Abstract Number: PB1018
Meeting: ISTH 2021 Congress
Background: Glycoprotein VI (GPVI) is the major signalling receptor for collagen on platelets and is a promising anti-thrombotic target. Dimerisation of this receptor is believed to have roles in both ligand binding and signalling, but the mechanisms of GPVI dimerisation remain poorly understood. We have previously raised a series of nanobodies against GPVI as novel probes to further study GPVI structure and function.
Aims: We aim to map the binding sites of the nanobodies on GPVI by crystallography and competition assays, and relate to function.
Methods: The ability of the nanobodies to inhibit GPVI in response to collagen was assessed using NFAT activation reporter assays, thrombus formation of whole blood under flow, and binding of recombinant GPVI to a collagen-coated surface. The most potent nanobody was co-crystallised with recombinant GPVI. NFAT reporter assays on a truncated GPVI mutant were performed to validate the novel GPVI dimer conformation.
Results: We show that three of the nanobodies inhibited collagen-induced GPVI signalling by >90 % and significantly reduced thrombus formation in whole blood in response to collagen. This inhibition was due to direct displacement of collagen binding. Solving the crystal structure of the most potent nanobody with GPVI revealed a binding epitope close to the collagen related peptide (CRP) binding site. In addition, GPVI adopted a novel domain-swapped dimer conformation, and through site directed mutagenesis, we demonstrate that the critical domain-swapped hinge region is required for GPVI signalling.
Conclusions: The solved nanobody bound crystal-structure reveals a domain-swapped GPVI dimer, which may represent a biologically active conformation. The inhibitory nanobodies provide new therapeutic frameworks for treating thrombosis.
To cite this abstract in AMA style:Slater A, Di Y, Clark J, Jooss N, Martin E, Alenazy F, Thomas M, Ariëns R, Herr A, Poulter N, Emsley J, Watson S. Structural Characterisation of GPVI in Complex with Nanobody 2 Generates a Domain-swapped GPVI Dimer: Could this Represent a Biologically Active Conformation? [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/structural-characterisation-of-gpvi-in-complex-with-nanobody-2-generates-a-domain-swapped-gpvi-dimer-could-this-represent-a-biologically-active-conformation/. Accessed December 7, 2021.
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