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

• Type of Material: Article
• Authors: Kayleigh M Voos, Emory University; Wenpeng Cao, Lehigh University; Nicholas A Arce, Emory University; Emily R Legan, Emory University; Yingchun Wang, Emory University; Asif Shajahan, University of Georgia; Parastoo Azadi, University of Georgia; John Lollar, Emory University; Frank X Zhang, Lehigh University; Renhao Li, Emory University
• Language: English
• Date: 2021-09-26
• Publisher: WILEY
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2021 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.
• Final Published Version (URL): http://dx.doi.org/10.1111/jth.15528
• Title of Journal or Parent Work: JOURNAL OF THROMBOSIS AND HAEMOSTASIS
• Volume: 20
• Issue: 1
• Start Page: 196
• End Page: 207
• Grant/Funding Information: This work was supported in part by NIH research grants (HL082808, HL143794, HL153986, and R24GM137782), NIH training grants (GM008602 and GM008367), and an infrastructure grant from Hemophilia of Georgia Center for Bleeding & Clotting Disorders of Emory; NAA was supported in part by NIH fellowship HL154656. ERL was supported in part by NIH fellowship HL149357.
• Keywords: A1 DOMAIN; COMPLEX; blood; Science & Technology; PLATELET GLYCOPROTEIN-IB; biomechanics; Hematology; protein stability; LINKED GLYCOSYLATION; neuraminidase; REGION; ADHESION; PROTEIN; Peripheral Vascular Disease; Life Sciences & Biomedicine; VONWILLEBRAND-FACTOR; von Willebrand factor; IX; Cardiovascular System & Cardiology

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.