Platelet signaling is triggered by MSD unfolding. (A) Representative histograms illustrating 5G6 binding in PRP treated with control IgG, 6B4, or AK2 under 0 dyn/cm2 (top) and 30 dyn/cm2 (bottom) of uniform shear. EDTA was added to prevent GPIbα shedding via metalloproteinases. (B) Graphs of fold increase (sheared/static) in 5G6 and WM23 binding for PRP treated with IgG, AK2, or 6B4. **P ≤ .01. (C) Plots of lifetimes of noted bonds as a function of force. Lifetimes were obtained by transforming the histograms of unbinding or unfolding forces using the method developed by Dudko et al.53 (D) Overlaid force-distance traces for pulling AK2, 11A8, or 6B4 on GPIb-IX. (E) Force distributions of MSD unfolding or unbinding between LBD mAbs and GPIb-IX. Loading rate was ∼10 pN/s. Error bars are Poisson noise. Asterisks indicate an underestimation of unbinding force resulting from strong bonds that were above the detection limit of our optical tweezers. FITC, fluorescein isothiocyanate.
