title: Plasmin Cleaves Von Willebrand Factor at K1491-R1492 in the A1-A2 Linker Region in a Shear- and Glycan-Dependent Manner In Vitro.
abstract: Previous studies have demonstrated a role for plasmin in regulating plasma von Willebrand factor (VWF) multimer composition. Moreover, emerging data have shown that plasmin-induced cleavage of VWF is of particular importance in specific pathological states. Interestingly, plasmin has been successfully used as an alternative to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) in a mouse model of thrombotic thrombocytopenic purpura. Consequently, elucidating the molecular mechanisms through which plasmin binds and cleaves VWF is not only of basic scientific interest but also of direct clinical importance. Our aim was to investigate factors that modulate the susceptibility of human VWF to proteolysis by plasmin.
  
  
  
  We have adapted the VWF vortex proteolysis assay to allow for time-dependent shear exposure studies. We show that globular VWF is resistant to plasmin cleavage under static conditions, but is readily cleaved by plasmin under shear. Although both plasmin and ADAMTS13 cleave VWF in a shear-dependent manner, plasmin does not cleave at the Tyr1605-Met1606 ADAMTS13 proteolytic site in the A2 domain. Rather under shear stress conditions, or in the presence of denaturants, such as urea or ristocetin, plasmin cleaves the K1491-R1492 peptide bond within the VWF A1-A2 linker region. Finally, we demonstrate that VWF susceptibility to plasmin proteolysis at K1491-R1492 is modulated by local N-linked glycan expression within A1A2A3, and specifically inhibited by heparin binding to the A1 domain.
  
  
  
  Improved understanding of the plasmin-VWF interaction offers exciting opportunities to develop novel adjunctive therapies for the treatment of refractory thrombotic thrombocytopenic purpura.
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Brophy
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Soracha E
authors.initials: SE
authors.lastname: Ward
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Thomas R
authors.initials: TR
authors.lastname: McGimsey
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Sonja
authors.initials: S
authors.lastname: Schneppenheim
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Clive
authors.initials: C
authors.lastname: Drakeford
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Jamie M
authors.initials: JM
authors.lastname: O'Sullivan
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Alain
authors.initials: A
authors.lastname: Chion
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.).
authors.forename: Ulrich
authors.initials: U
authors.lastname: Budde
authors.affiliation: From the Haemostasis Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Trinity College Dublin, Ireland (T.M.B., S.E.W., T.R.M.G., C.D., J.M.O., A.C., J.S.O.); Medilys Laborgesellschaft mbH, Department of Hämostaseology, Hamburg, Germany (S.S., U.B.); National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland (J.S.O.); and Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, Dublin (J.S.O.). jamesodonnell@rcsi.ie.
authors.forename: James S
authors.initials: JS
authors.lastname: O'Donnell
mesh_terms: D001665:Binding Sites; D005341:Fibrinolysin / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis; D013314:Stress, Mechanical; D013329:Structure-Activity Relationship; D013997:Time Factors; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D014841:von Willebrand Factor; D006493:Heparin; D005341:Fibrinolysin
keywords: glycosylation; heparin; plasmin; shear; von Willebrand factor