Biosynthesis of heparin. O-sulfation of the antithrombin-binding region

The antithrombin-binding region in heparin is a pentasaccharide sequence with the predominant structure GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-IdoA -(2-OSO3)-GlcNSO3(6-OSO3) (where GlcA and IdoA represent D-glucuronic and L-iduronic acid, respectively), in which the 3-O-sulfate residue on the internal glucosaminyl unit is a marker group for this particular region of the polysaccharide molecule. A heparin octasaccharide which contained the above pentasaccharide sequence was N/O-desulfated and re-N-sulfated and was then incubated with adenosine 3'-phosphate 5'-phospho[35S]sulfate in the presence of a microsomal fraction from mouse mastocytoma tissue. Fractionation of the resulting 35S-labeled octasaccharide on antithrombin-Sepharose yielded a high affinity fraction that accounted for approximately 2% of the total incorporated label. Structural analysis of this fraction indicated that the internal glucosamine unit of the pentasaccharide sequence was 3-O-35S-sulfated, whereas both adjacent glucosamine units carried 6-O-[35S]sulfate groups. In contrast, the fractions with low affinity for antithrombin (approximately 98% of incorporated 35S) showed no consistent O-35S sulfation pattern and essentially lacked glucosaminyl 3-O-[35S]sulfate groups. It is suggested that the 3-O-sulfation reaction concludes the formation of the antithrombin-binding region. This proposal was corroborated in a similar experiment using a synthetic pentasaccharide with the structure GlcNSO3(6-OSO3)-GlcA-GlcNSO3(6-OSO3)-Id oA (2-OSO3)-GlcNSO3(6-OSO3) as sulfate acceptor. This molecule corresponds to a functional antithrombin-binding region but for the lack of a 3-O-sulfate group at the internal glucosamine unit. The 35S-labeled pentasaccharide recovered after incubation bound with high affinity to antithrombin-Sepharose and contained a 3-O-[35S]sulfate group at the internal glucosamine residue as the only detectable labeled component. The use of this pentasaccharide substrate along with the affinity matrix provides a highly specific assay for the 3-O-sulfotransferase.     

Location of the antithrombin-binding sequence in the heparin chain

The antithrombin-binding region of heparin is a pentasaccharide sequence with the predominant structure -GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-Ido A(2-OSO3)- GlcNSO3(6-OSO3)-. By using the 3-O-sulfated glucosamine residue as a marker for the anti-thrombin-binding sequence, the location of this sequence within the heparin chain was investigated. Heparin with high affinity for antithrombin (HA-heparin) contains few N-acetyl groups located outside the antithrombin-binding region, and cleavage at such groups was therefore expected to be essentially restricted to this region. HA-heparin was cleaved at N-acetylated glucosamine units by partial deacetylation followed by treatment with nitrous acid at pH 3.9, and the resulting fragments with low affinity for anti-thrombin (LA-fragments) were recovered after affinity chromatography on immobilized antithrombin. The LA-fragments were further divided into subfractions of different molecular size by gel chromatography and were then analyzed with regard to the occurrence of the nonreducing terminal GlcA-GlcNSO3(3,6-di-OS-O3)- sequence. Such units were present in small, intermediate-sized as well as large fragments, suggesting that the antithrombin-binding regions were randomly distributed along the heparin chains. In another set of experiments, HA-heparin was subjected to limited, random depolymerization by nitrous acid (pH 1.5), and the resulting reducing terminal anhydromannose residues were labeled by treatment with NaB3H4. The molecular weight distributions of such labeled LA-fragments, determined by gel chromatography, again conformed to a random distribution of the antithrombin-binding sequence within the heparin chains. These results are in apparent disagreement with previous reports (Radoff, S., and Danishefsky, I. (1984) J. Biol. Chem. 259, 166-172; Rosenfeld, L., and Danishefsky, I. (1988) J. Biol. Chem. 263, 262-266) which suggest that the antithrombin-binding region is preferentially located at the nonreducing terminus of the heparin molecule.     

