LOW-MOLECULAR-WEIGHT DERMATAN SULFATE AS AN ANTITHROMBOTIC AGENT - STRUCTURE-ACTIVITY RELATIONSHIP STUDIES

A structure-activity relationship of low molecular weight dermatan sul fate was undertaken to understand better this new non-heparin, glycosa minoglycan-based antithrombotic agent. A dermatan sulfate prepared fro m bovine intestinal mucosa [average molecular weight (MWavg) 25,000], and currently in clinical trials as an antithrombotic agent, was used in this study. Dermatan sulfate was partially depolymerized using hydr ogen peroxide and copper(II) as catalyst to MWavg 5600 to obtain a low molecular weight dermatan sulfate. This low molecular weight dermatan sulfate was then fractionated by gel permeation chromatography to obt ain four subfractions having MWavg 7800, 5500, 4200 and 1950. The derm atan sulfate, low molecular weight dermatan sulfate and its subfractio ns showed substantially different optical rotations. The H-1-NMR spect roscopic analysis of dermatan sulfate samples showed some differences including increased content of GalpNAc4S6S residues and improved resol ution in ring resonances for low molecular weight dermatan sulfate fra ctions, primarily the result of reduced molecular weight and lowered h eterogeneity. Saccharide compositional analysis relied on chondroitin ABC lyase treatment followed by capillary electrophoresis. Polyacrylam ide gel-based oligosaccharide mapping was also performed by treating d ermatan sulfate samples with chondroitin B, AC and ABC lysases. These analyses showed increased amounts of sulfation as the MWavg decreased. In vitro bioassay showed maximum anti-Xa activity in the 4.2 kDa frac tion and maximum heparin cofactor II-mediated anti-IIa activity in the 5.5 kDa fraction. The in vivo antithrombotic activity of these fracti ons was measured using a modified Wessler stasis thrombosis model. The 4.2 kDa fraction showed greater antithrombotic activity than the othe r low molecular weight dermatan sulfate fractions, dermatan sulfate, a nd low molecular weight dermatan sulfate. This enhanced activity may r esult from several structural features of the 4.2 kDa fraction includi ng: a high content of 4,6- and 2,4-disulfated disaccharide sequences; the requirement of specific chain length; a change in the ratio of idu ronic to glucuronic acid; and the presence of chondroitin ABC lyase re sistant material.