Force-induced on-rate switching and modulation by mutations in gain-of-function von Willebrand diseases

Abstract

Mutations in the ultralong vascular protein von Willebrand factor (VWF) cause the common human bleeding disorder, von Willebrand disease (VWD). The A1 domain in VWF binds to glycoprotein Ibα (GPIbα) on platelets, in a reaction triggered, in part, by alterations in flow during bleeding. Gain-of-function mutations in A1 and GPIbα in VWD suggest conformational regulation. We report that force application switches A1 and/or GPIbα to a second state with faster on-rate, providing a mechanism for activating VWF binding to platelets. Switching occurs near 10 pN, a force that also induces a state of the receptor−ligand complex with slower off-rate. Force greatly increases the effects of VWD mutations, explaining pathophysiology. Conversion of single molecule k on (s −1 ) to bulk phase k on (s −1 M −1 ) and the k on and k off values extrapolated to zero force for the low-force pathways show remarkably good agreement with bulk-phase measurements.U nderstanding how force affects receptor and ligand binding and unbinding is a long-standing effort in mechanobiology (1-5). Bond dissociation rates typically increase under mechanical stress; however, bond stability can be enhanced through specialized mechanisms induced by force, including catch bonds and switching to a slip bond with a slower off-rate (flex bonds) (6, 7). Bond formation against an applied force has recently been measured (8). Force-regulated switching to a faster on-rate has not yet been reported for any receptor−ligand bond but would have important biological implications for adhesion in environments with high forces such as the circulation.At sites of vascular injury, hydrodynamic force in the bloodstream acting on von Willebrand factor (VWF) is pivotal in regulating the binding of the VWF A1 domain to GPIbα on platelets and commencing the crosslinking of platelets by VWF to form a platelet plug (9-11). VWF circulates in the form of long, disulfidebonded concatemers, with tens to hundreds of monomers, which mostly adopt a compact, irregularly coiled conformation during normal hemodynamics (12). At sites of hemorrhage, flow changes from shear to elongational. On transition from low to high shear and from shear to elongational flow, irregularly coiled molecules extend to a thread-like shape, and elongational (tensile) force is exerted throughout their lengths (13-16). Molecular elongation exposes the multiple A1 binding sites in VWF concatamers for multivalent binding to GPIbα (9,11,14,(16)(17)(18).In vivo, tensile force transmitted through VWF is applied to the N and C termini of individual domains, and could theoretically change A1 domain conformation before binding to GPIbα. Although this scenario has not yet been observed, single-molecule studies demonstrate two distinct force-dependent dissociation pathways (flex-bond behavior) of the wild-type (WT) A1-GPIbα complex, and thus suggest that two conformational states can be present after formation of the receptor−ligand complex (6).Mutations in VWF cause von Willebrand disease (VWD), the most common human heri...

Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping

“…The use of equilibrium binding models coupled with atomic force microscopy and thermodynamics of partial denaturation by urea was approximate, but over-estimated the energetics of conformational change within the native state ensemble [9]. Single molecule force spectroscopy used in conjunction with thermodynamic models gives apparent on and off rates as a function of force, but the thermodynamic linkage through the equilibrium, K 1 , between free states was not explicitly considered [44, 45]. The force-induced rate switching observed by Kim et.…”

“…al. could result from conformational selection due to thermodynamic linkage, but could also result from microscopic irreversibility in favor of the higher affinity state of A1 in the single-molecule construct [45], a complication that is not shared by analytical rheology.…”

Probing the structural dynamics of proteins and nucleic acids with optical tweezers

“…Kim et al studied how the binding of von Willebrand factor (VWF) to platelets is regulated by hydrodynamic forces in the vasculature, revealing that force activates platelet binding and increases the effects of disease-related mutations [51]. Especially fascinating is recent work probing the activity of molecular chaperones, which are required for the folding of many proteins but whose mechanisms remain poorly understood at the molecular level [52].…”

Ultrasensitivity of Cell Adhesion to the Presence of Mechanically Strong Ligands

“…The membrane-bound receptor proteins, called integrins, interact with the ECM and relay information about the extracellular environment to the cell interior and to the underlying actin cytoskeleton through interaction with other proteins [11,[17][18][19][20][21][22][23][24]. The spatial extent of the ECM communication with the actin cytoskeleton through integrins ranges from nano to micrometers with a force sensitivity that ranges from a few pico-Newton (pN) to a few hundreds of pN [12,[25][26][27][28][29][30][31]. Precise understanding of the underlying mechanisms requires techniques that are sensitive in these ranges.…”