Force-induced cleavage of single VWFA1A2A3 tridomains by ADAMTS-13.
A disintegrin and metalloprotease with a thrombospondin type 1 motifs 13 (ADAMTS-13) regulates hemostasis by cleaving the folded A2 domain of von Willebrand factor (VWF). The cleavage is regulated by 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 Ibalpha or antibodies to measure time, distance, and force. Structural destabilization of A1A2A3 was induced by 5- to 80-pN forces, manifesting as an abrupt molecular length increase distributed around 20 and 50 nm, probably because of 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 seconds at 20pN, then decreased (slip) with increasing force, independent of ADAMTS-13. The time required to rupture A1A2A3 exhibited a similar catch-slip behavior when pulled by glycoprotein Ibalpha but only slip behavior when pulled by antibody, which was progressively shortened by increasing concentration of ADAMTS-13 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 k(cat) of 2.9 (+/- 59) seconds and a K(d) 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.
Wu
Tao
T
Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Lin
Jiangguo
J
Cruz
Miguel A
MA
Dong
Jing-fei
JF
Zhu
Cheng
C
D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008956:Models, Chemical; D008958:Models, Molecular; D019038:Platelet Glycoprotein GPIb-IX Complex; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008562:Membrane Glycoproteins; D019038:Platelet Glycoprotein GPIb-IX Complex; C094990:adhesion receptor; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human