Biosynthesis of heparin. Enzymatic sulfation of pentasaccharides

Heparin-derived pentasaccharides with the general structures GlcN-GlcA/IdoA-GlcN-GlcA/IdoA-GlcN (where GlcA represents D-glucuronic acid and IdoA represents L-iduronic acid) and GlcNSO3-GlcA/IdoA-GlcNSO3-GlcA/IdoA- GlcNSO3 (where -NSO3 represents an N-sulfate group) were tested as exogenous sulfate acceptors in incubations with adenosine 3'-phosphate 5'-[35S]phosphosulfate and microsomal enzymes from a heparin-producing mouse mastocytoma. No transfer occurred to the N-unsubstituted pentasaccharide containing only L-iduronic acid, but the other three isomers incorporated various amounts of 35S, which was totally present in N-sulfate groups. After complete chemical N-sulfation, all four pentasaccharides served as acceptors in O-sulfotransferase reactions and incorporated from 20 to greater than 200 times as much radioactivity as did the nonsulfated parent compounds. The C-6 position of the internal glucosamine unit was labeled preferentially, irrespective of the structures of the adjacent hexuronic acid units. Significant 2-O-35S-sulfation of IdoA units occurred in both -IdoA-Glc-NSO3-GlcA- and -GlcA-GlcNSO3-IdoA- sequences, whereas no significant sulfation of GlcA residues was detected. The pentasaccharide GlcNSO3-GlcA-Glc-NSO3-GlcA-GlcNSO3 thus can be used as a selective substrate in assays for glucosaminyl-6-O-sulfotransferase activity. The antithrombin-binding region, essential for the blood anticoagulant activity of heparin, has been identified as a pentasaccharide sequence with the predominant structure GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-++ +IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R represents either a sulfate or an acetyl group and -OSO3 represents an O-sulfate/ester sulfate group, with locations of O-sulfate groups indicated in parentheses) (Lindahl U., Thunberg, L., B?ckstr?m, G., Riesenfeld, J., Nordling, K., and Bj?rk, I. (1984) J. Biol. Chem. 259, 12368-12376). The products of [35S]sulfate transfer to the pentasaccharide GlcNSO3-GlcA-GlcNSO3-IdoA-GlcNSO3 contained molecules with high affinity for antithrombin, corresponding to 0.3-0.5% of the total label. Structural analysis suggested the occurrence of O-[35S]sulfate groups at both C-6 of the nonreducing terminal glucosamine unit and C-3 of the internal glucosamine unit. No products with high affinity for antithrombin were formed from the pentasaccharides that had a different monosaccharide sequence than the binding region; and moreover, these oligosaccharides appeared unable to incorporate glucosaminyl 3-O-sulfate groups. These findings point to the importance of the uronic acid sequence in the generation of the antithrombin-binding region of heparin.     

Determination of the range in binding-site densities of rat skin heparin chains with high binding affinities for antithrombin.

Rat skin heparin proteoglycans vary markedly in the proportions of their constituent polysaccharide chains that have high binding affinity for antithrombin. As the proportion of such chains in a proteoglycan rises, their degree of affinity for antithrombin also increases [Horner (1987) Biochem. J. 244, 693-698]. The antithrombin-binding-site densities of such chains have now been determined, by measuring heparin-induced enhancement of the intrinsic fluorescence of antithrombin and by chemical analysis for the disaccharide sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate), which is unique to this site in heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555]. Antithrombin-binding-site density ranged from one to five sites per chain.     

