Molecular mechanisms of von Willebrand Factor mechanoregulation

Abstract

Von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. The potency to recruit platelets critically depends on the size of VWF multimers, which is regulated by a feedback mechanism involving shear-induced unfolding of the A2 domain in VWF and cleavage by the metalloprotease ADAMTS-13. We identified and functionally characterized a calcium-binding site in the A2 domain that modulates key aspects of VWF mechanoregulation by hydrodynamic forces. We determined the crystal structure of wild-type A2 with bound Ca2+. This structure showed that Ca2+ binds to a highly dynamic loop region preceding the scissile bond. Biophysical assays in conjunction with molecular dynamics simulations revealed the mechanism by which Ca2+ binding stabilizes the A2 domain in its native conformation and impedes proteolytic cleavage at low force. We used optical tweezers experiments to impose tensile force on individual A2 molecules. Mimicking hydrodynamic stretching forces in the blood stream, these experiments demonstrated that unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate. Calcium further accelerates refolding markedly, and permits active refolding under tensile load. Our data support a model by which Ca2+ binding provides A2 with the capabilities of a force-sensitive molecular switch in response to physiologically relevant hydrodynamic conditions. This mechanism provides a tension-dependent signal for proteolytic feedback inhibition regulating the thrombogenic potential of VWF multimers. Our crystal structure revealed that the calcium-binding site of the A2 domain is formed by the P9 to P2 residues immediately amino-terminal of the scissile bond. Several naturally occurring sequence variants of the A2 domain involve amino acid substitutions at or close to this calcium-binding site. We characterized the kinetics of proteolytic cleavage for an extensive set of P5-P1’ variants and investigated the mechanism by which naturally occurring A2 variants affect ADAMTS-13 cleavage efficiency. We demonstrate that specificity of ADAMTS-13 does primarily depend on the scissile bond motif, but rather on the P3 and P1 residues. Specifically, P3 interaction with its S3 subsite determines substrate affinity, while P2 supports productive binding of the scissile bond. Our data suggest that these features, in combination with previously characterized exosites in ADAMTS-13 curtail the high specificity of ADAMTS-13 for the substrate VWF. Furthermore, we show that a naturally occurring A2 variant implicated in type 2A VWD disrupts Ca2+ binding and the associated protective effect toward ADAMTS-13 cleavage. Our functional analysis underscores the relevance of Ca2+ binding by A2 in proteolytic processing of VWF by ADAMTS-13. Collectively, the data have significantly advanced our molecular understanding of force-dependent cleavage of VWF multimers by ADAMTS-13 and its regulation by hydrodynamic forces. Last, we present a novel method for the assembly of repetitive DNA constructs such as frequently employed in engineering of proteins for single-molecule force spectroscopy. We adapt concepts from combinatorial chemistry to permit in vitro assembly of DNA synthons into functional open reading frames. We show that this strategy significantly accelerates assembly of such constructs and anticipate that our method will facilitate engineering of modular protein encoding DNA sequences in general.