Functional Display of Platelet-Binding VWF Fragments on Filamentous Bacteriophage

Abstract

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.

Salmonella and Campylobacter : Antimicrobial resistance and bacteriophage control in poultry

“…There is a third, albeit less common, phage life cycle referred to as filamentous. This cycle uses the bacteria's machinery as do the other two cycles; however, filamentous systems do not result in cell death, rather continuous production and release of phages into the environment (Yee et al, 2013). The phages used for therapeutic purposes are usually strictly lytic (Wright et al, 2013) and mainly occupy three families of the order Caudovirales: Myoviridae, Siphoviridae, and Podoviridae (Wittebole et al, 2013).…”

Phage Display Broadly Identifies Inhibitor-reactive Regions in von Willebrand Factor

“…The sequences of all oligonucleotides (P1-P12, IDT DNA Technologies) are listed in Table 1. A FLAG tag (NH 2 -DYKDDDDK-COOH) was inserted into the phagemid pAY-E to facilitate specific elution of bound M13 with enterokinase[22]. Oligonucleotides P1 and P2 were annealed (65.0°C) and ligated into the NotI and SgrAI sites of pAY-E to introduce the FLAG tag.…”

“…Phage were finally collected by centrifugation at 20,000g at 4°C and resuspended in antibody selection buffer (TBS-T + 5% bovine serum albumin (BSA)). Phage concentrations were quantified by infecting XL1-Blue MRF’ cells as previously described[22].…”

The Flow Dependent Adhesion of von Willebrand Factor (VWF)-A1 Functionalized Nanoparticles in an in Vitro Coronary Stenosis Model

“…An expression plasmid was constructed to fuse an N-terminal myc tag, a C-terminal biotinylation site, and a C-terminal FLAG tag to the VWF A1 domain (P1254-L1460). The myc tag was fused to A1 by PCR amplification of VWF cDNA with primers P1, P2, and P3 using Herculase II (Agilent) [25]. The C-terminal fusions were subsequently added by PCR amplification of the myc-A1 fusion with primers P4, P5, P6, P7, and P8.…”