Thrombospondin-1 and ADAMTS13 competitively bind to VWF A2 and A3 domains in vitro.
BACKGROUND
ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motif. 13) is the major metalloprotease for VWF degradation. ADAMTS13 deficiency causes the accumulation of uncleaved VWF and might lead to a lethal thrombotic thrombocytopenic purpura (TTP). Thrombospondin-1 (TSP1) is considered as a reductase of VWF (von Willebrand factor) which can mildly downregulate the size of VWF by targeting on disulfide bond between VWF dimers. It was reported that TSP1 might protected VWF from cleaving by ADAMTS13, yet the underlying mechanism of this VWF protection has remained unknown.


METHODS
Full-length ADAMTS13 and different domains (A1,A2,A3) of human VWF were constructed and expressed respectively. The binding ability of TSP1 or ADAMTS13 with each VWF domain or full-length VWF was investigated by using enzyme linked immunosorbent assay. The inhibition of ADAMTS13 activities by the different concentrations of TSP1 were observed by western blot and residual-collagen binding assay (R-CBA) under the denaturing condition.


RESULTS
We found that ADAMTS13 interacted with the rVWF A1, A2, A3 domains and full-length VWF, while TSP1 also bound to three A domains, especially to A2 and A3 domains. We observed that TSP1 partially blocked ADAMTS13 binding to A2 domain, A3 domain and full length VWF. The results of our assays showed that TSP1 could restrain ADAMTS13 activity up to 70%.


CONCLUSIONS
Our study suggested that TSP1 played competitively inhibitory role in ADAMTS13 binding and cleaving of VWF, and the potential competition might happen within A2 and A3 domains.
Wang
Anyou
A
Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China.
Liu
Fang
F
Dong
Ningzheng
N
Ma
Zhenni
Z
Zhang
Jingyu
J
Su
Jian
J
Zhao
Yiming
Y
Ruan
Changgeng
C
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019700:Thrombospondin 1 / Q000378:metabolism*; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D019700:Thrombospondin 1; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human