Nanobodies to GPVI as alternative reagents for platelet spreading

Platelet spreading assays are widely used to assess platelet adhesion, cytoskeletal remodeling and receptor‑specific signaling, but current GPVI ligands such as Collagen Related Peptide (CRP‑XL) suffer from non‑defined structure, batch variability, and high cost. Nanobodies targeting GPVI offer an attractive alternative due to their defined stoichiometry and recombinant production. Here, we evaluated whether GPVI‑binding nanobodies can support GPVI‑dependent platelet spreading. Using automated image analysis, we compared spreading on monovalent Nb2, trivalent Nb2‑3 and CRP‑XL. Monovalent Nb2 supported full spreading in only a subset of platelets, with reduced adhesion compared to CRP‑XL. In contrast, trivalent Nb2‑3 induced platelet adhesion and spreading indistinguishable from CRP‑XL, with comparable morphology, actin organization and spread area at concentrations ≥0.1 µg mL-1. Spreading on Nb2‑3 was inhibited by soluble Nb2 and by Src kinase blockade, confirming GPVI‑dependent signaling, while the non‑blocking nanobody Nb28 had no inhibitory effect. Notably, Nb28 alone supported spreading, demonstrating that ligand‑induced GPVI clustering, rather than binding site location, drives activation. These findings show that Nb2‑3 is a potent and reproducible alternative to CRP‑XL, offering a well‑defined and standardizable reagent for GPVI‑dependent static platelet spreading assays.