Preparation Of Heparin Oligosaccharides In Two Different Techniques And Their Effects On HUVEC

Heparin, a sulfated polysaccharide, is a member of glycosaminoglycan family with heterogeneous structure and highly dispersed polymerization degree. In structure heparin is alternatively interlinked by uronic acid (iduronic acid and glucuronic acid) and glucosamine (including its derivatives) through 1,4-glycosidic bond. Furthermore, the major repeating disaccharide unit in heparin is -α-L-IdoA-2-SO₄-(1→4)-α-D-GlcNSO₃-6-SO₄-, which is also called trisulfated disaccharide unit. The mean relative molecular weight (M_r) of heparin ranges from 3 kDa to 30 kDa.Thrombosis is defined as a pathological process that some blood components formed embolus in the vessels, resulting in partially or completely vessel block, consequently blocks blood supply in certain circumstances. Thrombosis can be divided into arterial thrombosis and venous thrombosis according to their forming position. Comparatively, thrombo-embolism is a pathological process occurred when the thrombus fall off the vessel and cause blockage of blood flow if some vessels, resulting in corresponding organizations and (or) organ ischemia, hypoxia, necrosis (arterial thrombosis) and edema (venous thrombosis).It is traditionally accepted that thrombosis and coagulation both include two pathways, endogenous and exogenous. Tissue factor (TF), the extrinsic coagulation pathway initiating factor, can combine Fâ…§to form TF/â…¦a complex, activating the tissue factor pathway (also called exogenous coagulation pathway). This process generally occurs on the surface of vascular endothelial cells. In addition, TF also plays an important role in intrinsic coagulation pathway. Tissue factor pathway inhibitor (TFPI) is a new anticoagulant protein, the only active physiological substance that can inhibit the TF activity currently known. Thrombomodulin (TM) is a glycoprotein located on the surface of endothelial cell membrane, and playing a significant role in anticoagulation mechanisms via combining thrombin to form thrombin-thrombomodulin complex, and activate protein C to promote fibrinolysis and anticoagulant effects. The study of TF, TFPI, and TM has become a new hot spot in researching the coagulation mechanism, especially in studies of a variety of thrombotic diseases such as cardiovascular and cerebrovascular diseases, where great attention is particularly paid to.As a drug to prevent and control anticoagulant and thrombotic disease, heparin has a history of 70 years in clinical application. Its major anticoagulant effect is achieved by activating ATâ…¢. In addition, clinical research shows that in the treatment of thrombosis heparin also causes side effects such as bleeding and platelet reducing, whereas low molecular weight heparin oligosaccharides could reduce or eliminate these side effects.Heparin oligosaccharides are degraded heparin fragments with the average molecular weight between 1 kDa and 4 kDa. The current method of degrading heparin mainly consists of physical separation and chemical degradation. Chemical degradation method mainly includes nitrite degradation andβ-elimination. In order to further study antithrombotic mechanism of heparin oligosaccharides,β-elimination technique and improved nitrite degradation technique were both applied to prepare heparin oligosaccharides with unfractioned heparin, followed by Superdex 30 gel column chromatography to obtain graded oligosaccharide components for structure determination. The anti-FXa activity of separated heparin oligosaccharide components were also tested based on general pharmacology in this study. The effects of each molecular heparin oligosaccharide component on TF, TFPI, and TM secretion in HUVEC were determined, thus discussed the antithrombotic activity basis and mechanism of heparin oligosaccharides on molecular level.The results and conclusions obtained in this study are shown as follows. 1. Heparin oligosaccharides prepared byβ-elimination and nitrite degradation techniques, followed by Superdex 30 gel column chromatography separation and desalination, were demonstrated of high purity via polyacrylamide gel electrophoresis, thus obtaining relatively pure components of NO-1, NO3, NO-4, NO-5,β-3,β-4,β-5,β-6.2. The relative molecular weight of the fractioned components was determined by HPLC.3. The heparin molecular structures of major oligosaccharidesβ-5,β-6, NO-4, and NO-5 were determined by a combined analyzing approach of UV, infrared spectroscopy (IR), and nuclear magnetic resonance spectroscopy (1H-NMR, 13C-NMR).4. Anti-FXa activity of each oligosaccharide components was detected.5. Effects of oligosaccharide fractions on TF, TFPI, and TM secretion in HUVEC were also determined. The results show that, (1) Heparin oligosaccharides prepared by the mentioned two techniques can both greatly inhibit TF increasing in HUVEC induced by LPS, and the smaller the molecular weight, the stronger the inhibitory activity is. (2) Heparin oligosaccharides prepared by the mentioned two techniques can both increase TFPI secretion in HUVEC, and the smaller the molecular weight, the stronger the boosting activity is. (3) Heparin oligosaccharides prepared by the mentioned two techniques both increase TM expression in HUVEC, however, no significant enhancing difference was found between different molecular weights.