Affinity-fractionated porcine heparin was randomly scissioned by chemical techniques to give hexasaccharides, octasaccharides, decasaccharides, and mucopolysaccharide fragments of approximately 14 residues and approximately 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 these fractions greatly enhanced the rate of Factor Xa inactivation by antithrombin. Indeed, these species exhibited specific molar activities that ranged from 6.9% (hexaccharide) to 60.9% (decasaccharide) of that of the heparin fragment of approximately 16 residues. However, these 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 dissociation constants ranging from 5.5 X 10(-6) M to 2.9 X 10(-7) M. These data suggest that the region of the heparin molecule needed for catalyzing Factor Xa-antithrombin interaction is intimately related to the antithrombin binding domain. The smallest complex carbohydrate fragment that accelerated the inactivation of thrombin by antithrombin had approximately 14 residues. This fraction had an avidity for the protease inhibitor of 2.8 X 10(-7) M and specific molar activities of 140 units per mumol (thrombin neutralization) and 460 units per mumol (factor Xa inactivation). The largest heparin fragment examined contained approximately 16 residues. This fraction had an avidity for antithrombin of 2.4 X 10(-7) M and specific molar activities of 500 units per mumol (thrombin neutralization) and 560 units per mumol (Factor Xa inactivation). Detailed kinetic analyses showed that these two species are able to directly activate antithrombin to the same extent with respect to thrombin inhibition. However, the larger mucopolysaccharide fragment is also capable of approximating free enzyme with protease inhibitor.
Animals Chromatography, Affinity