Desialylation of O‐glycans activates von Willebrand factor by destabilizing its autoinhibitory module

Abstract

Background: The binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O-glycans in VWF are located around the A1 domain. Removing certain O-glycans in the flanking sequences by site-directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. Objectives: To understand if and how desialylation of O-glycans in the flanking sequences increases A1 activity. Methods: A recombinant AIM-A1 fragment encompassing VWF residues 1238-1493 and only O-glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment.Results: Asialo-AIM-A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen-deuterium exchange rates at residues near the secondary GPIbα binding site and the N-terminal flanking sequence. Asialo-AIM-A1 is less mechanically stable than sialo-AIM-A1, with its unstressed unfolding rate approximately 3-fold greater than the latter.Conclusions: Desialylation of O-glycans around A1 increases its activity by destabilizing the AIM.

Autoinhibitory module underlies species difference in shear activation of von Willebrand factor

“…Given the importance of O-glycans, 27,45,46 negatively charged amino acids on both VWF autoinhibition 47 and A1-LBD guidance, 48 an increase in positive charge may change the function of the AIM and facilitate the A1-LBD interaction.…”

“…Sequence differences result in the mouse AIM having more positive charge and possibly different O‐linked glycosylation, further increasing positive charge. Given the importance of O‐glycans, 27,45,46 negatively charged amino acids on both VWF autoinhibition 47 and A1‐LBD guidance, 48 an increase in positive charge may change the function of the AIM and facilitate the A1‐LBD interaction.…”

“…This AIM comprises discontinuous sequences flanking the A1 domain that is delimited by the 1272–1458 disulfide bond (Figure 1A). 26 While the AIM contains some intrinsically disordered sequences, it contains a certain folded structure that likely involves sialylated glycans 25,27 . It masks a part of the GPIbα binding site in the A1 domain.…”

“…26 While the AIM contains some intrinsically disordered sequences, it contains a certain folded structure that likely involves sialylated glycans. 25,27 It masks a part of the GPIbα binding site in the A1 domain. The AIM unfolds when sufficient tensile force is applied, thereby relieving the inhibition on A1 (Figure 1B).…”

The Role of the Complement System in the Pathogenesis of Infectious Forms of Hemolytic Uremic Syndrome

“…Fibrinogen desialylation reduces thrombin time in patients with liver disease, which is apparently due to increased aggregation of fibrin monomers [208,209]. Desialylation of plasma von Willebrand factor induces its binding to platelets [210]. Desialation of FVIIa promotes its active recognition by hepatocytes through the asialoglycoprotein receptor and accelerates its clearance from the bloodstream [211].…”