STRUCTURAL ASPECTS OF HEPARIN RESPONSIBLE FOR INTERACTIONS WITH VON-WILLEBRAND-FACTOR

Unfractionated heparin (UFH) binds von Willebrand factor (vWF) and inh ibits the vWF-platelet GP Ib interaction. For VWF, a heparin-binding d omain has been identified, but for heparin, the structures that confer such activity are unknown. To investigate this, UFH was depolymerized by methods that yield structurally distinct fragments. The glycosamin oglycans (GAGs) produced were separated into five groups of homogeneou s molecular weight (MW). Anti-Xa activity, VWF binding affinity, and v WF-dependent platelet agglutination were measured. Periodate oxidation but not heparinase digestion destroyed anti-Xa activity. At all MWs, periodate conferred greater VWF binding affinity and greater ability t o inhibit platelet agglutination than heparinase. As an example, at MW 6100, the binding IC50 was 100+/-19 mu mol/L for a periodate-derived GAG and 527+/-70 mu mol/L for a heparinase-derived GAG. At the same MW , the agglutination IC50 was 17+/-5 mu mol/L for periodate and 135+/-1 8 mu mol/L for heparinase. This suggests that the disaccharide GlcNS[6 S]-IdoA2S, destroyed by heparinase but not periodate, is crucial to he parin-vWF interactions. An MW dependency was also noted, with a minimu m dodecasaccharide required for activity inhibition. To further invest igate the heparin/vWF interaction, affinity fractionation of heparins was performed with an immobilized peptide derived from a heparin-bindi ng domain of vWF. Disaccharide analysis of high-affinity heparins reve aled an increased ratio of IdoA2S-GlcN[S/Ac]6S to IdoA2S-GlcN[S/Ac]. A ffinity fractionation of oligosaccharides (MW 3500) diminished the rel ative content of all disaccharides except IdoA2S-GlcNS6S, which was in creased. These data suggest that the disaccharide structures IdoA2S-Gl cNS6S and GlcNS6S-IdoA2S are crucial to heparin/vWF interactions. Unde rstanding the structural aspects that confer such activity may be usef ul in designing heparin-based antithrombotic drugs.