Probing ADAMTS13 substrate specificity using phage display.

Karl C Desch,Kristin Metzger,Colin Kretz,Jane Cheng,Andrew Yee,Nidhi Agrawal,David Ginsburg,Robert Gildersleeve,Toshiyuki Miyata

doi:10.1371/journal.pone.0122931

Karl C Desch, Kristin Metzger + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0122931

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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.

Highlights

Introduction vonWillebrand factor (VWF) is a multimeric plasma glycoprotein that functions as a critical regulator of hemostasis, both as a carrier for coagulation factor VIII and as a molecular bridge between circulating blood platelets and sites of vascular injury
Of the 260 phage clones screened by ELISA, 136 (52%) showed decreased relative cleavage compared to known wild-type VWF73 expressing phage controls
Previous investigations into ADAMTS13 substrate specificity have analyzed a limited set of amino acid substitutions within the vonWillebrand factor (VWF) A2 domain, this is the first report of an unbiased screen of ADAMTS13 specificity in a large library of VWF mutations

Summary

Introduction

Willebrand factor (VWF) is a multimeric plasma glycoprotein that functions as a critical regulator of hemostasis, both as a carrier for coagulation factor VIII and as a molecular bridge between circulating blood platelets and sites of vascular injury. Synthesized exclusively in endothelial cells and megakaryocytes, VWF is stored in endothelial Weibel-Palade bodies and platelet alpha granules and secreted upon activation[1]. The initial secreted molecules include the most highly multimeric and procoagulant form of VWF, termed ultra-large VWF. ADAMTS13 Substrate Specificity (UL-VWF)[2]. UL-VWF is rapidly cleaved upon secretion into the circulation by the metalloprotease ADAMTS13[3, 4]. ADAMTS13 deficiency results in an accumulation of UL-VWF and is associated with the development of thrombotic thrombocytopenic purpura (TTP), a life threatening thrombotic microangiopathy[5]

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