TY - JOUR
T1 - Functional characterization of a nanobody-based glycoprotein VI-specific platelet agonist
AU - Zivkovic, Minka
AU - the Thrombocytopathy in the Netherlands (TiN) Study Group
AU - the SYMPHONY consortium
AU - Pols - van Veen, Elisabeth
AU - van der Vegte, Vossa
AU - Sebastian, Silvie A.E.
AU - de Moor, Annick S.
AU - Korporaal, Suzanne J.A.
AU - Schutgens, Roger
AU - Urbanus, Rolf T.
AU - Jansen, Gerard
AU - Al Arashi, Wala
AU - Arisz, Ryanne
AU - Bierings, Ruben
AU - Boender, Johan
AU - Brands, Martijn
AU - Bredenoord, Annelien
AU - Bukkems, Laura
AU - Burdorf, Lex
AU - Cnossen, Marjon
AU - Goedhart, Tine
AU - Hazelzet, Jan
AU - Huisman, Elise
AU - Jansen, Nathalie
AU - Kruip, Marieke
AU - Laan, Sebastiaan
AU - Leebeek, Frank
AU - van Leeuwen, Nikki
AU - Lingsma, Hester
AU - de Maat, Moniek
AU - Meijer, Karina
AU - van Moort, Iris
AU - Mussert, Caroline
AU - Polinder, Suzanne
AU - Reitsma, Simone
AU - Romano, Lorenzo
AU - Schols, Saskia
AU - Uyl, Carin
N1 - Publisher Copyright: © 2024 The Author(s)
PY - 2024/10
Y1 - 2024/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85207106683&partnerID=8YFLogxK
U2 - 10.1016/j.rpth.2024.102582
DO - 10.1016/j.rpth.2024.102582
M3 - Article
C2 - 39512585
AN - SCOPUS:85207106683
SN - 2475-0379
VL - 8
JO - Research and Practice in Thrombosis and Haemostasis
JF - Research and Practice in Thrombosis and Haemostasis
IS - 7
M1 - 102582
ER -