Biosynthesis of heparin. Availability of glucosaminyl 3-O-sulfation sites

Heparin preparations isolated from pig intestinal mucosa and from bovine lung were fractionated with regard to affinity for antithrombin. The resulting fractions, with high (HA) or low (LA) affinity for the proteinase inhibitor, were analyzed by 13C NMR or by identification of di- and tetrasaccharides obtained through deaminative cleavage with nitrous acid. Structural differences between corresponding HA and LA fractions were essentially restricted to minor constituents, in particular 3-O-sulfated glucosamine units that occurred (1 or 2 residues/chain) in all HA preparations but were scarce or absent in LA heparin. The HA fractions also consistently showed higher contents of nonsulfated iduronic acid and, to a lesser extent, N-acetylated glucosamine units than the LA fractions. The two tetrasaccharide sequences, -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3- and -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3(6-OSO3)- , recently implicated as part of the acceptor site for glucosaminyl 3-O-sulfate groups (Kusche, M., B?ckstr?m, G., Riesenfeld, J., Petitou, M., Choay, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 15474-15484), were identified in mucosal LA heparin; it was calculated that the preparation contained approximately one potential acceptor site/polysaccharide chain. Yet this material did not yield any labeled HA components on incubation with adenosine 3'-phosphate 5'-phospho-[35S]sulfate in the presence of glucosaminyl 3-O-sulfotransferase, solubilized from a mouse mastocytoma microsomal fraction. The failure to incorporate any 3-O-sulfate groups could conceivably be explained by the occurrence of a D-glucuronic rather than L-iduronic acid unit linked at the reducing ends of the above tetrasaccharide sequences. Alternatively, 3-O-sulfation may be restricted by other, as yet unidentified, inhibitory structural elements that are preferentially expressed in polysaccharide sequences selected for the generation of LA heparin.     

Location of antithrombin-binding regions in rat skin heparin proteoglycans.

Rat skin heparin proteoglycan labelled biosynthetically with 35S was fractionated on a column of antithrombin-Sepharose into fractions with varying degrees of affinity for antithrombin. These were treated with NaOH to release heparin chains (Mr 60,000-100,000), by beta-elimination or incubated with serum to produce fragments of the same order of size as commercial heparin (Mr 5000-30,000), by endoglycosidase cleavage. Chains and fragments were then fractionated on antithrombin-Sepharose. The various fractions were deaminated with HNO2 at pH 1.5 followed by reduction with NaB3H4. Approx 90% of the incorporated 3H was associated with disaccharides. These were fractionated by high-performance ion-exchange chromatography. A unique minor component corresponding to the sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate) in the polysaccharide was found only in fractions with high affinity for antithrombin. The glucosamine residue linked to C-4 of this glucuronosyl unit was predominantly (or exclusively) N-sulphated rather than N-acetylated, pointing to a structural difference between the antithrombin-binding region of rat heparin and that of pig mucosal heparin. Calculations based on the distribution of the glucosaminyl 3-O-sulphate group showed that approximately two-thirds of the total antithrombin-binding regions present in the unfractionated material were accommodated by only 20% of the proteoglycan molecules, and by 10% of the polysaccharide chains. While most of the proteoglycan molecules thus lacked such regions (and hence affinity for antithrombin) a minor proportion of the polysaccharide chains contained on the average three binding regions per molecule. These findings support by direct chemical analysis an earlier proposal, based on anticoagulant activities of similar rat skin heparin fractions, that the distribution of antithrombin-binding sites in intact heparin proteoglycans is markedly non-random.     

Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.     

Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the acceleration of the reactions between antithrombin and thrombin or factor Xa

Oligosaccharides (10-20 monosaccharide units) with high affinity for antithrombin, as well as larger high-affinity heparin fractions (having relative molecular masses between 6,000 and 21,500), all markedly accelerated the inhibition of Factor Xa by antithrombin. Moreover, all high-affinity oligosaccharides and heparins enhanced, to a similar extent, the amount of free proteolytically modified antithrombin cleaved at the reactive bond by Factor Xa. In contrast, a minimum high-affinity heparin size of approximately 18 monosaccharide units was required to significantly accelerate the inactivation of thrombin by antithrombin and to enhance the production of modified antithrombin by this enzyme. All high-affinity fractions studied had similar affinities for antithrombin, as determined by fluorescence titrations. In competition experiments, binary complexes of antithrombin with octadecasaccharide or larger high-affinity heparins, but not with smaller oligosaccharides, displaced inactivated 125I-thrombin from matrix-linked low-affinity heparin. Moreover, similar binary complexes with 3H-labeled octadecasaccharide or larger chains, but not with smaller oligosaccharides, were capable of binding to matrix-linked inactivated thrombin. These results indicate that simultaneous binding of antithrombin and thrombin to high-affinity heparin is a prerequisite to the acceleration of the antithrombin-thrombin reaction and that the minimum heparin sequence capable of binding both proteins comprises approximately 18 monosaccharide units. Similar complex formation apparently is not required for the acceleration of the antithrombin-Factor Xa reaction.     

