treated for B-cell malignancies. The BTKis also suppressed adenosine triphosphate secretion, P-selectin expression, and platelet-neutrophil complex formation after FcRIIA cross-linking. Moreover, platelet aggregation in donor blood stimulated by sera from HIT patients was blocked by BTKis. A single oral intake of ibrutinib (280 mg) was sufficient for a rapid and sustained suppression of platelet FcRIIA Acipimox activation. Platelet aggregation by adenosine 5-diphosphate, arachidonic acid, or thrombin receptor-activating peptide was not inhibited. Thus, irreversible and reversible Rabbit Polyclonal to BRF1 BTKis potently inhibit platelet activation by FcRIIA in blood. This new rationale deserves testing in patients with HIT. Visual Abstract Open in Acipimox a separate window Introduction The platelet Fc receptor CD32a (FcRIIA) plays a central role in the pathogenesis of heparin-induced thrombocytopenia (HIT).1-4 HIT is observed in 0.2% to 0.3% of patients receiving heparin4 and is caused by immunoglobulin G (IgG) antibodies against new epitopes uncovered after association of Acipimox polyanionic heparin with platelet-factor 4 (PF4) secreted from platelets.1 The immune complexes bind to FcRIIA around the platelet surface with their Fc domain and cross-link the receptors, which induces platelet aggregation and secretion.1-4 Formation of procoagulant vesicles by activated platelets and tissue factor expression by activated monocytes triggers thrombin formation and thrombosis, that together with enhanced platelet clearance by splenic macrophages results in thrombocytopenia.1,2,4 Platelets carry 1000 to 4000 copies of FcRIIA (CD32a) per cell, the dominant compartment of this receptor in the body.2 FcRIIA is a type I transmembrane protein consisting of 2 extracellular Ig-like domains (similar to glycoprotein VI [GPVI]), a single transmembrane domain name, and a cytoplasmic tail that contains an immunoreceptor tyrosine-based activation motif (ITAM) domain Acipimox name with dual YXXL amino acid consensus sequences. Signaling through the platelet FcRIIA is similar to other ITAM receptors such as GPVI in platelets and the B-cell receptor in lymphocytes.3,5 Cross-linking of the FcRIIA by immune complexes induces ITAM phosphorylation by Src family kinases, probably Fyn and/or Lyn. Phosphorylated ITAM provides a docking site for the tandem SH2 domains of tyrosine kinase Syk, which recruits and phosphorylates LAT.6,7 This adapter molecule is important for recruitment and activation of PLC2 and PI3K.5,7 The latter enzyme (by generating phosphatidylinositol(3,4,5)-triphosphate that binds the PH domains of the homologous tyrosine kinases Bruton tyrosine kinase [BTK] and Tec) recruits these kinases to the plasma membrane allowing their tyrosine autophosphorylation in the SH3 domain and tyrosine phosphorylation by Lyn in the catalytic domain.5,8 After GPVI-mediated platelet activation by collagen, BTK and Tec activation supports PLC2 activation.6 BTK alone mediates platelet activation only after low-degree GPVI activation,9 whereas Tec compensates for the absence of BTK in signaling downstream of GPVI.10 PLC2 activation then generates the second messengers inositol-1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG), which release Ca2+ from intracellular stores and activate protein kinase C (PKC), respectively, causing platelet aggregation and secretion.11 After FcRIIA cross-linking, increased BTK and Tec phosphorylation has been demonstrated in human platelets,12 but their respective causative functions for Fc receptorCstimulated platelet activation are unknown. The current treatment of HIT patients relies on parenteral application of rapid-acting, non-heparin anticoagulants, such as the direct thrombin inhibitor argatroban or the antithrombin-dependent factor Xa inhibitor danaparoid.1,4 In the future, immediate dental anticoagulants like the element Xa inhibitors apixaban and rivaroxaban may be authorized.13 Inhibiting platelet FcRIIA signaling would stop an early important part of HIT pathogenesis not targeted up to now. We therefore researched the effect of BTK inhibitors (BTKis) on FcRIIA-induced platelet activation and examined the irreversible BTKis ibrutinib and acalabrutinib (authorized for the long-term treatment of varied B-cell malignancies and mantle cell lymphoma, respectively),14,15 zanubrutinib (BGB-3111) and tirabrutinib (ONO/GS-4059) (both with excellent results in medical tests of B-cell malignancies),16,17 evobrutinib (with results in a lately finished trial in multiple sclerosis),18 as well as the reversible extremely specific and powerful BTKi fenebrutinib (GDC-0853), created to focus on B cells and macrophages in autoimmune disorders (arthritis rheumatoid, lupus).19-21 We activated platelet FcRIIA in blood by antibody cross-linking, with anti-CD9 antibody, and with Strike sera, and measured BTKi effects on platelet activation and the forming of platelet-neutrophil complexes. Strategies and Components For information regarding components and strategies see supplemental Data. 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