TISSUE-PLASMINOGEN ACTIVATOR BINDING TO THE ANNEXIN II TAIL DOMAIN - DIRECT MODULATION BY HOMOCYSTEINE

Tissue plasminogen activator binds to endothelial cells via the calciu m-regulated phospholipid-binding protein annexin II, an interaction th at is inhibited by the prothrombotic amino acid homocysteine. We sough t to identify the tissue plasminogen activator binding domain of annex in II and to determine the mechanism of its modulation by homocysteine . Tissue plasminogen activator binding to immobilized annexin II was i nhibited by intact fluid phase annexin II but not by its ''core'' frag ment (residues 25-339). Two overlapping ''tail'' peptides specifically blocked 65-75% of binding. Localization of the tissue plasminogen act ivator binding domain was confirmed upon specific inhibition by the he xapeptide LCKLSL (residues 7-12). Expressed C9G annexin II protein fai led to support tissue plasminogen activator binding, while binding to C133G, C262G, and C335G was equivalent to that of wild type annexin II . Upon exposure to homocysteine, annexin II underwent a 135 +/- 4-Da i ncrease in mass localizing specifically to Cys(9) and a 60-66% loss in tissue plasminogen activator-binding capacity (I-50 = 11 mu M). Upon treatment of cultured endothelial cells with [S-35]homocysteine, the d ithiothreitol-sensitive label was recovered by immunoprecipitation wit h anti-annexin II IgG. These data provide a potential mechanism for th e prothrombotic effect of homocysteine by demonstrating direct blockad e of the tissue plasminogen activator binding domain of annexin II.