Page 237 - Haematologica - Vol. 105 n. 6 - June 2020

Coagulation & its Disorders
D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding
Małgorzata A. Przeradzka,1 Josse van Galen,1 Eduard H.T.M. Ebberink,1 Arie J. Hoogendijk,1 Carmen van der Zwaan,1 Koen Mertens,1
Maartje van den Biggelaar1 and Alexander B. Meijer1,2
1Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam and 2Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands
ABSTRACT
In the complex with von Willebrand factor (VWF) factor VIII (FVIII) is protected from rapid clearance from circulation. Although it has been established that the FVIII binding site resides in the N-terminal D’-D3 domains of VWF, detailed information about the amino acid regions that contribute to FVIII binding is still lacking. In the present study, hydrogen- deuterium exchange mass spectrometry was employed to gain insight into the FVIII binding region on VWF. To this end, time-dependent deu- terium incorporation was assessed in D’-D3 and the FVIII-D’-D3 complex. Data showed reduced deuterium incorporation in the D’ region Arg782- Cys799 in the FVIII-D’-D3 complex compared to D’-D3. This implies that this region interacts with FVIII. Site-directed mutagenesis of the six charged amino acids in Arg782-Cys799 into alanine residues followed by surface plasmon resonance analysis and solid phase binding studies revealed that replacement of Asp796 affected FVIII binding. A marked decrease in FVIII binding was observed for the D’-D3 Glu787Ala variant. The same was observed for D’-D3 variants in which Asp796 and Glu787 were replaced by Asn796 and Gln787. Site-directed mutagenesis of Leu786, which together with Glu787 and Cys789 forms a short helical region in the crystal structure of D’-D3, also had a marked impact on FVIII binding. The com- bined results show that the amino acid region Arg782-Cys799 is part of a FVIII binding surface. Our study provides new insight into FVIII-VWF com- plex formation and defects therein that may be associated with bleeding caused by markedly reduced levels of FVIII.
Introduction
The multimeric glycoprotein von Willebrand factor (VWF) acts as a carrier pro- tein for coagulation factor VIII (FVIII) in the circulation.1 In the complex with VWF, FVIII is protected from rapid clearance from plasma.2,3 Multiple amino acid substi- tutions have been identified in VWF that impair FVIII-VWF complex formation. The associated reduced plasma levels of FVIII can result in the bleeding disorder referred to as von Willebrand disease type 2 Normandy (VWD type 2N).4 Most of the aberrant mutations in VWF involve substitutions of amino acid residues that have been proposed to affect the structural integrity of VWF.5,6 These substitutions provide therefore only limited information about the identity of the FVIII binding site on VWF.
Distinct protein domains can be identified in the primary amino acid sequence of VWF. These domains are arranged in the order: D’-D3-A1-A2-A3-D4-B-C1-C2-C2- CK.7 Zhou et al. have refined the domain organization within VWF. For D’-D3, they proposed that these domains can be further divided into TIL’-E’-VWD3-C8_3-TIL3- E3 subdomains.8 In plasma, VWF circulates as an ensemble of multimeric proteins of varying size. In these multimers, the VWF monomers are head-to-head and tail- to-tail connected via disulphide bridges between two D3 domains and two CK domains.9 FVIII also comprises multiple domains that together constitute a light chain of the domains A3-C1-C2 and a heavy chain comprising the domains A1-A2-
Ferrata Storti Foundation
Haematologica 2020 Volume 105(6):1695-1703
Correspondence:
ALEXANDER B. MEUJER
s.meijer@sanquin.nl
Received: March 20, 2019. Accepted: September 25, 2019. Pre-published: September 26, 2019.
doi:10.3324/haematol.2019.221994
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/106/6/1695
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