OC 23.4 - Functional characterization of a nanobody based GPVI specific platelet agonist

Background: Glycoprotein VI (GPVI) is a platelet specific collagen receptor required for platelet activation on extravascular collagen during hemostasis. Platelet reactivity towards collagen is always assessed during diagnostic workup of platelet disorders. GPVI can be activated by inducing receptor clustering with its ligands collagen, synthetic cross-linked collagen-related peptide (CRP-XL), or the snake venom tetramer protein convulxin. However, these agonists are hard to standardize and difficult to produce. Nanobodies are small recombinant proteins consisting of the complementarity determining region of camelid heavy-chain antibodies. They are highly stable, easy to produce and ideal candidates for developing a stable GPVI agonist for diagnostic assays.

Aims: To develop a stable nanobody-based GPVI agonist.

Methods: Lama glama were immunized with recombinant extracellular domains of GPVI and a phage display library was generated. Nanobodies were selected, produced as monomers or dimers in Escheria coli 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 (PRP) and whole blood FACS analysis.

Results: A nanobody with high affinity for recombinant GPVI was selected, optimized for expression and produced. FACS analysis confirmed the nanobody reacted with human GPVI on platelets. The nanobody fully inhibited CRP-XL induced P-selectin expression and fibrinogen binding in whole blood and attenuated collagen induced platelet aggregation in PRP. Neither monomers or dimers resulted in platelet activation in LTA or flow cytometry settings at concentrations ≥ 1 μM. However, tetramers of the nanobody caused full platelet aggregation in LTA, as well as P-selectin expression and fibrinogen binding in whole blood FACS analysis, with an EC50 of 2.3 ± 0.1 nM.

Conclusion(s): Nanobody tetramers to GPVI act as strong platelet agonists and may serve as stable GPVI agonists in diagnostic assays.

This research was funded by NWO-NWA grant number 1160.18.038.