Abstract
The multimeric plasma glycoprotein von Willebrand factor (VWF) is exceptionally rich in cysteine, and its structure is largely determined by inter- and intramolecular disulfide bonding. Additionally, VWF was shown to contain unpaired cysteine residues potentially affecting protein function. The significance of free thiols on the surface of plasmatic VWF has been confirmed previously with respect to platelet binding under pathologically high shear stress. Furthermore their potential involvement in functional VWF self-association occurring at elevated shear stress has been suggested.
Objective of the present study was to investigate whether free thiol groups of plasma VWF contribute to the physiological VWF function under high physiological arterial shear stress conditions. Furthermore, we aimed to elucidate possible underlying mechanisms involved in this regulation.
Free and accessible thiol groups of plasma-derived VWF were blocked with N-ethylmaleimide (NEM). Derivatization was followed by detailed structural and functional examination including multimer analysis (MMA) and Fourier transform infrared spectroscopy (FTIR). Functional differences between the NEM-derivatized sample and the control sample were detected using an in vitro flow chamber system with respect to VWF-mediated platelet adhesion to collagen. Interactions with collagen type III and platelet glycoprotein (GP)Ib receptor were investigated using surface plasmon resonance (SPR). Identification of accessible cysteine residues was accomplished using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation as well as mass spectrometry.
Blocking free thiol groups provoked substantial loss of VWF activity with respect to platelet recruitment to collagen type III under flow. The lowered platelet adhesion to collagen type III was shown to be a combined effect of inhibition of (i) the initial VWF binding to collagen type III as well as (ii) VWF-platelet GPIb interaction. Free thiol groups were accessible for derivatization solely on the surface of coiled multimers. Domain incorporation studies revealed a high level of derivatization in VWF N- and C-terminus. This was in accordance with the mass spectrometric analysis, where 19 MPB-derivatized peptides, predominantly located at the N- and C-terminus, could be identified.
Blocking free thiol groups in VWF significantly impaired mediation of platelet adhesion under physiological shear stress conditions. This result suggests an essential functional role of free thiol groups in VWF regarding binding to subendothelial matrix as well as platelet recruitment.
Solecka:Octapharma AG: Employment. Fuchs:Octapharma AG: Employment. Kannicht:Octapharma AG: Employment.
Author notes
Asterisk with author names denotes non-ASH members.
