Mutational Constraints on Local Unfolding Inhibit the Rheological Adaptation of von Willebrand Factor

Abstract

Unusually large von Willebrand factor (VWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease, two rare mutations (G1324A and G1324S) within the platelet GPIb␣ binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding affinity is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to His 1322 across the ␤2-␤3 hairpin in the GPIb␣ binding interface, which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIb␣ binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

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

“…For example, type 2B gain-of-function mutations in and around the α2 helix thermodynamically and kinetically destabilize the native state forcing the conformational selection, K 1 , in favor of A1 T (a shift in equilibrium) or misfold the A1 domain so that the thermodynamic flux is shunted through the induced fit pathway through A1 W [9, 24, 31]. Conversely, a type 2M loss-of-function G1324S mutation in the β2-β3 hairpin stabilizes the A1 domain by constraining the conformational flexibility of the domain so that A1 becomes kinetically trapped in the A1 W state [34]. Enhanced dynamics caused by type 2M VWD mutations in stable secondary structure elements, like the α3 helix, misfold A1 and abolish platelet adhesive function [23] potentially leading to completely off-pathway conformational states.…”

“…The most hydrophobic helix (0.214) of A1, α3, is a probable candidate structure since it remains ordered and protected from exchange in both disulfide-intact and RCAM A1. Alternatively, and perhaps more likely, platelet-type VWD mutations in this opposable thumb may kinetically destabilize GPIbα leading to an enhanced affinity as has been observed for type 2B VWD mutations (R1306Q and I1309V) in the natively folded conformation of the A1 domain [23, 34]. …”

The Von Willebrand Factor A1–Collagen III Interaction Is Independent of Conformation and Type 2 Von Willebrand Disease Phenotype

“…This process of initiating the platelet plugs necessary to stop bleeding is driven by the VWF A1 domain binding to the platelet receptor GPIbα and, in part, the cooperative adhesion of the A1 and A3 domains to subendothelial collagen. The rheological biophysics of platelet adhesion to the A1 domain under shear flow is highly dependent on the flexibility of local secondary structure within the native state [1]. However, this dynamic adaptation to blood flow becomes disadvantageous in von Willebrand disease (VWD) when mutations induce misfolding of A1 in both gain- (2B) and loss-of-function (2M) phenotypes [2].…”

“…Evidence for a collagen-induced conformational change of A1 stems from the observation that the reactivity of A1 to “ conformationally specific” monoclonal antibodies was significantly reduced when bound to type III collagen [7]. A thermodynamically stable and conformationally rigid type 2M loss-of-function VWD variant (G1324S) [1], did not display a loss of antibody response upon binding collagen [7], but the epitopes of the particular antibodies used that would yield structural insight into the proposed conformational changes are not known [7, 8]. Evidence against a collagen-induced enhancement of GPIbα binding to VWF originates more than two decades ago from competition experiments which demonstrated that recombinant GPIbα inhibits the binding of VWF to collagen [9].…”

Combined effects of two mutations in von Willebrand disease 2M phenotype

“…The native state of VWF‐A1 domain is highly flexible; platelet adhesion under shear flow is highly dependent on it 24. Certain mutations can induce changes in thermodynamic and kinetic stability, resulting in the local misfolding of the VWF‐A1 domain in secondary structures that affects GP1bα binding, and results in loss‐of‐function phenotype (VWD2M) 25.…”