| Heparin, a sulfated polysaccharide, belonging to the family of glycosaminoglycans, is present on the surface of mammalian cells in large quantities. Heparin has numerous important biological activities, associated with its interaction with diverse proteins. It is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade.Due to the structural diversity and molecular weight polydispersity of heparin, it may has narrow therapeutic window, low bioavailability, short half-life. In addition, heparin can cause bleeding and induced thrombocytopenia (HIT), osteoporosis and other side effects. These defects limit the clinical application of heparin. Therefore, low molecular weight heparin (LMWH) was developed as a new anticoagulant and antithrombotic drug,. These drugs have advantages of short chains, less binding sites, high bioavailability, long half-life, and a good medical outlook.This paper studied on the preparation of low molecular weight heparin. By (3-elimination method, we studied on preparation route of LMWH. Though changing the reaction time and controlling of the degradation time, the reaction of esterification rate was optimized. When the reaction time was 25 hours, the esterification rate was 13.97% and the range of molecular weight from 2000Da to 8000Da accounted for 71.1%. When the degradation time was 1.5 hours, the range of molecular weight from 2000Da to 8000Da accounted for 66.5%.As an anticoagulant drugs, low molecular weight heparin have many advantages.But in the preparation process, some antithrombin-binding pentasaccharides, which is essential for anticoagulant activity, can be destroyed. With N-,6-O-and 3-O-sulfo groups of the pentasaccharide heparin can bind with AT-â…¢, and demonstrate anticoagulant activity.The 3-O-sulfo group is believed to be more important for the specific intereaction with AT-â…¢. Therefore, this paper focuses on the increased activity of low molecular weight heparin modified by enzyme reaction.By the means of 3-O-sulfo group modified, it is can increase the level of sulfation and the quantity of antithrombin-binding pentasaccharide. Two classes enzymes would be used in the reaction of modification.One class is 3-O-sulfotransferase (3-OST-I) and the other is aryl sulfotransferase (AST-IV). These two recombinant plasmids were transformed into E.coliBL21 (DE3), and they was expressed as a fusion and purified using a Ni-NTA Agarose Fast Flow. By the determination of enzyme activity, the specific activity of the enzyme can be determined.By chromogenic substrate method, the anti-FXa activity of modified low molecular weight heparin was determined. While it was controlled with the unmodified low molecular weight heparin.After determination of FXa activity percent, the low molecular weight heparin semi-inhibition rate can be calculated.As the results showed that comparation of unmodified and modified Fragmin, Innohep and Lovenox, IC50 values were decreased by 3.3,8.7 and 2.4 times. It was indicated that the anticoagulantant activity was greatly enhanced.Finally, through the degrataion of Heparinase we discussed the oligosaccharide fragment distribution of modified heparin. In the excess amount of enzyme, heparin can be completely degraded to disaccharide.But after 3-OST-I modified the glucosidic bond between glucosamine sulfate and 3-O-sulfo glucuronic acid can not be cut off by heparinase. The result of experiment was the ratio of tetrasaccharide of modified heparin was increased by 8.2 times. Thus we can conclude that the modified heparin has more antithrombin-binding pentasaccharides than that of unmodified heparin.
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