Mutual A domain interactions in the force sensing protein von Willebrand factor

Abstract

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Coarse-Grain Modeling of Shear-Induced Binding between von Willebrand Factor and Collagen

“…In a recent collection of work, binding between the A1 domain in VWF and platelets in blood was explored experimentally; authors of those works presented strong evidence supporting a mechanism for reaction in which the A1 domain could only bind with a platelet if the adjacent A2 domain was sufficiently elongated (11,12). In separate work, it has been instead postulated that flanking residues to A1 undergo some structural change that unmasks A1 and enables binding to platelets (4).…”

“…In the works exploring interactions between A1 domains and blood platelets (11,12), it was pointed out that, though elongation of the A2 domain was required for reaction between the two, it was equally important that the A2 domain be not too greatly elongated. The reason is that VWF multimers are subject to scission by the species ADAMTS13 at exposed A2 domains.…”

“…Focus herein is on A3-collagen binding; nonetheless, it is interesting to note recent evidence advanced that the A2 domain in VWF blocks the A1 domain in equilibrium or low-shear conditions, thereby mediating A1-platelet binding (10). With sufficient shear forces, dissociation of A1 and A2 occurs-accompanied by partial A2 domain unfolding-to permit A1-platelet interaction (11,12). In separate work examining A1-platelet binding, it was found that N-and C-terminal residues that flank the A1 domain mask it and impede binding with platelets; this suggests that shear-induced activation of A1 binding may be associated with an externally applied force altering the molecular structure in the vicinity of the residues (and A1), effectively unmasking the A1 domain for binding (13).…”

Endothelial Cell von Willebrand Factor Secretion in Health and Cardiovascular Disease

“…Atomic force micrographs have demonstrated at the single molecule level that under static conditions, vWF assumes a globular conformation, whereas, under high shear flow, vWF turns into an extended chain format [16] that forms ultra-large strings to which platelets bind to initiate clot formation at sites of vascular damage [25] and, when shear stress is above 30.000 s À1 , factor VIII is released from its carrier protein to provide factor VIII to the coagulation cascade [138]. We now realize that, while ultra-large MW (molecular weight) vWF is essential for the normal hemostasis, this multimeric array should not become too large because it alters the thrombotic propensity [15,16,133,134,[138][139][140][141][142][143][144][145][146][147].…”

Internal Tensile Force and A2 Domain Unfolding of von Willebrand Factor Multimers in Shear Flow

“…Although the A2 domain is not directly responsible for vWF/platelet binding, a recent experimental work found that the A2 domain specifically binds to the active conformation of the A1 domain and effectively blocks interactions between the A1 domain and platelet GPIbα [20]. Another work employed both experiments and simulations in tandem and also reported specific A1/A2 domain binding that inhibits vWF/platelet binding; they found that an induced stretching force caused detachment of the two domains, thereby exposing the A1 domain binding site for GPIbα [21]. Clearly then, examination of internal vWF dynamics, namely A2 domain dynamics, is crucial for understanding vWF's role in physiological processes at large.…”