Binding of von Willebrand factor cleaving protease ADAMTS13 to Lys-plasmin(ogen)

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• The metalloprotease ADAMTS13 is crucial for regulating platelet aggregation by cleaving von Willebrand factor (VWF) multimers.
• Understanding proteins that bind ADAMTS13 is key to uncovering mechanisms of microvascular thrombosis.

Purpose of the Study:

• To identify proteins that bind to ADAMTS13.
• To characterize the binding interaction between ADAMTS13 and its identified binding partners.

Main Methods:

• Yeast two-hybrid screening using specific domains of ADAMTS13 as bait.
• Domain truncation analysis of ADAMTS13 and plasminogen.
• Elastase digestion of plasminogen.
• Biacore assays for binding affinity measurements.
• Specific activity assays for protease function.

Main Results:

• Lys-plasminogen, but not intact Glu-plasminogen, was identified as an ADAMTS13-binding protein.
• The Cys-rich and spacer domains of ADAMTS13 and the kringle 5 and protease domains of plasminogen are key binding sites.
• Lys-plasminogen binds ADAMTS13 with a K(d) of 1.9 × 10(-7) M, significantly higher affinity than Glu-plasminogen.
• Both ADAMTS13 and Lys-plasmin retain activity after complex formation.

Conclusions:

• ADAMTS13 specifically binds Lys-plasminogen, suggesting a novel regulatory role.
• This interaction may localize ADAMTS13 and Lys-plasminogen to sites of thrombus formation or vascular injury.
• The binding facilitates the localization of these proteases within the activated fibrinolytic system during thrombosis.