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Blood First Edition Paper, prepublished online November 6, 2009; DOI 10.1182/blood-2009-03-210369. This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Google Scholar Articles by Wu, T. Articles by Zhu, C. PubMed PubMed Citation Articles by Wu, T. Articles by Zhu, C. Social Bookmarking                   What's this?  Previous Article  |  Next Article  Submitted March 12, 2009; accepted October 8, 2009. Force-induced cleavage of single VWF A1A2A3-tridomains by ADAMTS-13 Tao Wu1, Jiangguo Lin2, Miguel A. Cruz3, Jing-Fei Dong3 and Cheng Zhu4,4 1 Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, United States; 2 Institute of Biomechanics and School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China; 3 Cardiovascular Sciences-Thrombosis, Department of Medicine, Baylor College of Medicine, Houston, TX, United States; 4 Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, United States * Corresponding author; email: cheng.zhu{at}bme.gatech.edu Abstract A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs 13 (ADAMTS-13) regulates hemostasis by cleaving a cryptic peptide bond inside the folded A2 domain of von Willebrand factor (VWF). This cleavage is regulated mechanically by hemodynamic forces as it occurs in flowing blood. We tested the hypothesis that force-induced A2 domain unfolding facilitates cleavage using atomic force microscopy to pull single VWF A1A2A3-tridomain polypeptides by platelet glycoprotein (GP) Ib or antibodies to measure time, distance, and force. Structural destabilization of A1A2A3 was induced by 5-80 pN forces, manifesting as an abrupt molecular length increase distributed around 20 and 50 nm, likely due to uncoupling A1A2A3 (or partially unfolding A2) and fully unfolding A2, respectively. Time required to destabilize A1A2A3 first increased (catch), reaching a maximum of 0.2 s at 20 pN, then decreased (slip) with increasing force, independent of ADAMTS-13. Time required to rupture A1A2A3 exhibited similar catch-slip behavior when pulled by GPIb but only slip behavior when pulled by antibody, which was progressively shortened by increasing concentration of ADAMTS-13 and by decreasing force after (but not before) structural destabilization, indicating that cleavage of A2 requires the force-induced A2 unfolding. Analysis with a model for single-substrate trimolecular enzymatic kinetics estimated a cleavage rate kcat of 2.9 ± 0.59 s-1 and a Kd of 5.6 ± 3.4 nM for ADAMTS-13/A1A2A3 binding. These findings quantify the mechanical regulation of VWF cleavage by ADAMTS-13 at the level of single A1A2A3-tridomain. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

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