Hepain Quality Control And The Generation Of A Novel Low Molecular Weight Heparin

Heparin and heparan sulfate are linear polysaccharides consisting of repeating disaccharide units of uronic acid–(1 4)-D-glucosamine. This disaccharide building block has varied structures with N-sulfo, O-sulfo and N-acetyl derivatives. The primary structure of the polysaccharide chain is complicated with apparently randomly organized sequences of these disaccharide units. Heparin was discovered in 1916 and has been used as a clinical anticoagulant since 1935. Heparin is second to insulin of the most popularly used natural medicine. China produces more than half of the total raw heparin used worldwide. A major contamination crisis, leading to the death of more than one hundred patients, has resulted in an increased interest in determining the structure of heparin. However, there is currently no way to directly characterize the structure and sequence of heparin. A project is underway with the goal of replacing animal sourced heparin with a bioengineered heparin. The structure of this bioengineered heparin must be the same as natural heparin. Current methods are not capable of analyzing heparin’s complicated sequence. Thus, a search for new methods of sequencing heparin is underway.This thesis introduces the properties of heparin and enzymes that act on heparin, the heparanases. Four aspects addressed in this thesis are: (1) the structure of heparin; (2) the characteristics of heparinases; (3) heparin digestion by heparinases; and (4) the generation of a novel low molecular weight heparin.The structure of heparin is complicated and there is no ideal method that can directly characterize heparin. Current studies on the structure and sequence of heparin relie on the digestion of heparin into different sizes of oligosaccharides using heparinases. The characteristic action pattern of heparinase 1 acting on heparin is discussed.There have been many studies on bacterial heparinases, also called heparin lyases. The work in this thesis expands these studies by examining the specificities and action patterns of heparanases. Many of the results presented within this thesis are similar to those of previous reports. It is interesting that heparinase 1 and heparinase 2 show identical action patterns on oligosaccharide substrates, particularly substrate-dependant exolytic action patterns. A new selectivity for heparinase 3 was observed. Heparinase 3 selectively digests certain carbohydrate linkages depending on their placement within an oligosaccharide chain.An additional major part of this thesis involves the digestion of 7 pharmaceutical heparins using heparanases. The resulting products were compared by qualitative and quantitative analysis using purified oligosaccharide standards. These heparin oligosaccharide mapping experiments could be used to distinguish the heparins of different origins. This new method can also be used to analyze the structure and sequence of synthetic heparin.On the basis of these studies on heparin structure and the characteristic action patterns and specificities of heparinases, a novel low molecular weight heparin was produced using heparinase 3.The potential value and importance of this complicated polysaccharide are discussed and new directions of heparin research are described.