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 -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...

Anticoagulant heparan sulfate: structural specificity and biosynthesis

“…The necessary size for an oligosaccharide to inhibit thrombin activity is much longer (14-20 saccharides) than the pentasaccharide that is required to bind AT and inhibit factor Xa (23). A synthetic hexadecasaccharide (SR123781) that displays excellent anticoagulant activity against thrombin in vitro and in vivo has been produced (20).…”

Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-O-sulfate group.

“…Tetrasaccharide c lacks the terminal sugar at the nonreducing end of pentasaccharide a. Tetrasaccharide d lacks the terminal residue from the reducing end of pentasaccharide a. A mixture of tetrasaccharides obtained by gel filtration (Oosta et al, 1981) was also tested. Virtually identical results were obtained when these heparin species were assayed on rat aortic and calf aortic SMC.…”

“…These experiments also indicated that both the N-and O-sulfates of heparin were necessary for antiproliferative activity, although the N-sulfates could be replaced with acetyl groups without significant loss of activity. Both the hexasaccharide fragment and the Ndesulfated, re-N-acetylated heparin are devoid of anticoagulant activity (Kazatchkine et al, 1981;Oosta et al, 1981), thus providing a chemical basis for the difference between the antiproliferative and anticoagulant activities of this glycosaminoglycan.In this paper, we have further analyzed the structural determinants of the antiproliferative effect of heparin. These experiments were done using synthetically prepared and therefore chemically defined heparin oligosaccharides.…”

Carbohydrate post-glycosylational modifications

“…For example, a pentasaccharide sequence HexA-GlcN(NS)-HexA-GlcN(NS)-IdoA (2S) (Figure 2a) has been identified to be specific for fibroblast growth factor 2 (FGF2) binding, 32 another pentasaccharide sequence of heparin GlcN(6S)-GlcA-GlcN(NS,3S)-IdoA(2S)-GlcN(NS) (Figure 2b) is responsible for antithrombin III (AT-III, a serine protease inhibitor that blocks thrombin and factor Xa in the coagulation cascade) binding, 33 although a longer sequence (14-20 saccharide units) is required to accelerate the AT-III/thrombin interaction and inhibit thrombin activity. 34 Based on the structure of the AT-binding pentasaccharide, a new synthetic pentasaccharide anticoagulant under the trade name of Arixtra was approved by the FDA (Figure 2c). 33b A synthetic hexadecasaccharide (SR123781) that displays excellent anticoagulant activity against thrombin in vitro and in vivo was produced (Figure 3).…”