Xu, R.-G. orcid.org/0000-0003-0774-112X, Tiede, C. orcid.org/0000-0003-0280-4005, Calabrese, A.N. orcid.org/0000-0003-2437-7761 et al. (13 more authors) (2024) Affimer reagents as tool molecules to modulate platelet GPVI-ligand interactions and specifically bind GPVI dimer. Blood Advances, 8 (15). pp. 3917-3928. ISSN 2473-9529
Abstract
Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5′-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM) > Discovery & Translational Science Dept (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Synthetic Biology (Leed) The University of Leeds > Faculty of Biological Sciences (Leeds) > Faculty of Biological Sciences Office (Leeds) |
Funding Information: | Funder Grant number BBSRC (Biotechnology & Biological Sciences Research Council) BB/W000237/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 13 Aug 2024 12:51 |
Last Modified: | 11 Sep 2024 14:58 |
Status: | Published |
Publisher: | American Society of Hematology |
Identification Number: | 10.1182/bloodadvances.2024012689 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:215990 |