Regular Article Thrombospondin-1 and ADAMTS13 competitively bind to VWF A2 and A3 domains in vitro Anyou Wang a , Fang Liu b , Ningzheng Dong a , Zhenni Ma a , Jingyu Zhang a , Jian Su a , Yiming Zhao a , Changgeng Ruan a, ⁎ 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 b Department of Medicine, Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, 63110, USA abstract article info Article history: Received 16 December 2009 Received in revised form 13 July 2010 Accepted 14 July 2010 Available online 11 August 2010 Keywords: Thrombospondin-1 von Willebrand factor ADAMTS13 Introduction: 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. Materials and 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. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved. Introductions Von Willebrand factor (VWF) is, one of largest multimeric glycoprotein in peripheral blood, synthesized and secreted from endothelial cells. It plays a very important role in blood coagulation and primary hemostasis by carrying coagulation factor VIII and initiating platelets adhesion. A mature VWF monomer has 2050 amino acids and contains 5 domains in the order D’-D3-A1-A2-A3-D4- B1-B2-B3-C1-C2-CK. Investigations on the structure and function of VWF disclose that multiple binding sites within VWF A domains contribute to VWF's hemostatic function. VWF captures circulating platelets through the interaction between A1 domain and platelet GPIb/IX/V receptors complex on one hand, and on the other hand via A3 domain binding to collagen in the subendothelium matrix [1–3]. High shear stress caused by blood flow unfolds VWF and fully exposes these binding sites in order to facilitate the formation of VWF-rich platelets thrombus. The major regulation of this kind of thrombus formation is conducted by a VWF specific metalloprotease, ADAMTS13, which cleaves VWF into two smaller and less adherent multimers at the Y1605-M1606 peptidyl bond in VWF A2 domain, thus to prevent the thrombus formation [4–6]. A severe deficiency of ADAMTS13 activity due to congenital or acquired reasons causes ultra-large VWF increasing in peripheral blood, and high risk of a lethal disease, thrombotic thrombocytopenic purpura (TTP) [7,8]. Recently studies show that ADAMTS13 cleave VWF after directly interacting with some potential binding sites (Residues 1641-1659, residues 1597-1623, residue 1609-1623) within A2 domain [9,10]. Besides, A1 and A3 domains also are considered as the docking site of ADAMTS13, especially A3 domain shows high affinity to ADAMTS13 bead under flow condition [11]. Since the interactions between ADAMTS13 and VWF are so important that any protein inhibits ADAMTS13 binding to VWF might influence the ADAMTS13 activity. According to Pimanda's report, thrombospondin-1 (TSP1) binds to A3 domain like ADAMTS13 does, but its relationship with ADAMTS13 Thrombosis Research 126 (2010) e260–e265 Abbreviations: ADAMTS13, a disintegrin-like and metalloprotease with thrombospon- din type 1 repeats motif. 13; TSP1, thrombospondin-1; TTP, thrombotic thrombocytopenic purpura; VWF, von Willebrand factor; R-CBA, residual-collagen binding assay. ⁎ Corresponding author. Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China. Tel./fax: +86 512 65123356. E-mail address: changgengruan@hotmail.com (C. Ruan). 0049-3848/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.07.009 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres