Tissue Plasminogen Activator Binding to the Annexin II Tail Domain

Abstract

Tissue plasminogen activator binds to endothelial cells via the calcium-regulated phospholipid-binding protein annexin II, an interaction that is inhibited by the prothrombotic amino acid homocysteine. We sought to identify the tissue plasminogen activator binding domain of annexin II and to determine the mechanism of its modulation by homocysteine. Tissue plasminogen activator binding to immobilized annexin II was inhibited by intact fluid phase annexin II but not by its "core" fragment (residues 25-339). Two overlapping "tail" peptides specifically blocked 65-75% of binding. Localization of the tissue plasminogen activator binding domain was confirmed upon specific inhibition by the hexapeptide LCKLSL (residues 7-12). Expressed C9G annexin II protein failed 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 increase in mass localizing specifically to Cys 9 and a 60 -66% loss in tissue plasminogen activator-binding capacity (I 50 ‫؍‬ 11 M). Upon treatment of cultured endothelial cells with [35 S]homocysteine, the dithiothreitolsensitive label was recovered by immunoprecipitation with anti-annexin II IgG. These data provide a potential mechanism for the prothrombotic effect of homocysteine by demonstrating direct blockade of the tissue plasminogen activator binding domain of annexin II.

Annexin II Is Present on Renal Epithelial Cells and Binds Calcium Oxalate Monohydrate Crystals

“…Although Ax-V and Ax-II share similar calcium-binding motifs, as do all members of the annexin family (35), they differ in other important aspects, most notably in the composition of the NH 2 -terminus. Ax-II has been demonstrated on the surface of many cell types, where it can serve receptor-like functions for molecules, including Tissue Plasminogen Activator (37). In addition, the NH 2 -terminus of Ax-II can bind a p11 dimer and become linked to another Ax-II molecule via its NH 2 tail, a process that can mediate aggregation of membrane vesicles (35).…”

Factors Affecting S-Homocysteinylation of LDL Apoprotein B

“…These Hcy-mediated posttranslational modifications may have important functional consequences. In vitro studies have shown that homocysteinylation of the Cys 9 residue of annexin II, the endothelial cell surface docking protein for tissue plasminogen activator, inhibits binding (26 ). Hcy also binds the fibrinbinding domain of plasma fibronectin in vitro, inhibiting its ability to bind fibrin (24 ), and binds factor Va in vitro, making it resistant to inactivation by activated protein C (27 ).…”

Serine Protease Inhibitors: Novel Therapeutic Targets for Stroke?

“…1). Although there is no clear evidence of an obligatory interaction between t-PA and a membrane-bound receptor to initiate proteolysis, t-PA can interact with the cell surface by means of the cal cium-regulated phospholipid-binding protein annexin II (Hajjar et a!., 1998). The expression of annexin II has been detected in the developing murine CNS (Hamre et a!., 1995), but its distribution in the adult brain remains to be established.…”