Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their gamma-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10 beta 1-beta 3 ((MEMKN)-M-23 ISALK HGKNN LRFKF RGIKI QVL(60)) bound to immobilized pro thrombin, and mutants that contained ssu10 beta 10-beta 12 ((SFYNL)-S-174 DLRSK LKFKY MGEVI ESKQI KDIEV NLK207) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to pro thrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants. (C) 2017 Elsevier Inc. All rights reserved.