Cleavage of von Willebrand factor by ADAMTS-13 in vitro: Effect of temperature and barium ions on the proteolysis kinetics

Paolo Perutelli, Stefano Amato, Angelo C. Molinari

Research output: Contribution to journalArticlepeer-review

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°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°C, in the presence or absence of barium ions, by measuring the binding of VWF to collagen. Complete VWF proteolysis was observed at 37°C in the presence of BaCl2, while about 25% VWF still bound to collagen when BaCl 2 supplementation was omitted. Proteolysis kinetics at 22 and 4°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.

Original languageEnglish
Pages (from-to)607-611
Number of pages5
JournalBlood Coagulation and Fibrinolysis
Volume16
Issue number8
Publication statusPublished - Nov 2005

Keywords

  • ADAMTS-13
  • Metalloprotease
  • von Willebrand factor
  • von Willebrand factor-cleaving protease

ASJC Scopus subject areas

  • Hematology

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