The domain swap is required for signaling but not ligand binding. (Ai-ii) Crystal structures of the nanobody bound and original unbound GPVI structures, respectively, highlighting the region of the C-C′ hinge loop (orange) deleted for mutation studies. The domain-swapped structure contains 2 GPVI subunits colored in green and cyan, whereas only 1 subunit is shown for the original GPVI structure. The domain-swapped D2 domain is labeled D2a and D2b for the N- and C-terminal D2 regions, respectively. (Bi) Solid-phase binding assay comparing the dose-dependent binding of GPVI-Fc and GPVI-Fc ΔC-C′ to a collagen and CRP-coated surface. Respective EC₅₀ values of 42 and 294 nM were calculated for GPVI and GPVI ΔC-C′ binding to collagen and 2 and 9 nM for CRP, respectively. (Bii) Binding of GPVI-Fc and GPVI-Fc ΔC-C′ to a collagen and CRP-coated surface and subsequent displacement by increased concentrations of Nb2. GPVI (500 nM) was used for collagen, and 100 nM GPVI was used for CRP. Data are normalized against the highest and lowest concentrations of Nb2 and are expressed as percent binding. Respective IC₅₀ values of 268 and 51 nM were calculated for GPVI and GPVI ΔC-C′ binding to collagen and 132 and 17 nM for CRP. Data represent mean values of 3 experiments ± SD. (C) NFAT reporter assay of DT40 cells transfected with the FcR-γ chain and either full-length GPVI wild type or hinge mutant. Luciferase activity was reported for nonstimulated and CRP- and collagen-stimulated cells. Data represent mean values of 5 experiments performed in triplicate ± SD, and values are normalized against the respective basal levels. A Student 2-tailed t test was used to determine significance between basal and stimulated values.