Binding of ADAMTS13 to von Willebrand factor.
ADAMTS13, a metalloprotease, cleaves von Willebrand factor (VWF) in plasma to generate smaller, less thrombogenic fragments. The interaction of von Willebrand factor with specific ADAMTS13 domains was characterized with a binding assay employing von Willebrand factor immobilized on a plastic surface. ADAMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was approximately 4 h. Binding to von Willebrand factor was similar with either recombinant ADAMTS13 or normal plasma ADAMTS13; plasma from a patient who lacked ADAMTS13 activity showed no binding. The stoichiometry of binding was one ADAMTS13 per two von Willebrand factor monomers, and the K(d) was 14 nm. The ADAMTS13 metalloprotease and disintegrin domains did not bind VWF detectably. ADAMTS13 truncated after the first thrombospondin type 1 repeat bound VWF with a K(d) of 206 nm, whereas ADAMTS13 truncated after the spacer domain had a K(d) of 23 nm, which is comparable with that of full-length ADAMTS13. Truncation after the eighth thrombospondin type 1 repeat reduced the binding affinity by approximately 3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by approximately 2-fold. Therefore, the spacer domain is required for ADAMTS13 binding to von Willebrand factor. The first thrombospondin repeat also affects binding, and the C-terminal thrombospondin type 1 and CUB domains of ADAMTS13 may modulate this interaction.
Majerus
Elaine M
EM
Department of Medicine and Howard Hughes Medical Institute, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
Anderson
Patricia J
PJ
Sadler
J Evan
JE
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019699:Thrombospondins / Q000737:chemistry; D013997:Time Factors; D014841:von Willebrand Factor / Q000378:metabolism*
D007074:Immunoglobulin G; D011994:Recombinant Proteins; D019699:Thrombospondins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human