Anticoagulant therapy by injection of heparin, a naturally occurring glycosaminoglycan is widely used to prevent pulmonary emboli or postoperative deep vein thrombosis. Due to heterogeneity in commercial heparins and its adverse side effects such as bleeding, thrombocytopenia and osteoporosis attempts have been made to find new antithrombic agents. A pentosan polysulfate, a sodium salt of polysulfated xylan (SP-54) marketed by Benechemie, Munich, West Germany is currently used in Europe as a replacement for heparin. This compound is not as effective as heparin and its mechanism of action remains obscure. Studies in our laboratory have shown that xylan sulfates prepared by sulfation of xylans isolated from plant materials are effective anticoagulants both in vivo and in vitro. The LD50's of these compounds were similar to heparin and were hundred fold higher than the effective dosage suggesting a wide margin of safety in clinical application. This proposal is concerned with studies on the mechanism of anticoagulant action of sulfated xylans and will include the following: 1) Kinetic analysis of antithrombin III (AT-III)-thrombin or heparin cofactor II (HC-II)-thrombin interactions in absence of or in the presence of sulfated xylans. 2) Determination of binding constants for the interactions of sulfated xylans with AT-III by studying the fluorescence enhancement due to perturbation of key tryptophan residue of AT-III or by following the enhancement of specific bands of the aromatic amino acids in the circular dichroism (CD) spectra of AT-III in the near-UV region. 3) Studies of the complexation of 125I-thrombin with AT-III or HC- II in presence of sulfated xylans using ex-in vivo human plasma. 4) A comparison of anticoagulant properties of xylan sulfates in control human plasma versus AT-III depleted plasma. 5) Determination of the binding sites of xylan sulfates with AT-iii, thrombin or HC-II using protein modification reagents. The proposed study will give an insight into the mechanism by which sulfated xylans modify coagulation and may lead to the development of a selective anticoagulant.
