Destabilization of the von Willebrand factor A2 domain under oxidizing conditions investigated by molecular dynamics simulations

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

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Molecular Dynamics Simulated Unfolding of von Willebrand Factor A Domains by Force

Cellular and Molecular Bioengineering ◽

10.1007/s12195-009-0051-0 ◽

2009 ◽

Vol 2 (1) ◽

pp. 75-86 ◽

Cited By ~ 14

Author(s):

Wei Chen ◽

Jizhong Lou ◽

Cheng Zhu

Keyword(s):

Molecular Dynamics ◽

Von Willebrand Factor ◽

A Domains ◽

Von Willebrand ◽

Willebrand Factor

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Long-ranged Protein-glycan Interactions Stabilize von Willebrand Factor A2 Domain from Mechanical Unfolding

Scientific Reports ◽

10.1038/s41598-018-34374-y ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 4

Author(s):

Chuqiao Dong ◽

Jumin Lee ◽

Seonghoon Kim ◽

Whitney Lai ◽

Edmund B. Webb ◽

...

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand ◽

A2 Domain ◽

Willebrand Factor

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A Novel Quali-Quantitative Defect of VWF

Blood ◽

10.1182/blood-2021-147899 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1032-1032

Author(s):

Tom Van De Berg ◽

Alice Todaro ◽

Joyce van Beers ◽

Kanin Wichapong ◽

Floor Heubel-Moenen ◽

...

Keyword(s):

Molecular Dynamics ◽

Amino Acid ◽

Genetic Analysis ◽

Molecular Dynamics Simulations ◽

Mrna Level ◽

Bleeding Tendency ◽

Unknown Significance ◽

Dynamics Simulations ◽

Von Willebrand

Abstract Background Von Willebrand Factor (VWF) is a multimeric protein largely involved in both primary and secondary hemostasis. The diagnosis and classification of von Willebrand Disease (VWD) patients can be challenging. In this poster we explore the genetic defects and their structural consequences in a VWD patient with a disproportionately high bleeding phenotype for her VWD severity. A 31-year old female, initially diagnosed with VWD type 1, presented herself with a bleeding tendency (ISTH-BAT 13) disproportionate to the severity of her VWD with antigen levels of 36%. Additional analysis showed decreased FVIII-binding at 28%. The combination of both quantitative and functional defects of VWF was an indication for further genetic analysis in order to better define the subtype of VWD. Aims Genetic and structural analysis of VWF in a patient with a disproportionally high bleeding phenotype with regard to a mild decrease in VWF antigen. Methods Routine laboratory analysis for VWD was performed. Genetic screening was performed by exome sequencing of hemostasis related genes. VWF mRNA analysis was carried out by RT-PCR and Sanger sequencing. The X-ray structure of furin in complex with a peptide-based inhibitor (PDB ID: 6YD7) was used as a template to construct furin-VWF (759HR(R760S)SKRS764) complex. The derived structures (furin in complex with WT/R760S-VWF) were subjected to molecular dynamics (MD) simulations (200ns) and binding free energy (BFE) calculations by using standard parameters and protocols implemented in AMBER20 program. Results Routine analysis showed PFA-ADP and PFA EPI >300 seconds, VWF:ACT of 37% with a VWF:AG of 36%. Collagen binding and FVIII-binding were 46% and 28% respectively. Genetic analysis of the VWF gene disclosed 2 heterozygous variants of unknown significance (VUS): c.2771 G>A (exon 21, p.Arg924Gln) has a 1-2.5% population prevalence and has been previously described in type 1 and 2N VWD. The other VUS (c.2278 C>A; exon 17) is a novel mutation predicting a major amino acid substitution (p.Arg760Ser) in the D2-domain of VWF. Sequencing of exons 17 and 21 in the patient's VWF mRNA revealed homozygosity for the mutated allele at both mutation sites, indicating that the two variants are in cis and that the 'normal' allele is not expressed at mRNA level. Molecular dynamics simulations of the novel c.2278 C>A mutation (Arg760Ser) predicts a markedly decreased binding of furin to its VWF binding site, possibly decreasing or preventing VWF pro-peptide cleavage. This in turn has been shown to lead to reduced FVIII-binding of VWF. Conclusion Genetic analysis shows one polymorphism (c.2771 G>A) and one variation of unknown significance (c.2278 C>A) in the patient's VWF-gene. The polymorphism is known to be of low pathogenicity. The c. 2278 C>A mutation was not known in any of the mutation databases and is a novel VWF mutation. Both mutations were shown to be present on the same allele. As the wild-type allele was not expressed on mRNA level, all of the patient's VWF protein includes both amino acid substitutions. Modeling and molecular dynamics simulations show a markedly decreased affinity of furin to its cleavage site on the VWF protein due to the Arg760Ser substitution, likely resulting in a persistent pro-peptide binding to the mature VWF protein. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain

Blood Advances ◽

10.1182/bloodadvances.2018030122 ◽

2019 ◽

Vol 3 (7) ◽

pp. 957-968 ◽

Cited By ~ 4

Author(s):

Changjie Zhang ◽

Anju Kelkar ◽

Sriram Neelamegham

Keyword(s):

Von Willebrand Factor ◽

Ex Vivo ◽

Association Studies ◽

Apolipoprotein A1 ◽

Platelet Glycoprotein ◽

Functional Domain ◽

Self Association ◽

Von Willebrand ◽

A2 Domain ◽

Willebrand Factor

Abstract von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues (“Lock-VWF”) reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain (“ΔA1 proteins”) suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

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