Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.