Dataset Information

Structure and Function of Recombinant versus Plasma-Derived von Willebrand Factor and Impact on Multimer Pharmacokinetics in von Willebrand Disease.

ABSTRACT:

Background

Recombinant von Willebrand factor (rVWF, vonicog alfa) is a purified VWF concentrate produced from Chinese hamster ovary cells. rVWF is not exposed to the VWF-cleaving protease ADAMTS13 and so is not subject to proteolytic degradation of large (L) and ultra-large (UL) VWF multimers by that enzyme.

Purpose

To compare the structure and function of rVWF with the human plasma-derived VWF [pdVWF] concentrates Haemate P^®/Humate-P^®, Voncento^®, Wilate^®/Eqwilate^®, and Wilfactin^®/Willfact^®; to investigate the relationship between VWF multimeric pattern and VWF:ristocetin cofactor (VWF:RCo) activity through population pharmacokinetic (PK) modeling in patients with severe von Willebrand disease (VWD) treated with rVWF.

Methods

Analyses included VWF:RCo activity, VWF:collagen-binding activity, VWF:platelet glycoprotein Ib receptor binding, factor VIII (FVIII) binding capacity, and VWF-mediated platelet adhesion under flow conditions. VWF multimeric structure was determined by agarose gel electrophoresis. Population PK models describing the activity-time profile of small, medium, and L/UL multimers following intravenous administration of rVWF in patients with severe VWD were developed.

Results

Findings demonstrate that rVWF contains a non-degraded VWF multimer pattern including the UL multimers not present in pdVWF concentrates. rVWF displayed higher specific platelet-binding activity, and faster mediation of platelet adhesion to collagen under shear stress versus pdVWF concentrates. rVWF also demonstrated higher FVIII binding capacity than Haemate P^®, Voncento^® and Wilate^®. Modeling provided evidence that VWF:RCo activity in patients with severe VWD treated with rVWF is associated with L/UL VWF multimers in the circulation.

Conclusions

Findings suggest that the L and UL multimers preserved in rVWF contribute to high biological activity and might be important for providing hemostatic efficacy.

SUBMITTER: Gritsch H

PROVIDER: S-EPMC9673800 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Structure and Function of Recombinant versus Plasma-Derived von Willebrand Factor and Impact on Multimer Pharmacokinetics in von Willebrand Disease.

Gritsch Herbert H Schrenk Gerald G Weinhappl Nina N Mellgård Björn B Ewenstein Bruce B Turecek Peter L PL

Journal of blood medicine 20221114

<h4>Background</h4>Recombinant von Willebrand factor (rVWF, vonicog alfa) is a purified VWF concentrate produced from Chinese hamster ovary cells. rVWF is not exposed to the VWF-cleaving protease ADAMTS13 and so is not subject to proteolytic degradation of large (L) and ultra-large (UL) VWF multimers by that enzyme.<h4>Purpose</h4>To compare the structure and function of rVWF with the human plasma-derived VWF [pdVWF] concentrates Haemate P<sup>®</sup>/Humate-P<sup>®</sup>, Voncento<sup>®</sup>, ...[more]

PMID: 36405429

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Project description:The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.

Dataset Information

Structure and Function of Recombinant versus Plasma-Derived von Willebrand Factor and Impact on Multimer Pharmacokinetics in von Willebrand Disease.

Background

Purpose

Methods

Results

Conclusions

Publications

Structure and Function of Recombinant versus Plasma-Derived von Willebrand Factor and Impact on Multimer Pharmacokinetics in von Willebrand Disease.

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