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

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.