title: Cleavage of von Willebrand factor by ADAMTS-13 in vitro: effect of temperature and barium ions on the proteolysis kinetics.
abstract: The multimeric size of von Willebrand factor (VWF) is regulated by the specific cleaving metalloprotease, ADAMTS-13. Laboratory assays for ADAMTS-13 are useful for identifying severe protease-deficient activity. ADAMTS-13 activity is currently assayed by prolonged dialysis of plasma at 37 degrees C in low-ionic-strength denaturing buffer. We investigated the effect of temperature and divalent cation supplementation on the kinetics of VWF proteolysis by ADAMTS-13 in vitro. Proteolysis was monitored for 24 h at 37, 22, and 4 degrees C, in the presence or absence of barium ions, by measuring the binding of VWF to collagen. Complete VWF proteolysis was observed at 37 degrees C in the presence of BaCl2, while about 25% VWF still bound to collagen when BaCl2 supplementation was omitted. Proteolysis kinetics at 22 and 4 degrees C was slower but complete, even in the absence of added barium. A subphysiological temperature might influence the proteolysis kinetics by determining minor variations of the ADAMTS-13 structure, or further modification of the VWF substrate. We describe a simple procedure to analyse the kinetics of VWF proteolysis that is suitable for routine diagnostic use. Furthermore, we offer new insight into the biochemistry of ADAMTS-13.
authors.affiliation: Hemostasis and Thrombosis Unit, Hematology and Oncology Department, IRCCS G. Gaslini, Genova, Italy. paoloperutelli@ospedale-gaslini.ge.it
authors.forename: Paolo
authors.initials: P
authors.lastname: Perutelli
authors.forename: Stefano
authors.initials: S
authors.lastname: Amato
authors.forename: Angelo C
authors.initials: AC
authors.lastname: Molinari
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000172:deficiency; D000071120:ADAMTS13 Protein; D017609:Barium Compounds / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D014841:von Willebrand Factor / Q000737:chemistry*
chemical_list: D017609:Barium Compounds; D002712:Chlorides; D007477:Ions; D014841:von Willebrand Factor; C024986:barium chloride; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human