title: 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.
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Yee
authors.forename: Fen-Lai
authors.initials: FL
authors.lastname: Tan
authors.forename: David
authors.initials: D
authors.lastname: Ginsburg
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D017931:DNA Primers; D005822:Genetic Vectors; D016296:Mutagenesis; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014841:von Willebrand Factor
references.citation: Biotechnology (N Y). 1993 Jun;11(6):709-13
references.pmid: 7763674
references.citation: J Biol Chem. 1986 Jan 5;261(1):381-5
references.pmid: 2934387
references.citation: Chem Rev. 2005 Nov;105(11):4056-72
references.pmid: 16277371
references.citation: J Biol Chem. 2012 Apr 27;287(18):14579-85
references.pmid: 22431729
references.citation: J Biol Chem. 2002 Jun 14;277(24):22063-72
references.pmid: 11943773
references.citation: Arterioscler Thromb Vasc Biol. 1996 Jan;16(1):64-71
references.pmid: 8548428
references.citation: Arterioscler Thromb Vasc Biol. 2000 Oct;20(10):2303-8
references.pmid: 11031219
references.citation: Arterioscler Thromb Vasc Biol. 2000 May;20(5):1347-53
references.pmid: 10807753
references.citation: Nat Struct Mol Biol. 2005 Feb;12(2):152-9
references.pmid: 15665869
references.citation: Nature. 2010 Aug 19;466(7309):992-5
references.pmid: 20725043
references.citation: Proc Natl Acad Sci U S A. 1983 Mar;80(5):1463-6
references.pmid: 6402781
references.citation: Circulation. 2007 Dec 4;116(23):2678-86
references.pmid: 18025536
references.citation: J Biol Chem. 1995 Aug 18;270(33):19668
references.pmid: 7642656
references.citation: Annu Rev Biochem. 1998;67:395-424
references.pmid: 9759493
references.citation: J Biol Chem. 1993 Oct 5;268(28):21238-45
references.pmid: 8407961
references.citation: J Biol Chem. 2000 Mar 17;275(11):7539-46
references.pmid: 10713059
references.citation: Biochemistry. 1994 Mar 29;33(12):3643-8
references.pmid: 8142362
references.citation: Blood. 2012 Jul 5;120(1):214-22
references.pmid: 22517896
references.citation: Science. 2002 Aug 16;297(5584):1176-9
references.pmid: 12183630
references.citation: Arterioscler Thromb Vasc Biol. 1998 Feb;18(2):200-7
references.pmid: 9484984
references.citation: Blood. 2006 Sep 15;108(6):1903-10
references.pmid: 16772609
references.citation: Nat Med. 2009 Jun;15(6):665-73
references.pmid: 19465929
references.citation: J Biol Chem. 2004 May 28;279(22):23327-34
references.pmid: 15039442
references.citation: J Biol Chem. 1998 Apr 24;273(17):10396-401
references.pmid: 9553097
references.citation: Thromb Res. 2008;121(4):519-26
references.pmid: 17614124
references.citation: J Biol Chem. 2006 Feb 24;281(8):4699-707
references.pmid: 16373331
references.citation: J Biol Chem. 2000 Jun 23;275(25):19098-105
references.pmid: 10764791
references.citation: J Mol Biol. 2008 Oct 24;382(5):1211-27
references.pmid: 18706916