TISSUE PLASMINOGEN ACTIVATOR BINDING TO ANNEXIN II TAIL DOMAIN DIRECT MODULATION

组织型纤溶酶原激活剂通过 钙调节磷脂结合蛋白膜联蛋白II， 由血栓前氨基酸抑制的相互作用 同型半胱氨酸 我们试图确定组织纤溶酶原激活剂 膜联蛋白II的结合结构域，并确定其机制， 通过同型半胱氨酸调节。 组织纤溶酶原激活剂结合 固定化膜联蛋白II被完整的液相膜联蛋白II抑制 而不是通过其“核心”片段（残基25-339）。 两个重叠 “尾”肽特异性阻断65-75%的结合。 定位 组织型纤溶酶原激活物结合域的表达得到证实， 通过六肽LCKLSL（残基7-12）的特异性抑制。 表达的C9 G膜联蛋白II蛋白不能支持组织纤溶酶原 激活剂结合，而与C133 G、C262 G和C335 G的结合是 相当于野生型膜联蛋白II。 在暴露于 同型半胱氨酸，膜联蛋白II经历了135 +/- 4-Da的质量增加 特异性定位于Cys 9，组织中60-66%的损失 纤溶酶原激活物结合能力。 在处理培养的 内皮细胞与[35 S]同型半胱氨酸，二硫苏糖醇敏感 通过用抗膜联蛋白II IgG免疫沉淀回收标记物。 这些数据提供了一个潜在的机制，促血栓形成的影响 同型半胱氨酸通过证明直接阻断组织 膜联蛋白II的纤溶酶原激活物结合结构域。 Hajjar，K.A.， 毛里湖Jacovina，A.T. Zhong，F.，米尔扎大学，Padovan，J.C.，查特， B.T. J. Biol. Chem. 1998，Apr 17 273（16）9987-9993，

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 Cys9 and a 60-66% loss in tissue plasminogen activator-binding capacity. Upon treatment of cultured endothelial cells with [35S]homocysteine, the dithiothreitol-sensitive 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. Hajjar, K.A., Mauri, L. Jacovina, A.T. Zhong, F., Mirza U.A., Padovan, J.C., Chait, B.T. J. Biol. Chem. 1998, Apr 17 273(16) 9987-9993,