title: Probing ADAMTS13 substrate specificity using phage display.
abstract: Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.
authors: Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: Karl C
authors: KC
authors: Desch
authors: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: Colin
authors: C
authors: Kretz
authors: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: Andrew
authors: A
authors: Yee
authors: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: Robert
authors: R
authors: Gildersleeve
authors: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: Kristin
authors: K
authors: Metzger
authors: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: Nidhi
authors: N
authors: Agrawal
authors: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: Jane
authors: J
authors: Cheng
authors: Howard Hughes Medical Institute, Ann Arbor, Michigan, United States of America; Department of Internal Medicine and Human Genetics, University of Michigan, Ann Arbor, Michigan, United States of America.
authors: David
authors: D
authors: Ginsburg
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000595:Amino Acid Sequence; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D059748:Proteolysis; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D019151:Peptide Library; D014841:von Willebrand Factor; D051722:ADAM Proteins; C502939:ADAMTS13 protein, mouse; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human