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Poster Networking

Platelets and Megakaryocytes

PB0816 - Generation of new Affimers that modulate platelet GPVI-ligand interactions and selectively target GPVI dimer

Monday, June 24, 2024
13:45 – 14:45 ICT
Room: Exhibition Hall
Background: Glycoprotein VI (GPVI) is a collagen/fibrin(ogen) receptor on platelets. Recent studies using inhibitors highlighted important structure/function relationships. Affimers are small protein alternatives to antibodies, and can be used as tool to study protein structure/function.

Aims: Investigate whether Affimers modulate GPVI function, map their binding sites on GPVI and analyse Affimer-GPVI interactions at the molecular level.

Methods: GPVI-binding Affimers were screened by phage-display. Modulation of GPVI-ligand interaction was studied using ELISA. Effect of cross-linked collagen relate peptide (CRP-XL) on platelet function was tested by aggregation assays. Affimer binding to monomeric/dimeric GPVI was studied by ELISA, SPR, microscale thermophoresis (MST), flow cytometry and native mass spectrometry. Affimer binding sites on GPVI were mapped by hydrogen deuterium exchange mass spectrometry (HDX-MS). Affimer structures were modelled by AlphaFold and their interactions with GPVI were visualized using molecular docking.

Results: Among the identified Affimers, D18, M17 and D22 bound GPVI dimer with nM affinities. These Affimers inhibited GPVI-collagen/CRP-XL/fibrinogen interaction and CRP-XL induced platelet aggregation. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17 upon platelet activation by CRP-XL. Mapping of binding sites revealed that while D22 binds a site that overlaps with nanobody2 (Slater et al. Blood 2021) on the D1 domain, M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site. D18 targets a new region on D2 domain. We also found that D18 is a stable non-covalent dimer and forms a stable complex with dimeric GPVI via 2:2 stoichiometry.

Conclusion(s): Affimers modulate GPVI function and bind different sites on GPVI. D18 is GPVI dimer-specific and could be used as tool to detect GPVI dimerization in platelets. A dimeric epitope regulating GPVI-ligand interaction was identified, which could be used for developing novel anti-thrombotic inhibitors selectively targeting GPVI dimer in platelets.