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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We have utilized circular dichroism spectroscopy to examine the interaction of antithrombin with heparin-derived oligosaccharides and mucopolysaccharides of various sizes. Our studies demonstrate that the various complexes exhibit two major types of chiral absorption spectra. The first of these patterns is seen when octasaccharide, decasaccharide, dodecasaccharide, or tetradecasaccharide fragments bind to the protease inhibitor. The circular dichroism spectra of these complexes when compared to the spectrum of free antithrombin show several distinguishing characteristics. On the one hand, there is a marked general increase in positive chiral absorption that is maximal at 296 and 288 nm and 290 and 282.5 nm. These observations indicate perturbation of "buried" and "exposed" tryptophan residues. On the other hand, a significant augmentation in circular dichroism that peaks at 269.5 and 263 nm is noted. These findings are probably due to the summed positive and negative contributions arising from tryptophan residue(s), disulfide bridge(s), and phenylalanine residue(s). Given that these heparin fragments are able to accelerate factor Xa-antithrombin interactions but not thrombin-antithrombin interactions, the above spectral transitions must be associated with either the binding of a critical domain of the oligosaccharides to the protease inhibitor or the "activation" of the protease inhibitor with respect to factor Xa neutralization. The second ofthese patterns is apparent when octadecasaccharide, low molecular weight heparin (6,500), and high molecular weight heparin (22,000) interact with antithrombin. The circular dichroism spectra of these complexes compared to the spectrum of free protease inhibitor are similar to the first pattern except for changes within the 292-to 282-nm and 275-to 255-nm regions. The subtraction ofthe first pattern from the second pattern reveals a shallow negative band between 300 and 275 nm with potential negative minima at 290 and 283 nm as well as a deep negative band between 275 and 255 nm with possible negative minima at 268 and 262 nm. This chiral absorption profile is most likely to arise from conformational changes of a disulfide bridge(s). However, we cannot completely exclude the possibility that the above circular dichroism difference curve might be explained on the basis of transitions originating from a tryptophan residue(s). Given our method for generating the above data, these spectral alterations must be associated with the binding of a second critical domain of the mucopolysaccharide to antithrombin that is required for rapid complex formation with thrombin or the activation of the protease inhibitor with respect to the neutralization of the latter enzyme.A small fraction of all heparin preparations binds tightly to antithrombin and is responsible for the anticoagulant activity of the mucopolysaccharide (1, 2). A unique tetrasaccharide sequence that contains two nonsulfated uronic acid residues and a N-acetylglucosamine group (3, 4) as well as a 3,6-disulfated gl...