title: Bruton's Tyrosine Kinase mediates platelet receptor-induced generation of microparticles: a potential mechanism for amplification of inflammatory responses in rheumatoid arthritis synovial joints.
abstract: Platelet microparticles (pMPs) are small membrane-coated vesicles that are released from the plasma membrane upon platelet activation. In the joint fluid of patients with rheumatoid arthritis, pMP can interact with and activate fibroblast-like synoviocytes (FLS), which are important effector cells that mediate both immune activation and joint destruction. The signaling process by which engagement of glycoprotein VI (GPVI), a surface glycoprotein receptor for collagen which is expressed on platelets, triggers pMP generation is poorly understood, but has been suggested to involve Spleen Tyrosine Kinase (SYK), best known as an upstream activator of Bruton's Tyrosine Kinase (BTK) in B cells. In this study, we showed that activation of human platelets triggered by convulxin or collagen, specific ligands for GPVI receptor, or alternatively by antibody-mediated cross-linking of another platelet receptor, C type lectin-like receptor 2 (CLEC2), resulted in phosphorylation of BTK and downstream effector, phospholipase Cγ2 (PLCγ2). A potent and selective BTK inhibitor, RN486, inhibited GPVI- or CLEC2-mediated PLCγ2 phosphorylation and pMP production in a dose-dependent manner. BTK is also an essential effector of B cell receptor (BCR)-induced B cell signaling. Consistent with the biology, the IC50s of BTK inhibitors with varying potencies in a BCR-dependent B cell activation marker assay correlated with those in the GPVI-mediated PLCγ2 phosphorylation. In a co-culture system consisting of human primary synovial FLS and activated human platelets, convulxin stimulation resulted in elevated production of pro-inflammatory cytokines, IL-6 and IL-8, an effect which was dose-dependently blocked by RN486. The effects are specific as RN486 abrogated platelet aggregation induced by GPVI ligands but not by other platelet surface receptor agonists. Taken together, our data further support the potential therapeutic utility of BTK inhibitors in RA therapy, by inhibiting GPVI-mediated platelet activation and thus subsequent amplification of inflammation driven by pMP-induced FLS cytokines production.
authors.affiliation: Hoffmann-La Roche Inc., pRED, Pharma Research & Early Development, Inflammation Discovery and Therapeutic Area, Nutley, NJ 07110, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hsu
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Gu
authors.forename: Seng-Lai
authors.initials: SL
authors.lastname: Tan
authors.forename: Satwant
authors.initials: S
authors.lastname: Narula
authors.forename: Julie A
authors.initials: JA
authors.lastname: DeMartino
authors.forename: Cheng
authors.initials: C
authors.lastname: Liao
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D001172:Arthritis, Rheumatoid / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology; D001792:Blood Platelets / Q000378:metabolism*; D002384:Catalysis; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D018920:Coculture Techniques; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D037181:Lectins, C-Type / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D013585:Synovitis / Q000378:metabolism*
chemical_list: D015850:Interleukin-6; D016209:Interleukin-8; D037181:Lectins, C-Type; D010980:Platelet Membrane Glycoproteins; D047428:Protein Kinase Inhibitors; D011947:Receptors, Antigen, B-Cell; C098341:platelet membrane glycoprotein VI; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D051966:Phospholipase C gamma