Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex

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 GPIba 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 Â 10 À3 s À1 . 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 À1 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.

New insights into glycoprotein Ibα desialylation-mediated platelet clearance

“…This current study demonstrates that retention of glycans but the removal of sialic acid has a similar force-reducing effect, and in 76% of measurements no unfolding event was observed. Though this might point to a low level of unfolding it is consistent with prior models of an MSD which is natively unfolded and, perhaps surprisingly, occurs at the same frequency as in overstretching of unglycosylated MSD [14], suggesting a need for these modifications, in part or in entirety, for MSD stability.…”

Unaccompanied mechanosensory domain mediates low expression of glycoprotein Ibα: implications for Bernard‐Soulier syndrome

“…The MSD is not stable because deglycosylation or low concentration of urea significantly destabilizes or unfolds it . Thus it is conceivable that, without accompanying GPIbβ and GPIX, the unstable or unfolded MSD may induce unfolded protein response (UPR) in the cell.…”

Structure‐function of platelet glycoprotein Ib‐IX

“…10 The MSD unfolds under a continuous pulling force of ~15 pN. 11,14,52 Although the precise boundary of the MSD is not settled, estimates based on mutagenesis and fitting of single-molecule data suggest that the MSD spans about 60 residues. Recent studies indicate that this domain is structured and quasi-stable, and its stability is altered by O-glycosylation therein.…”

“…The stretch closest to the platelet membrane contains the quasi-stable mechanosensory domain (MSD), the structure of which remains to be determined. 10,11 The MSD contains the shedding cleavage site by ADAM17, the physiological sheddase of GPIbα. [12][13][14] The extracellular domains of GPIbβ and GPIX are smaller than that of GPIbα; each contains one LRR sequence.…”