INTERACTION BETWEEN YERSINIA-PESTIS YOPM PROTEIN AND HUMAN ALPHA-THROMBIN

YopM, a 41.5 kDa virulence protein of Yersinia pestis is believed to h ave an antiinflammatory role in bubonic plague. It has been shown prev iously that YopM binds human alpha-thrombin but not prothrombin and in hibits thrombin-induced platelet aggregation in vitro. In the present studies we carried out crosslinking reactions between purified YopM an d alpha-thrombin or its blocked form FPR-alpha-thrombin in the presenc e of various competitors to identify where on thrombin YopM binds. We found that thrombin cleaves YopM at the C-terminus, indicating that th is part of YopM must interact with thrombin's catalytic site. Hirudin, a 65 amino acid natural thrombin inhibitor, prevents both the YopM de gradation and the formation of a ca. 75 kDa crosslinking complex betwe en YopM and alpha-thrombin. A similar effect is observed when hirugen, a short peptide corresponding to hirudin's C-terminus (amino acids 58 -64), is used as a synthetic thrombin inhibitor. A 15 bp long specific oligonucleotide known to block alpha-thrombin successfully competes w ith YopM for the thrombin-binding site, whereas a control, scrambled s equence aptamer does not. As these competitors block a fibrinogen bind ing site (also called anion binding exosite I), our crosslinking data indicate that YopM binds not only to the active site of alpha-thrombin but also to the abeI.