The fine structure of the glycosaminoglycan heparin is to be characterized by a range of chemical, physical, and enzymological techniques. These will include enzymic and chemical degradation of both free and bound heparins, high-field NMR spectroscopy, mass spectrometry, and X-ray crystallography. Heparin acts biologically in anti-thrombotic, anti-coagulant, and anti-lipemic roles. The present studies will concentrate on those aspects of the molecule crucial to the thrombin--antithrombin III (AT-III) interaction. Heparin is a heterogeneous proteoglycan whose gross polysaccharide repeating-unit consists of a highly sulfated, uronic acid--hexosamine disaccharide. This disaccharide shows high structural variability within the heparin chain and its specific topology dictates the binding to certain proteins through which heparin exerts its effect. Gross structure, functionality, and conformational aspects of this unit may all be significant in the mode of action of heparin. This investigation will focus, therefore, on the fine structure as well as the secondary and tertiary conformations within the thrombin and AT-III binding-regions of heparin preparations that show high anti-thrombin activity. Some of the heparin used will be commercial material, but most will be isolated from fresh tissues via a mild, non-degradative technique developed in this laboratory. This information will be of value for development of new heparionoids from readily available, inexpensive starting materials. This project will add new information to enhance our understanding of the mechanisms of action of heparin. Heparin is important in anticoagulant, antithrombic, and anti-lipemic therepy and may also be useful in the prophylaxis of atherosclerosis. The increased in cost of heparin and the fluctuation in its availability has placed a high priority on the search for an accessible non-toxic, synthetic replacement. The basis for understanding heparin and its role must necessarily be the unravelling of the structure--function relationships in native heparin, including elucidation of the fine structure and secondary and tertiary conformations of the active regions of heparin.
