Abstract
In platelets, the glycoprotein (GP) Ib-IX receptor complex senses blood shear flow and transmits the mechanical signals into platelets. Recently, we have discovered a juxtamembrane mechanosensory domain (MSD) within the GPIbα subunit of GPIb-IX. Mechanical unfolding of the MSD activates GPIb-IX signaling into platelets, leading to their activation and clearance. Using optical tweezer-based single-molecule force measurement, we herein report a systematic biomechanical characterization of the MSD in its native, full-length receptor complex and a recombinant, unglycosylated MSD in isolation. The native MSD unfolds at a resting rate of 9 × 10(-3) s(-1). Upon exposure to pulling forces, MSD unfolding accelerates exponentially over a force scale of 2.0 pN. Importantly, the unfolded MSD can refold with or without applied forces. The unstressed refolding rate of MSD is ∼17 s(-1) and slows exponentially over a force scale of 3.7 pN. Our measurements confirm that the MSD is relatively unstable, with a folding free energy of 7.5 k(B)T. Because MSD refolding may turn off GPIb-IX's mechanosensory signals, our results provide a mechanism for the requirement of a continuous pulling force of >15 pN to fully activate GPIb-IX.