Research and Practice in Thrombosis and Haemostasis (Oct 2024)

Functional characterization of a nanobody-based glycoprotein VI-specific platelet agonist

  • Minka Zivkovic,
  • Elisabeth Pols - van Veen,
  • Vossa van der Vegte,
  • Silvie A.E. Sebastian,
  • Annick S. de Moor,
  • Suzanne J.A. Korporaal,
  • Roger E.G. Schutgens,
  • Rolf T. Urbanus,
  • Erik Beckers,
  • Michiel Coppens,
  • Jeroen Eikenboom,
  • Louise Hooimeijer,
  • Gerard Jansen,
  • Roger Schutgens,
  • Rolf Urbanus,
  • Minka Zivkovic,
  • Emile van den Akker,
  • Wala Al Arashi,
  • Ryanne Arisz,
  • Lieke Baas,
  • Ruben Bierings,
  • Maartje van den Biggelaar,
  • Johan Boender,
  • Anske van der Bom,
  • Mettine Bos,
  • Martijn Brands,
  • Annelien Bredenoord,
  • Laura Bukkems,
  • Lex Burdorf,
  • Jessica Del Castillo Alferez,
  • Michael Cloesmeijer,
  • Marjon Cnossen,
  • Mariëtte Driessens,
  • Jeroen Eikenboom,
  • Karin Fijnvandraat,
  • Kathelijn Fischer,
  • Geertje Goedhart,
  • Tine Goedhart,
  • Samantha Gouw,
  • Rieke van der Graaf,
  • Masja de Haas,
  • Lotte Haverman,
  • Jan Hazelzet,
  • Shannon van Hoorn,
  • Elise Huisman,
  • Nathalie Jansen,
  • Alexander Janssen,
  • Sean de Jong,
  • Sjoerd Koopman,
  • Marieke Kruip,
  • Sebastiaan Laan,
  • Frank Leebeek,
  • Nikki van Leeuwen,
  • Hester Lingsma,
  • Moniek de Maat,
  • Ron Mathôt,
  • Felix van der Meer,
  • Karina Meijer,
  • Sander Meijer,
  • Stephan Meijer,
  • Iris van Moort,
  • Caroline Mussert,
  • Hans Kristian Ploos van Amstel,
  • Suzanne Polinder,
  • Diaz Prameyllawati,
  • Simone Reitsma,
  • Eliza Roest,
  • Lorenzo Romano,
  • Saskia Schols,
  • Roger Schutgens,
  • Rolf Urbanus,
  • Carin Uyl,
  • Jan Voorberg,
  • Huan Zhang,
  • Minka Zivkovic

Journal volume & issue
Vol. 8, no. 7
p. 102582

Abstract

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Background: Glycoprotein (GP)VI is a platelet-specific collagen receptor required for platelet activation during hemostasis. Platelet reactivity toward collagen is routinely assessed during diagnostic workup of platelet disorders. GPVI can be activated by inducing receptor clustering with suspensions of fibrillar collagen or synthetic cross-linked collagen-related peptide (CRP-XL). However, these suspensions are poorly standardized or difficult to produce. Nanobodies are small recombinant camelid-derived heavy-chain antibody variable regions. They are highly stable, specific, and ideal candidates for developing a stable GPVI agonist for diagnostic assays. Objectives: Develop a stable nanobody-based GPVI agonist. Methods: Nanobody D2 (NbD2) was produced as dimers and purified. Tetramers were generated via C-terminal fusion of dimers with click chemistry. Nanobody constructs were functionally characterized with light transmission aggregometry (LTA) in platelet-rich plasma and whole blood flow cytometry. Diagnostic performance was assessed in patients with inherited platelet function disorders with LTA and flow cytometry. Results: NbD2 was specific for human platelet GPVI. Dimers did not result in platelet activation in LTA or flow cytometry settings and fully inhibited CRP-XL-induced P-selectin expression and fibrinogen binding in whole blood and attenuated collagen-induced platelet aggregation in platelet-rich plasma. However, NbD2 tetramers caused full platelet aggregation, as well as P-selectin expression and fibrinogen binding. NbD2 tetramers were able to discriminate between inherited platelet function disorder patients and healthy controls based on fibrinogen binding, similar to CRP-XL. Conclusion: Nanobody tetramers to GPVI induce platelet activation and can be used to assess the GPVI pathway in diagnostic assays.

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