Anticoagulantly active heparin from clam (Mercenaria mercenaria)

Heparin was isolated from Mercenaria mercenaria by ion-exchange chromatography and was fractionated into two distinct populations with immobilized antithrombin. The high-affinity glycosaminoglycan accelerated dramatically the inhibition of purified human factors IIa and Xa via purified human antithrombin. Specific anti-factor IIa and anti-factor Xa activities were 363 and 348 U.S.P. units/mg, respectively. The highly active clam heparin exhibited a molecular weight of approximately 18,000 and contained approximately 2.5 sulfate groups per disaccharide. The intrinsic fluorescence of purified human antithrombin was enhanced in the presence of the high-affinity invertebrate glycosaminoglycan to an extent comparable to the level induced by vertebrate heparin. In addition, the critical tetrasaccharides containing 3-O-sulfated glucosamine residues, which constitute part of the unique antithrombin-binding domain of mammalian heparin, were also detected in high-affinity Mercenaria heparin.     

Binding of platelet factor 4 to heparin oligosaccharides.

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.     

1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site

Heparin inhibits blood coagulation by binding to the protease inhibitor antithrombin, thus promoting inactivation of the protease "factors" of the coagulation cascade mechanism. The article provides an overview of the studies, by different research groups, that led to the structural elucidation of the antithrombin-binding region of heparin. These studies were triggered by the finding that only a fraction of heparin molecules were capable of binding with high affinity to antithrombin, and further that this fraction essentially accounted for the anticoagulant activity of the unfractionated material. Oligosaccharides obtained by selective, partial depolymerization of heparin were fractionated on immobilized antithrombin, and the smallest high-affinity molecules recovered were subjected to structural analysis, in conjunction with various modification steps. The results were interpreted in terms of a binding site for antithrombin constituted by a pentasaccharide sequence with an internal unique 3-O-sulfated glucosamine unit, in addition to sugar residues and sulfate groups present elsewhere also in the polysaccharide. The structure/function relations deduced were verified by chemical synthesis of several pentasaccharide variants with the predicted bioactivities.     

The effect of a reducing-end extension on pentasaccharide binding by antithrombin

Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific pentasaccharide domain in a two-step mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHG'H'). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths >/=0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.     

Thrombin-inhibitory activity of whale heparin oligosaccharides

Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase. The analytical data indicated that the proportions of trisulfated disaccharide (IdUA(2S)alpha 1----4GlcNS(6S)) and disulfated disaccharide (UA1----4GlcNS(6S)) increased with the manifestation of high thrombin-inhibitory activity, while that of monosulfated disaccharide (UA1----4GlcNS) decreased. The present observations, together with those so far reported, suggest that the presence of the former structural elements, specifically IdUA(2S)alpha 1----4GlcNS(6S), as well as the antithrombin III-binding pentasaccharide at the proper positions in the molecules of whale heparin oligosaccharides is essential for the manifestation of high inhibitory activity for thrombin in the presence of antithrombin III. The structural bases for the manifestation of the anticoagulant activity of whale and porcine heparins and their oligosaccharides are also discussed.     

Kinetic analysis of the role of zinc in the interaction of domain 5 of high-molecular weight kininogen (HK) with heparin

