Selective Btk inhibition by PRN1008/PRN473 blocks human CLEC-2, and PRN473 reduces venous thrombosis formation in mice

Christopher W Smith, Joana Campos, Helena C Brown, Natalie J Jooss, Vanesa-Sindi Ivanova, Maan Harbi, Lourdes Garcia-Quintanilla, Sian Jossi, Marisol Perez-Toledo, Kieran Rookes, Alexander Brill, Lindsay N Theodore, Tim Owens, Jacob LaStant, Matthew C Foulke, Shin Mukai, Michelle Francesco, Michael Storek, Alexandra Hicks, Claire LangrishPhilip A Nunn, Adam F Cunningham, Abhi Chauhan, Mark R Thomas, Steve P Watson, Phillip L R Nicolson*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Platelet C-type lectin-like receptor 2 (CLEC-2) is a hem-immunoreceptor tyrosine-based activation motif-containing receptor that has a critical role in venous thrombosis but minimal involvement in hemostasis. CLEC-2 can be blocked by Btk inhibitors. Treatment with ibrutinib is associated with increased bleeding due to off-target inhibition of Src family kinases (SFKs). Patients with X-linked agammaglobulinemia (XLA) who lack Btk, however, do not bleed, suggesting selective Btk inhibition as a viable antithrombotic strategy. We assessed the effects of selective Btk inhibitors PRN1008 (rilzabrutinib) and PRN473 on platelet signaling and function mediated by CLEC-2 and glycoprotein-VI. We used healthy donors and XLA platelets to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects of PRN473 in vivo. PRN1008 and PRN473 potently inhibited CLEC-2-mediated platelet activation to rhodocytin. No off-target inhibition of SFKs was seen. PRN1008 treatment of Btk-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, likely reflecting inhibition of Tec. No effect on G protein-coupled receptor-mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin-positive vessels after Salmonella infection and the presence of IVC thrombosis after vein stenosis. The potent inhibition of human platelet CLEC-2 and reduced thrombosis in in vivo models, together with the lack of off-target SFK inhibition and absence of bleeding reported in rilzabrutinib-treated patients with immune thrombocytopenia, suggest Btk inhibition as a promising antithrombotic strategy.
Original languageEnglish
Pages (from-to)5557-5570
Number of pages14
JournalBlood advances
Volume8
Issue number21
DOIs
Publication statusPublished - 12 Nov 2024

Keywords

  • Lectins, C-Type/metabolism
  • Animals
  • Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors metabolism
  • Humans
  • Mice
  • Venous Thrombosis/etiology drug therapy metabolism
  • Blood Platelets/metabolism drug effects
  • Agammaglobulinemia/drug therapy
  • Disease Models, Animal
  • Protein Kinase Inhibitors/pharmacology therapeutic use
  • Genetic Diseases, X-Linked/drug therapy
  • Platelet Membrane Glycoproteins/metabolism antagonists & inhibitors
  • Pyrimidines/pharmacology therapeutic use
  • Membrane Glycoproteins

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