Binding of ERp5 and ERp5-AGHA to αIIbβ3 and directly to the β3 subunit. (A) Binding of αIIbβ3 to immobilized ERp5 or ERp5-AGHA was measured in the presence or absence of MnCl₂ (Mn⁺⁺; 2 mM), in the presence of EDTA (5 mM), or in the absence of added αIIbβ3 (background), as indicated. Bound αIIbβ3 was detected with an anti-CD41 conformation-independent antibody. ERp5 (black) and ERp5-AGHA (gray) binding to αIIbβ3 were compared by nonparametric Student t tests: no Mn⁺⁺ vs Mn⁺⁺ and no Mn⁺⁺ vs background,***P < .0001. There is no significant difference between no Mn⁺⁺ vs EDTA. Results are the average of 3 experiments. (B) Binding of ERp5 (100 nM) or ERp5-AGHA (100 nM) to αIIbβ3 (black) or glycoprotein (GP)Ibα (gray), both coated at 20 nM on 96-well plates in the absence of Mn⁺⁺ or in the absence of bound ERp5 or ERp5-AGHA (background; white). Bound ERp5 or ERp5-AGHA was detected with anti-ERp5 antibody. ERp5 binding to αIIbβ3 vs GPIba, ERp5-AGHA binding to αIIbβ3 vs GPIbα, ERp5 binding to αIIbβ3 vs background, and ERp5-AGHA binding to αIIbβ3 vs background, **P < .005. Binding of ERp5 or ERp5-AGHA to GPIbα vs background was not statistically significant. Results are the average of 3 experiments. Surface plasmon resonance was used to determine the binding constant for ERp5 with recombinant β3 integrin tagged with calmodulin in the presence (C) or absence of Mn⁺⁺ (D). ERp5 was coated on the Biacore chip. Lines represent the fitted curves of ERp5 at concentrations of 0, 0.33, 0.66, 1.31, 2.63, 5.25, 10.25, 21, and 42 µM. The K_D values for ERp5 interaction with the β3 subunit in the presence and absence of Mn⁺⁺ were 21.7 μM and 19.1 μM, respectively. ERp5 did not bind to calmodulin alone. n.s., not significant; RU, relative units.