Joint Research for Heparin-Platelet Interaction

用合成方法证实了肝素中与血小板结合的关键序列(简称NS6S-I2S)。也就是说，含有NS6S-I2S的合成模型二糖显示出比肝素酶I消化的双糖更高的血小板亲和力，尽管两者每个分子含有相同数量的硫酸盐。目前正在使用肝素酶I消化的肝素和合成的寡糖以及核磁共振波谱结合分子建模来进一步评估肝素中与血小板结合的基本序列和聚簇或聚合物效应，并使用类似的结合竞争方法来确定肝素与von Willebrand因子的结合结构。有趣的是，相同的关键二糖(NS6S-I2S)被发现对结合现象至关重要。为了检测和分离完整血小板表面的肝素结合蛋白(S)，我们开发了一种新的异双功能光交联剂(AA-D)。该交联剂成功地标记了药用肝素和[~3H]-肝素，而不丧失肝素的抗凝、抗Xa活性。与卵蛋白相比，AA-D标记的[~3H]-肝素对抗凝血酶III具有高度特异性的交联力，这是通过最新的成像技术([~3H]-BAS系统)进行分析的。然后将该程序应用于检测和分离完整血小板表面的肝素结合蛋白。仅用[~3H]-BAS系统中的显像板照射3天，观察到约100和115 kDa的两条带，表明完整的血小板表面存在真正的肝素结合蛋白。目前正在对这些蛋白质进行提纯和氨基酸序列分析。

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.