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.
One of the key morphological changes associated with the nerve growth factor (NGF)-induced differentiation of rat adrenal pheochromocytoma (PC-12) cells is the growth of axon-like processes called neurites. A growing body of evidence suggests that this process may be dependent upon plasmin, a serine protease generated from plasminogen (Plg) by either urokinase Plg activator (u-PA) or tissue Plg activator (t-PA). Prior work in our laboratory has identified annexin II (Ann-II) as a coreceptor for Plg and t-PA that promotes and localizes plasmin generation near the cell surface. In the present study, we report a 3-9-fold increase in Ann-II protein and message levels in NGF-treated PC-12 cells. Message stability and nuclear run-on assays suggest that this induction occurs at the level of gene transcription. Neurite outgrowth assays on and within a three-dimensional matrix demonstrate the inhibition of NGF-induced PC-12 cell differentiation by polyclonal and monoclonal antibodies directed against Ann-II as well as by the overexpression of antisense Ann-II mRNA. Neuritogenesis is also impaired by ␣ 2 -plasmin inhibitor, antibodies directed against t-PA and u-PA, and ⑀-aminocaproic acid, a lysine analog that inhibits Plg activation and the binding of Plg to Ann-II. Plasmin generation assays reveal a 2-fold increase in plasmin production on NGF-treated PC-12 cells, which can be blocked by a polyclonal antibody directed against the tail region of Ann-II. From these data, we conclude that Ann-II is transcriptionally up-regulated by NGF and that Ann-II-mediated plasmin generation may play an important role during neurite development in the differentiating PC-12 cell.
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