Flow induces loop-to-β-hairpin transition on the β-switch of platelet glycoprotein Ibα

Abstract

Interaction of glycoprotein Ib␣ (GPIb␣) with von Willebrand factor (VWF) initiates platelet adhesion to injured vascular wall to stop bleeding. A major contact between GPIb␣ and VWF involves the ␤-switch region, which is a loop in the unliganded GPIb␣ but switches to a ␤-hairpin in the complex structure. Paradoxically, flow enhances rather than impedes GPIb␣-VWF binding. Gain-offunction mutations (e.g., M239V) in the ␤-switch reduce the flow requirement for VWF binding, whereas loss-of-function mutations (e.g., A238V) increase the flow requirement. These phenomena cannot be explained by crystal structures or energy calculations. Herein we demonstrate that the ␤-hairpin is unstable without contacting VWF, in that it switches to a loop in free molecular dynamics simulations. Simulations with a novel flow molecular dynamics algorithm show that the loop conformation is unstable in the presence of flow, as it switches to ␤-hairpin even without contacting VWF. Compared with the wild-type, it is easier for the M239V mutant but harder for the A238V mutant to switch to ␤-hairpin in the presence of flow. These results elucidate the structural basis for the two mutants and suggest a regulatory mechanism by which flow activates GPIb␣ via inducing a loop-to-␤-hairpin conformational transition on the ␤-switch, thereby promoting VWF binding. flow molecular dynamics ͉ Platelet-type von Willebrand disease ͉ von Willebrand factor ͉ conformational change ͉ mechanical sensing B inding of glycoprotein Ib␣ (GPIb␣) to von Willebrand factor (VWF) initiates a multistep platelet adhesion and signaling cascade of the hemostatic process (1, 2). Dysfunction of GPIb␣-VWF interaction may cause bleeding disorders such as von Willebrand diseases (VWD) (3). A rapid kinetic on-rate of GPIb␣-VWF binding is required for flowing platelets to tether to injured vessel wall. Counterintuitively, however, GPIb␣-VWF binding is enhanced by blood flow, despite the fact that increasing flow shortens contact time for molecular interaction (4). A minimum flow is required for platelets to tether to VWF (4, 5). The kinetic rates of GPIb␣-VWF binding and their mechanical regulation by flow can be altered by structural variations. Mutations may require higher or lower flows for platelet to tether to VWF compared with the wild-type (WT) GPIb␣ (6). The former is referred to as a gain-of-function (GOF) mutant, whereas the latter is a loss-of-function (LOF) mutant. GOF mutations G233V and M239V naturally occur in some patients with platelet-type (PT) VWD (5,7,8).GPIb␣ consists of a glycosylated N-terminal domain (GPIb␣N), a long mucin stalk, a transmembrane domain, and a cytoplasmic tail (9, 10). The binding site for VWF resides on GPIb␣N, which contains eight tandem leucine-rich repeats (Fig. 1A) (11). The monomeric subunit of VWF consists of multiple copies of A, B, C, and D type domains (12). The binding site for GPIb␣ resides on the A1 domain ( Fig. 1 B, cyan) (13). Regions of GPIb␣N at each end of the leucine-rich repeats bind, respectively, to the top and botto...

Advances in Experiments and Modeling in Micro- and Nano-Biomechanics: A Mini Review

“…For example, the instability of GPIba b-hairpin without vWF is demonstrated by a free MDS as a spontaneous transition to a structureless loop. 59 But for vWF-A1/GPIba complex, MDS exhibits its conformational flexibility and the stabilizing electrostatic interactions between these two proteins. 40 The unfolding of the central b-sheet of vWF-A2 is proposed to start from its edges and then propagate into its center.…”

“…55 SMD simulations on unbinding of receptor from its ligand, such as P-selectin glycoprotein ligand 1 (PSGL-1) from P-selectin and glycoprotein Iba (GPIba) from vWF-A1, have provided insights into the molecular mechanism underlying catch-bond. 32,40,58,103 Flow MDS was inspired from FC assay and first carried out by Lou and Zhu 59 and further improved by Chen et al…”

“…1 Today, MDS is routinely applied in investigating the respective mechano-chemical coupling and mechanical properties of biomolecules at the single molecular level. 1 Variable simulation protocols and analysis methods such as free, 59 steered, 78 and flow molecular dynamics, 59 have been developed to manifest the interesting properties of biomolecules.…”

The Transmembrane Domains of β and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain

“…Furthermore, a number of investigations have shown that the single bond between the GP Ib-IX complex and vWf cannot be maintained under high shear force imposed on either platelets or the GP Ib-IX-expressing CHOs. In addition, vWf is a large multimeric molecule possessing multiple sites that can interact with GP Ib␣; therefore, GP Ib-IX complexes could be clustering on vWf, which allows for resistance to the shear force through the anchorage of the GP Ib-IX complex to the platelet cytoskeleton (3)(4)(5). Along this line, a GEM-based localization to concentrate larger numbers of GP Ib␣ molecules should facilitate such multivalent binding.…”

“…FcR␥ (16) and Fc␥RIIA (17,18)). One of the features of cell membranes is the existence of specialized glycosphingolipidenriched membranes (GEMs), 3 to which not only a number of receptors, in platelets, such as GP Ib-IX complex (19), Fc␥RIIA (19), P 2 X 1 (20), and P 2 Y 12 (21), but also various kinases, phosphatases, and adaptor proteins (22,23) are localized. Therefore, one would expect that upon ligand-receptor interaction, the receptor-associated GEMs can act as a platform or carrier to support a transmembrane signal, as well as the interplay of different signals elicited by various GEM-associated receptor-ligand interactions, helping them bind in an efficient, controlled, and synergistic manner.…”