Previous investigations have shown that HK and its light chain bind heparin, preventing the enhancement of antithrombin inhibition of thrombin and potentiating the inhibition of plasma kallikrein by antithrombin. We found that both HK and HKa bound heparin, but HK exhibited a greater affinity. We therefore localized the binding sites for heparin on HK. HK domains 5 and 6 of the light chain as well as domain 3 from the heavy chain, expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli, were tested for binding to immobilized heparin by surface plasmon resonance using a BiaCore 2000 instrument. GST-D5, but not GST-D3, GST-D6, or GST, bound to heparin when the recombinant domains were present at a concentration of 70 nM. To localize more precisely the amino acid sequences on D5, both of the subdomains, histidine-glycine-rich GST-(K420-D474) and histidine-glycine-lysine-rich GST-(H475-S626), were expressed and tested for binding to immobilized heparin. The K(d) was much lower for GST-(K420-D474) than for GST-(H475-S626) in the presence or absence of Zn(2+). GST-(K420-D474) was effective in decreasing the rate of inactivation of thrombin by antithrombin in the presence of heparin and Zn(2+), while GST-(H475-S626) had no effect. We conclude that the binding of heparin to HK is a complex function of Zn(2+) interacting with histidines in the sequence K420-D474 to create high-affinity binding sites. HK has the potential to be an important modulator of heparin therapy.     

Heparin/Heparan sulfate domains in binding and signaling of fibroblast growth factor 8b

The role of heparin and heparan sulfate in the binding and signaling of fibroblast growth factors (FGFs) has been subject to intense investigation, but the studies have largely been confined to two species (FGF1 and FGF2) of the family with approximately 20 members. We have investigated the structural requirements for heparin/heparan sulfate in binding and activation of FGF8 (splice variant b). We present evidence that the minimal FGF8b-binding saccharide domain encompasses 5-7 monosaccharide units. The N-, 2-O-, and 6-O-sulfate substituents of heparin/heparan sulfate (HS) are all involved in the interaction, preferentially in the form of trisulfated IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3)) disaccharide constituents. These structural characteristics resemble those described earlier for FGF1. By contrast, the saccharide structures required for the biological activity of FGF8b differed significantly from those characteristic for FGF1 and FGF2. Experiments with cells lacking active HS indicated that extended >/=14-mer heparin domains were needed to enhance cell proliferation and Erk phosphorylation by FGF8b, whereas in cells stimulated with FGF1 or FGF2 the corresponding responses were achieved by much shorter, 6-8-mer, oligosaccharides. Furthermore, still longer domains were needed to activate FGF8b in cells with "non-optimal" FGF receptor expression. Collectively, our data suggest that the heparin/HS structures enhancing the biological activity of FGFs were influenced by the FGF species involved as well as by the cellular composition of FGF receptors.     

Processing of macromolecular heparin by heparanase

Heparanase is an endo-glucuronidase expressed in a variety of tissues and cells that selectively cleaves extracellular and cell-surface heparan sulfate. Here we propose that this enzyme is involved also in the processing of serglycin heparin proteoglycan in mouse mast cells. In this process, newly synthesized heparin chains (60-100 kDa) are degraded to fragments (10-20 kDa) similar in size to commercially available heparin (Jacobsson, K. G., and Lindahl, U. (1987) Biochem. J. 246, 409-415). A fraction of these fragments contains the specific pentasaccharide sequence required for high affinity binding to antithrombin implicated with anticoagulant activity. Rat skin heparin, which escapes processing in vivo, was used as a substrate in reaction with recombinant human heparanase. An incubation product of commercial heparin size retained the specific pentasaccharide sequence, although oligosaccharides (3-4 kDa) containing this sequence could be degraded by the same enzyme. Commercial heparin was found to be a powerful inhibitor (I50 approximately 20 nM expressed as disaccharide unit, approximately 0.7 nM polysaccharide) of heparanase action toward antithrombin-binding oligosaccharides. Cells derived from a serglycin-processing mouse mastocytoma expressed a protein highly similar to other mammalian heparanases. These findings strongly suggest that the intracellular processing of the heparin proteoglycan polysaccharide chains is catalyzed by heparanase, which primarily cleaves target structures distinct from the antithrombin-binding sequence.     

