Multiple functional domains of the heparin molecule.

Abstract

Affinity-fractionated porcine heparin was randomly scissioned by chemical techniques to give hexasaccharides, octasaccharides, decasaccharides and mucopolysaccharide fragments of .apprxeq. 14 residues and .apprxeq. 16 residues that were able to complex with the protease inhibitor. Direct measurements of the kinetic behavior of the hexasaccharides, octasaccharides and decasaccharides showed that the fractions enhanced the rate of Factor Xa inactivation by antithrombin. The species exhibited specific molar activities that ranged from 6.9% (hexasaccharide) to 60.9% (decasaccharide) of that of the heparin fragment of .apprxeq. 16 residues. The oligosaccharides exhibited essentially no ability to accelerate thrombin-antithrombin interactions. The avidity of the hexasaccharides, octasaccharides and decasaccharides for the protease inhibitor increased as a function of size with the respective Kd ranging from 5.5 .times. 10-6 M to 2.9 .times. 10-7 M. The region of the heparin molecule needed for catalyzing Factor Xa-antithrombin interaction is related to the antithrombin binding domain. The smallest complex carbohydrate fragment that accelerated the inactivation of thrombin by antithrombin had .apprxeq. 14 residues. This fraction had an avidity for the protease inhibitor of 2.8 .times. 10-7 M and specific molar activities of 140 U/.mu.mol (thrombin neutralization) and 460 U/.mu.mol (factor Xa inactivation). The largest heparin fragment examined contained .apprxeq. 16 residues. This fraction had an avidity for antithrombin of 2.4 .times. 10-7 M and specific molar activities of 500 U/.mu.mol (thrombin neutralization) and 560 U/.mu.mol (Factor Xa inactivation). Detailed kinetic analyses showed that the 2 species are able to directly activate antithrombin to the same extent with respect to thrombin inhibition. The larger mucopolysaccharide fragment is capable of approximating free enzyme with protease inhibitor.