What a polyclonal antibody sees in von Willebrand factor

Tan, Fen Lai; Ginsburg, David

doi:10.1016/j.thromres.2007.05.015

Cited by 13 publications

(24 citation statements)

References 28 publications

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“…2A). According to recent work, this is because most of the immunoglobulin fraction of the antibody used detects one of the two dominant epitopes within the C‐terminal fragment [17]. Bands of similar mobility were detected upon treatment of rVWF with human and rabbit plasma, and to a lesser extent also upon treatment with plasma from monkeys, dogs and pigs.…”

Section: Resultsmentioning

confidence: 99%

Species‐dependent variability of ADAMTS13‐mediated proteolysis of human recombinant von Willebrand factor

Váradi¹,

Rottensteiner²,

Vejda

et al. 2009

Journal of Thrombosis and Haemostasis

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Summary. Background: von Willebrand factor (VWF) is composed of a series of multimers, the sizes of which are regulated by the plasma metalloprotease ADAMTS13. Objective: Proteolysis of human recombinant VWF (rVWF) by ADAMTS13 present in the plasma of different species typically used as preclinical animal models was investigated to evaluate the efficacy and safety of rVWF. Methods: Degradation of rVWF was studied in vitro under moderate denaturing conditions and was monitored by multimer analysis, residual collagen binding, and immunoblot analysis. In vivo cleavage was determined by administration of rVWF to cynomolgus monkeys, rabbits and VWF‐deficient mice and subsequent analysis of plasma samples by immunoblot. Plasma ADAMTS13 levels were determined with a synthetic human VWF peptide (FRETS‐VWF73). Results: From the animals tested, only rabbit plasma was as efficient as human plasma in proteolysing rVWF in vitro. Mouse plasma virtually failed to cleave rVWF. Administration of human rVWF resulted in ADAMTS13‐specific cleavage products in rabbits and, to a lesser extent, in cynomolgus monkeys at various doses of rVWF. Virtually no cleavage occurred in mice. ADAMTS13 activity levels in rabbit and monkey plasma were similar to those in human plasma and were not significantly altered upon infusion of rVWF up to very high doses, indicating that rVWF did not lead to an exhaustion of endogenous ADAMTS13 in both species. Conclusions: The differences in susceptibility to cleavage of rVWF by different species need to be considered when interpreting the physiology of human rVWF from results of tests in animal models.

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Section: Resultsmentioning

confidence: 99%

Species‐dependent variability of ADAMTS13‐mediated proteolysis of human recombinant von Willebrand factor

Váradi¹,

Rottensteiner²,

Vejda

et al. 2009

Journal of Thrombosis and Haemostasis

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“…Construction of a filamentous phage display wild type VWF (wtVWF) cDNA fragment library containing ∼7.7×10 6 independent clones with VWF cDNA fragments ranging in size from ∼100 bp to ∼700 bp has been previously described [14]. The size of VWF cDNA fragments cloned into the phagemid allowed expression and display of peptide lengths (∼33 aa to ∼233 aa) sufficient to encompass the intramolecular C1272–C1458 cystine loop (187 aa) of the A1 domain.…”

Section: Methodsmentioning

confidence: 99%

“…Individually rescued phage in 2.5% BSA/PBS were then incubated with the immobilized formalin-fixed platelets at 22°C for 30 min in the presence of 2 U/mL botrocetin. Bound phage were detected with HRP conjugated, monoclonal anti-M13 filamentous phage coat protein pVIII antibody as previously described [14]. The phagemid inserts from ELISA positive clones were PCR amplified and Sanger sequenced with primers (5′-CAACGTGAAAAAATTATTATTCGC-3′ and 5′-GTAAATGAATTTTCTGTATGAGG-3′).…”

Section: Methodsmentioning

confidence: 99%

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Functional Display of Platelet-Binding VWF Fragments on Filamentous Bacteriophage

2013

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von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254–A1461, which encompasses the C1272–C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

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“…As recently shown, the immunogenicity of VWF in rabbits and mice is determined by a limited number of domains on the surface of the human VWF [9]. Perhaps these immunogenic domains of VWF were present in FVIII preparation and, therefore, resulted in VWF-specific antibody response in mice.…”

Section: Introductionmentioning

confidence: 99%

Von Willebrand Factor-Specific Antibodies Developing upon Treatment of FVIII-Deficient Mice with Different FVIII Preparations

Kallas

Kuuse²,

Maimets³

et al. 2008

Acta Haematol

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What a polyclonal antibody sees in von Willebrand factor

Abstract: Taken together with previous monoclonal antibody epitope mapping studies, our results suggest that a limited number of exposed domains on the surface of the human VWF protein may be the primary determinants of immunogenicity.

Cited by 13 publications

References 28 publications

Species‐dependent variability of ADAMTS13‐mediated proteolysis of human recombinant von Willebrand factor

Species‐dependent variability of ADAMTS13‐mediated proteolysis of human recombinant von Willebrand factor

Functional Display of Platelet-Binding VWF Fragments on Filamentous Bacteriophage

Von Willebrand Factor-Specific Antibodies Developing upon Treatment of FVIII-Deficient Mice with Different FVIII Preparations

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