Joint Research for Heparin-Platelet Interaction

通过人工合成方法确认肝素与血小板结合的关键二氯化序列（简称NS6S-I2S）。也就是说，含有NS6S-I2S的合成模型双糖比肝素酶i消化的双糖表现出更高的血小板亲和力，尽管它们每个分子含有相同数量的硫酸盐。目前正在利用肝素酶I消化和合成的寡糖，以及核磁共振波谱结合分子模型，进一步评估肝素与血小板结合的必需序列，以及评估聚类或聚合物效应。采用类似的结合竞争方法确定肝素中血管性血友病因子的结合域结构。有趣的是，同样的关键双糖（NS6S-I2S）被发现对这种结合现象至关重要。为了检测和分离完整血小板表面的肝素结合蛋白，我们研制了一种新的异双功能光交联试剂（AA-D）。交联剂成功地标记了药物肝素以及[^3H] -肝素，而不失去肝素的抗凝抗xa活性。与卵清蛋白相比，AA-D标记的[^3H] -肝素对抗凝血酶III具有高度特异性的交联能力，采用最新的成像技术（[^3H] -BAS系统）进行了分析。然后将该方法应用于完整血小板表面肝素结合蛋白的检测和分离。仅在[^3H] -BAS系统中使用成像板暴露3天，就观察到约100和115 kDa的2条条带，表明完整的血小板细胞表面存在真正的肝素结合蛋白。这些蛋白质现在正在被纯化和分析它们的氨基酸序列。

The key disaachride sequence (abbreviated as NS6S-I2S) in heparin for the binding to platelets was confirmed by the synthetic approach. That is the synthetic model disaccharide containing NS6S-I2S demonstrated a higher platelet affinity than the heparinase I-digested disaccharide although they both contain the same number of sulfates per molecule.Further evaluation for the determination of essential sequence in heparin for the binding to platelets and evaluation of the clustering or polymer effect are now under examination using heparinase I digested and synthetic oligosaccharides, as well as NMR spectroscopy combined with molecular modeling.The similar binding competitive approach was applied to determine the binding domain structure in heparin for von Willebrand factor. Interestingly, the same key disaccharide (NS6S-I2S) was found to be crucial for the binding phenomena.To detect and isolate the heparin-binding protein (s) on the intact platelet surface, we have developed a new heterobifunctional photo-crosslinking reagent (AA-D). The cross-linker was succeeded to label pharmaceutical heparins as well as the [^3H] -heparin without losing heparin's anticoagulant anti-Xa activity. The AA-D labelled [^3H] -heparin possessed highly specific cross-linking potency to antithrombin III compared with ovalbumin, which was analyzed by the newest imaging technology ([^3H] -BAS system). This procedure were then applied to the detection and isolation of heparin-binding proteins on the surface of intact platelets. By only a three-days exposure using a imaging plate in the [^3H] -BAS system, 2 bands at about 100 and 115 kDa were observed, suggesting real heparin binding proteins on the intact platelet cell surfaces. These proteins are now being purified and analyzed for their amino acid sequence.