Binding of von Willebrand factor cleaving protease ADAMTS13 to Lys-plasmin(ogen).
The metalloprotease ADAMTS13 affects platelet adhesion and aggregation through depolymerization of von Willebrand factor (VWF) multimers. Identification of ADAMTS13-binding proteins would reveal the hitherto unrecognized mechanisms underlying microvascular thrombus. To identify ADAMTS13-binding proteins, we performed a yeast two-hybrid screen using the Cys-rich and spacer domains of ADAMTS13, the critical regions for the binding and cleavage of VWF, as a bait region. We identified Lys-plasminogen, an amino-terminal truncated form of plasminogen, as the binding protein to ADAMTS13. Intact Glu-plasminogen did not bind to ADAMTS13. Active-site blocked Lys-plasmin bound to ADAMTS13. Domain truncation of ADAMTS13 and elastase digest of plasminogen indicated that the Cys-rich and spacer domains of ADAMTS13 and the kringle 5 and protease domains of plasminogen served as the main binding sites. Biacore measurements revealed that Lys-plasminogen bound to ADAMTS13 with a K(d) of 1.9 ± 0.1 × 10(-7) M and Glu-plasminogen exhibited a significantly lower affinity to ADAMTS13. Specific activity measurements revealed that ADAMTS13 and Lys-plasmin were still active even after the binary complex was formed. The binding of ADAMTS13 to Lys-plasminogen may play an important role to localize these two proteases at sites of thrombus formation or vascular injury where the fibrinolytic system is activated.
Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, 5-7-1 Fujishirodai, Suita, Osaka 5658565, Japan. shin@cc.kogakuin.ac.jp
Yongchol
Y
Shin
Masashi
M
Akiyama
Koichi
K
Kokame
Kenji
K
Soejima
Toshiyuki
T
Miyata
D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000818:Animals; D005341:Fibrinolysin / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D034421:Sus scrofa; D020798:Two-Hybrid System Techniques; D014841:von Willebrand Factor / Q000378:metabolism*
D055767:Immobilized Proteins; D050505:Mutant Proteins; D010446:Peptide Fragments; C018998:lysyl-plasminogen; D014841:von Willebrand Factor; D010958:Plasminogen; D005341:Fibrinolysin; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human