硫酸皮肤素衍生物对炎性肠病患者血小板表面分子的影响

Effects of Dermatan Sulfate Derivatives on Platelet Surface Molecules in Inflammatory Bowel Disease Patients

目的研究硫酸皮肤素(DS)衍生物对炎性肠病(IBD)患者血小板表面P-选择素及活性蛋白C(APC)的影响。方法用氯磺酸磺化制备多硫酸化DS(PSDS)。测定其1H-NMR和13C-NMR光谱确定主要双糖单位。用过氧化氢降解DS和PSDS,所得片段用凝胶过滤色谱分离。用ELISA法测定DS衍生物对血小板表面P-选择素的影响,用底物显色法测定其对APC活性的影响。结果组成DS和PSDS链的主要双糖单位分别为IdoA-1→3-GalNAc-4-SO3和IdoA-2SO3-1→3-GalNAc4,6-diSO3。与二磷酸腺苷(ADP)组和IBD组相比,DS及其衍生物都具有抑制P-选择素表达的作用(P<0.01),但DS寡糖(DSOSs)和PSDS寡糖(PSDSOSs)没有差别。APC活性实验表明,DS及其衍生物均能提高APC活性。活性最强的DSOS是相对分子质量(Mr)为4 825的寡糖,APC活性由106.5%±11.5%升高到181.8%±22.3%(P<0.01)。随着Mr降低,DSOSs活性逐渐降低。PSDS提高APC活性的作用显著强于DS,使APC活性提高到205.2%±22.1%(P<0.01)。所有的PSDSOSs的活性都强于具有相当Mr的DSOSs活性。随着Mr降低PSDSOSs活性逐渐增强,活性最强的是Mr为2 749的PSDSOS3,APC活性提高到331.2%±27.8%(P<0.01),然后其活性逐渐降低。结论所有DS及其衍生物都有显著抑制血小板表面P-选择素表达的作用,但这种作用与DSMr分子量和硫酸化程度无关。DS及其衍生物对APC的作用在分子水平上具有复杂的机制,与DS的Mr、硫酸化程度以及非均一性成分有关。Mr相同时,DS硫酸化程度越高,APC活性提高作用越强。

Objective To study the influence of dermatan sulfate （DS） derivatives on platelet surface P-selectin and activated protein C （APC） of patients with inflammatory bowel disease （IBD）. Methods Polysulfated DS （PSDS） was prepared with chlorosulfonic acid and the main disaccharide units were determined with IH-NMR and 13C-NMR. DS and PSDS were degraded with n202 and separated with gel filtration chromatography. The influence of DS derivatives on platelet surface P-selectin was determined with ELISA and the effect on APC was determined by chromogenic substrate assay. Results The main polymer chains of DS and PSDS were nespectively composed of repeating disaccharide units of IdoA-1 -, 3-GalNAc-4-SO3 and IdoA-2SO3-1 -, 3-GalNAc4, 6-diSO3. Both DS and DS derivatives appeared to inhibit the expression of P-selectin compared with adenosine diphosphate （ADP） and IBD groups （P 〈 0.01）, while DS oligosaccharides （DSOSs） and PSDS oligosaccharides （PSDSOSs） didn＇t.The test with the activity of APC showed that DS and its derivatives could improve the activity of APC, and the DSOS with a Mr of 4 825 could most improve the activity of APC from 106.5 %±11.5 % to 181.8 % 22.3 % （P 〈 0.01 ）. The activity of DSOSs was decreased along with the reduction of M PSDS could make the activity of APC as high as 205.2 % ±22.1%（P 〈 0.01）, which was more than DS. The activity of PSDSOSs was increased with the reduction of Mr. The PSDSOS with a Mr of 2749 had the most activity and it could make the activity of APC as high as331.2 % ±27.8 % （P 〈 0.01）. Conclusion DS and its derivatives can apparently inhibit the expression of platelet surface P-selectin regardless of their Mr and extent of sulfation. The influence of DS and its derivatives on the activity of APC at molecular level relates to their Mr extent of sulfation and heterogeneity. With the same Mr, the more sulfation of DS, the more improved activity of APC.