Affinity-fractionated porcine heparin was randomly scissioned by chemical techniques to give hexasaccharides, octasaccharides, decasaccharides, and mucopolysaccharide fragments of -14 residues and -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% (hexasaccharide) to 60.9% (decasaccharide) ofthat ofthe heparin fragment of 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 -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 jamol (thrombin neutralization) and 460 units per jAmol (factor Xa inactivation). The largest hepran fragment examined contained -16 residues. This fraction had an avidity for antithrombin of 2.4 X 10-7 M and specific molar activities of 500 units per jsmol (thrombin neutralization) and 560 units per jtmol (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.Only a small fraction ofa given heparin preparation binds tightly to antithrombin, and this fraction is largely responsible for its anticoagulating activity (1, 2). Evidence has been provided that a unique tetrasaccharide sequence containing two nonsulfated uronic acid moieties is found almost exclusively in the highly active heparin, and it has been suggested that this portion of the mucopolysaccharide must represent a binding site that is recognized by antithrombin (3, 4). These findings have been confirmed by others (5). In this communication, we show that relatively small oligosaccharides can complex tightly with the protease inhibitor and dramatically accelerate Factor Xa-antithrombin but not thrombin-antithrombin interactions. We also show that larger mucopolysaccharide fragments are essential for approximating enzyme with protease inhibitor or activating antithrombin such that it can rapidly neutralize thrombin. Based on these data, we propose that heparin possesses multiple structural domains that modulate different functions of antithrombin. M...

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