title: Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.
abstract: The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.
authors.affiliation: Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1, Tanabe-Dori, Mizuho-ku, Nagoya 467-8603, Japan. s-itoh@phar.nagoya-cu.ac.jp
authors.forename: Saotomo
authors.initials: S
authors.lastname: Itoh
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Yokoyama
authors.forename: Go
authors.initials: G
authors.lastname: Kamoshida
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Hiromi
authors.initials: H
authors.lastname: Okada
authors.forename: Takemasa
authors.initials: T
authors.lastname: Takii
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tsuji
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujii
authors.forename: Hideki
authors.initials: H
authors.lastname: Hashizume
authors.forename: Kikuo
authors.initials: K
authors.lastname: Onozaki
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000276:immunology* / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D001777:Blood Coagulation / Q000276:immunology; D002118:Calcium / Q000276:immunology / Q000378:metabolism; D003030:Coagulase / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D011516:Prothrombin / Q000276:immunology* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000276:immunology* / Q000378:metabolism; D018089:Superantigens / Q000276:immunology* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013552:Swine; D013917:Thrombin / Q000276:immunology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003030:Coagulase; D007106:Immune Sera; D018089:Superantigens; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D013917:Thrombin; C022654:staphylothrombin; D015951:Factor Xa; D002118:Calcium
keywords: Bacterial Toxins; Blood Coagulation Factors; Coagulation Factors; Prothrombin; Staphylococcal Superantigen-like Protein; Staphylococcus aureus