Binding of heparin fractions and other polysulfated polysaccharides to plasma fibronectin: effects of molecular size and degree of sulfation of polysaccharides

Heparin was divided into four fractions on fibronectin-Sepharose. The higher affinity fraction for fibronectin was larger in molecular size, higher in sulfate content and higher in affinity for anti-thrombin III. Together with these heparin fractions, the following three series of heparin samples were examined to compare the affinity for fibronectin-Sepharose: four fractions separated on Sephadex G-100; five fractions separated on antithrombin III-Sepharose, and six partially and completely N-desulfated heparins. The result showed that the affinity of heparin for fibronectin was dependent exclusively on its molecular size, and that an appropriate level of sulfate content in heparin (1.9-2.4 mol/disaccharide) was essential for the affinity. The sulfated preparations of glycosaminoglycans (heparan sulfate, dermatan sulfate and chondroitin 4-sulfate) and neutral polysaccharides (amylose and dextran) having higher sulfate content than heparin were found to display higher affinity for fibronectin than heparin. This suggested that highly sulfated polysaccharides showed potent affinity irrespective of their polysaccharide structure. The sulfated chondroitin 4-sulfate having a sulfate content and molecular size comparable to those of heparin was inferior to heparin with respect to affinity. A competitive dissociation experiment indicated that heparin and other polysulfated polysaccharides share a common binding site on the fibronectin molecule.     

The region of antithrombin interacting with full-length heparin chains outside the high-affinity pentasaccharide sequence extends to Lys136 but not to Lys139

The interaction of a well-defined pentasaccharide sequence of heparin with a specific binding site on antithrombin activates the inhibitor through a conformational change. This change increases the rate of antithrombin inhibition of factor Xa, whereas acceleration of thrombin inhibition requires binding of both inhibitor and proteinase to the same heparin chain. An extended heparin binding site of antithrombin outside the specific pentasaccharide site has been proposed to account for the higher affinity of the inhibitor for full-length heparin chains by interacting with saccharides adjacent to the pentasaccharide sequence. To resolve conflicting evidence regarding the roles of Lys136 and Lys139 in this extended site, we have mutated the two residues to Ala or Gln. Mutation of Lys136 decreased the antithrombin affinity for full-length heparin by at least 5-fold but minimally altered the affinity for the pentasaccharide. As a result, the full-length heparin and pentasaccharide affinities were comparable. The reduced affinity for full-length heparin was associated with the loss of one ionic interaction and was caused by both a lower overall association rate constant and a higher overall dissociation rate constant. In contrast, mutation of Lys139 affected neither full-length heparin nor pentasaccharide affinity. The rate constants for inhibition of thrombin and factor Xa by the complexes between antithrombin and full-length heparin or pentasaccharide were unaffected by both mutations, indicating that neither Lys136 nor Lys139 is involved in heparin activation of the inhibitor. Together, these results show that Lys136 forms part of the extended heparin binding site of antithrombin that participates in the binding of full-length heparin chains, whereas Lys139 is located outside this site.     

Affinity chromatography of sulphated polysaccharides separately fractionated on antithrombin III and heparin cofactor II immobilized on concanavalin A—Sepharose

Three sulphated polysaccharides, dermatan sulphate, fucan and heparin, were fractionated according to their affinity towards antithrombin III (ATIII) and heparin cofactor II (HCII), the two main physiological thrombin (IIa) inhibitors. Both inhibitors were immobilized on concanavalin A—Sepharose, which binds to the glycosylated chains of the proteins while the protein-binding site for the polysaccharide remains free. Each polysaccharide was fractionated into bound and unbound fractions either for ATIII or HCII. The eluted fractions were tested for their ability to catalyse and interactions. The possible presence of a unique binding site for ATIII and HCII, on each sulphated polysaccharide, was also studied.     

Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2.

Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (> or =0.5 m NaCl required for elution) contained an internal IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3))-IdoUA(2-OSO(3))-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1. FGF-2 was shown to bind to a mono-O-sulfated HS 6-mer carrying a single internal IdoUA(2-OSO(3))-unit. However, a di-O-sulfated -IdoUA(2-OSO(3))-GlcNSO(3)-IdoUA(2-OSO(3))-trisaccharide sequence within a HS 8-mer gave stronger binding. These findings show that not only the number but also the positions of individual sulfate groups determine affinity of HS for FGFs. Our findings support the notion that FGF-dependent processes can be modulated in vivo by regulated expression of distinct HS sequences.