Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2 and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand

Abstract

Background: Clustering of the glycoprotein receptors GPVI, CLEC-2, FcγRIIA and PEAR1 leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signalling cascades. GPVI, CLEC-2 and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase (PI3K). Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. Objectives: We have raised nanobodies against each of these receptors and multimerised them to identify the minimum number of epitopes to achieve robust activation of human platelets. Methods: Divalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilise a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerise the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, ATP secretion and protein phosphorylation were analysed using standardised methods. Results: Multimerisation of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2 and PEAR1 stimulated powerful and robust platelet aggregation, secretion and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared to the other nanobody-based ligands, despite a sub-nanomolar binding affinity. Conclusions: The multivalent nanobodies represent a series of standardised, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.