A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor

Abstract

Summary Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261–1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238–1472. Both proteins contain the VWF A1 domain (residues 1272–1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1β2 and β3α2, reported markedly reduced HDX than their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

Delimiting the autoinhibitory module of von Willebrand factor

“…To further verify the coupling of the flanking residues to the A1 domain and delimit the range of the AIM in rVWF 1238–1493 , we conducted a similar HDX analysis of rVWF 1238–1493 as performed previously with rVWF 1238–1472 and rVWF 1261–1472 . With this approach, the deuterium exchange of the backbone proton on the amino acid chain of rVWF 1238–1493 was evaluated as a function of time by the use of a tandem mass spectrometer that can sequence the peptide fragments.…”

“…Expression and purification of rVWF 1261–1472 , rVWF 1238–1472 , rVWF 1238–1461 and rVWF 1271–1493 were performed with the same protocol as previously described . rVWF 1238–1493 was purified with a two‐step purification protocol.…”

“…ELISA, analytical ultracentrifugation and HDX mass spectrometry were performed as described previously .…”

“…However, studies on isolated or recombinant A1 fragments have shown that those A1 fragments containing residues flanking the A1 domain are as inactive as full‐length VWF under low‐shear conditions . However, those A1 fragments containing fewer flanking residues have high hemostatic activity in the absence of shear or agonist treatment . In addition, a recent study showed that elongation of VWF is not sufficient to promote the binding of GPIbα when the tension force is below a certain threshold .…”

“…rVWF 1261–1472 and rVWF 1238–1472 , varying in the length of flanking residue sequences and showing different binding affinities for GPIbα . HDX results suggested that the residues flanking the A1 domain in rVWF 1238–1472 (i.e., residues 1238–1271 and residues 1459–1472) bound the A1 domain cooperatively, forming a discontinuous autoinhibitory module (AIM) . The binding of AIM residues to the A1 domain provided a mechanism for the different GPIbα‐binding activities between rVWF 1261–1472 and rVWF 1238–1472 .…”