Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

Castro-Núñez, Lydia; Bloem, Esther; Boon-Spijker, Mariëtte; Zwaan, Carmen van der; Biggelaar, Maartje van den; Mertens, Koen; Meijer, Alexander B.

doi:10.1074/jbc.m112.400572

Cited by 12 publications

(19 citation statements)

References 40 publications

(48 reference statements)

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“…2 Previously, we have employed an primary amine-directed chemical foot printing foot printing approach on the FVIII -VWF complex and established that Lys773 contributes to FVIII binding. 23 This methodology has proven to be particularly powerful in the identification of protein interaction sites. 34 Applying HDX-MS on the FVIII -D'-D3 complex showed reduced deuterium incorporation in amino acid region Arg782-Cys799 in presence of FVIII ( Figure 2).…”

Section: Discussionmentioning

confidence: 99%

“…The efficiency by which the D'-D3 variants were able to compete with VWF for FVIII binding was assessed using a competitive binding assay as also employed in previous studies. 22,23 FVIII was incubated with immobilized VWF in the presence of increasing concentrations of the D'-D3 variants. Residual FVIII binding to immobilized VWF was assessed using an antibody against FVIII that does not interfere with the complex formation between FVIII and VWF ( Figure 4).…”

Section: Spr Analysis Reveals That Charged Residues In D' Region Arg7mentioning

confidence: 99%

“…22 Using a primary amine-directed chemical foot printing approach combined with mass spectrometry analysis, we have further demonstrated that Lys773 contributes to FVIII binding. 23 In the present study, we have now employed HDX-MS combined with site-directed mutagenesis and protein binding studies to further explore the FVIII binding regions on VWF. The combined results showed that D' domain region Arg782-Cys799 is part of the FVIII binding interface.…”

Section: Introductionmentioning

confidence: 99%

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D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding

et al. 2019

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In the complex with Von Willebrand Factor, factor VIII (FVIII) is protected from rapid clearance from the circulation. Although it has been established that the FVIII binding site resides in the N-terminal D'-D3 domains of Von Willebrand Factor, 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 Von Willebrand Factor. To this end, time-dependent deuterium incorporation was assessed in D'-D3 and the FVIII -D'-D3 complex. Data showed reduced deuterium incorporation in 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-individual 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 combined results show that amino acid region Arg782-Cys799 is part of a FVIII binding surface. Our study provides new insight into FVIII -Von Willebrand Factor complex formation and defects therein that may be associated with bleedings caused by markedly reduced levels of FVIII.

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Section: Discussionmentioning

confidence: 99%

Section: Spr Analysis Reveals That Charged Residues In D' Region Arg7mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding

et al. 2019

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“…15 Multiple biochemical methods and the location of VWD type 2N mutations implicate the VWF D9 domain (S764-A865) as the site for FVIII docking. [16][17][18][19][20] However, a S764-R1035 proteolytic fragment binds FVIII with markedly reduced affinity (K D 5 48.5 nM) compared with longer VWF fragments. 21,22 Several approaches have attempted to engineer FVIII with an extended plasma half-life (t 1/2 ).…”

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confidence: 99%

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

Yee

Gildersleeve

et al. 2014

Blood

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• The D9D3 domains of VWF are sufficient to stabilize FVIII in vivo.• The prolongation of VWF D9D3 survival in vivo by Fc fusion elevates FVIII levels in the setting of VWF but not FVIII deficiency.Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D9D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf 2/2 mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D9D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t 1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf 2/2 mice. Although the VWF D9D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D9D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D9D3 and VWF D9D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D9D3/D9D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding. (Blood. 2014; 124(3):445-452)

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“…The binding site for FVIII is located in the amino-terminal D’D3 region, spanning residues 764–1035 46,47. In a recent study, Castro-Nunez and coworkers used an approach of mass spectrometer-assisted footprinting to discover that VWF residues Ser-764 and Lys-773 seem to be directly involved in the binding of FVIII 48. The complementary binding site in FVIII has also been identified, involving residues at both the amino- and carboxyterminal regions of the FVIII light chain 49,50…”

Section: The Classical Functions Of Vwf: Fviii Bindingmentioning

confidence: 99%

On the Versatility of Von Willebrand Factor

Lenting¹

2013

Mediterr J Hematol Infect Dis

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Von Willebrand factor (VWF) is a large multimeric protein, the function of which has been demonstrated to be pivotal to the haemostatic system. Indeed, quantitative and/or qualitative abnormalities of VWF are associated with the bleeding disorder Von Willebrand disease (VWD). Moreover, increased plasma concentrations of VWF have been linked to an increased risk for thrombotic complications. In the previous decades, many studies have contributed to our understanding of how VWF is connected to the haemostatic system, particularly with regard to structure-function relationships. Interactive sites for important ligands of VWF (such as factor VIII, collagen, glycoprotein Ibα, integrin αIIbβ3 and protease ADAMTS13) have been identified, and mutagenesis studies have confirmed the physiological relevance of the interactions between VWF and these ligands. However, we have also become aware that VWF has a more versatile character than previously thought, given its potential role in various non-hemostatic processes, like intimal thickening, tumor cell apoptosis and inflammatory processes. In the presence review, a summary of our knowledge on VWF structure-function relationships is provided in the context of the “classical” haemostatic task of VWF and in perspective of pathological processes beyond haemostasis.

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Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

Cited by 12 publications

References 40 publications

D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding

D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

On the Versatility of Von Willebrand Factor

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