Delimiting the autoinhibitory module of von Willebrand factor

Abstract

Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

Conformation of the von Willebrand factor/factor VIII complex in quasi-static flow

“…For example, the A1 domain elongates in shear flow into a conformation with high affinity for GPIbα ( 18 ). An autoinhibitory module has been identified that flanks and binds the A1 domain, deletion of which activates the A1 domain ( 63 , 64 ), suggesting that shear flow produces dissociation of the autoinhibitory module. Additionally, shear flow has been reported to promote unfolding of the VWF A2 domain and drive self-association of VWF ( 59 ).…”

Cleavage by MMP‐13 renders VWF unable to bind to collagen but increases its platelet reactivity

“…Cleaved VWF also supported slower platelet rolling than intact VWF, indicating that cleaved VWF adopts a conformation that supports a longer or stronger interaction with platelets. The A1 domain of VWF forms an autoinhibitory module that masks the A1 domain . Cleavage of VWF just prior to the A1 domain may release it from protection by the D3 domain and/or destabilize the N‐terminus of the A1 domain to expose the GPIbα binding site .…”

Plasma kallikrein structure reveals apple domain disc rotated conformation compared to factor XI

“…Hydrogen deuterium exchange mass spectrometry (HDX‐MS) was performed largely as described on a Waters HDX system with nanoAcquity UPLC (Waters, Milford, MA, USA) and Micromass Q‐ToF Premier mass spectrometer. Samples were measured in tandem using the same buffers to minimize the difference in back exchange.…”