Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PLoS One

Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.

Published: May 2014

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.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760814PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073518PLOS

Publication Analysis

Top Keywords

vwf
8
phage library
8
vwf protein
8
formalin-fixed platelets
8
rounds affinity
8
cystine loop
8
functional display
4
display platelet-binding
4
platelet-binding vwf
4
vwf fragments
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!