Specificity of the basic side chains of Lys114, Lys125, and Arg129 of antithrombin in heparin binding

The anticoagulant polysaccharide heparin binds and activates the plasma proteinase inhibitor antithrombin through a pentasaccharide sequence. Lys114, Lys125, and Arg129 are the three most important residues of the inhibitor for pentasaccharide binding. To elucidate to what extent another positively charged side chain can fulfill the role of each of these residues, we have mutated Lys114 and Lys125 to Arg and Arg129 to Lys. Lys114 could be reasonably well replaced with Arg with only an approximately 15-fold decrease in pentasaccharide affinity, in contrast to an approximately 10(5)-fold decrease caused by substitution with an noncharged amino acid of comparable size. However, a loss of approximately one ionic interaction on mutation to Arg indicates that the optimal configuration of the network of basic residues of antithrombin that together interact with the pentasaccharide requires a Lys in position 114. Replacement of Lys125 with Arg caused an even smaller, approximately 3-fold, decrease in pentasaccharide affinity, compared with that of approximately 400-fold caused by mutation to a neutral amino acid. An Arg in position 125 is thus essentially equivalent to the wild-type Lys in pentasaccharide binding. Substitution of Arg129 with Lys decreased the pentasaccharide affinity an appreciable approximately 100-fold, a loss approaching that of approximately 400-fold caused by substitution with a neutral amino acid. Arg is thus specifically required in position 129 for high-affinity pentasaccharide binding. This requirement is most likely due to the ability of Arg to interact with other residues of antithrombin, primarily, Glu414 and Thr44, in a manner that appropriately positions the Arg side chain for keeping the pentasaccharide anchored to the activated state of the inhibitor.     

Binding of heparin to human antithrombin III activates selective chemical modification at lysine 236. Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of antithrombin III

A new water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC), was used to identify lysine residues of antithrombin III which participate in the binding of heparin. Antithrombin, modified with S-DABITC in the presence and absence of low molecular weight heparin (Mr 5000) was reduced, carboxymethylated, and digested with trypsin. The digest was analyzed by high-performance liquid chromatography and monitored at 465 nm. In the absence of heparin, four major colored peptides (T1, T2, T3, and T4) were identified. When antithrombin was preincubated with heparin (2-fold by weight), followed by S-DABITC modification, the recovery of peptide T4 remained unchanged, but the recoveries of T1, T2, and T3 were reduced by 93, 86, and 98%, respectively. In addition, a new colored peptide, TA, appeared. Amino acid sequencing of peptides T1, T2, T3, and TA localized S-DABITC modification sites as Lys-136, Lys-125, Lys-107, and Lys-236, respectively. Thus, binding of heparin to human antithrombin diminished S-DABITC modification at Lys-107, Lys-125, and Lys-136, but at the same time enhanced S-DABITC modification at Lys-236. This phenomenon was further characterized by varying the molar ratio of heparin/antithrombin (from 0.04 to 20). The shielding of Lys-125 and Lys-136 was inversely proportional to the activation of Lys-236. At a heparin/antithrombin molar ratio of 1, the extent of shielding of Lys-125 and Lys-136 and the unmasking of Lys-236 were 25-33%. This shielding-unmasking effect correlated with enhanced antithrombin inhibition of thrombin. We conclude that Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of human antithrombin and that binding of heparin to antithrombin causes a conformational change of antithrombin that leads to the exposure of Lys-236 for S-DABITC modification.     

Tryptophan residue at the heparin binding site in antithrombin III

Chemical modifications have demonstrated that the ultraviolet difference spectrum produced when heparin interacts with antithrombin III is due primarily to changes in the tryptophan environment. This is based on the observation that this spectrum could be abolished by treatment of antithrombin III with dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide but not with tetranitromethane. The tryptophan-modified antithrombin III is still capable of binding to thrombin even when it has lost 85% of heparin cofactor activity. A marked decrease in reactivity of tryptophan residues is observed when modification is carried out in the presence of heparin. Evidence is presented that tryptophan is in the heparin binding site.     

Role of tryptophan 49 in the heparin cofactor activity of human antithrombin III.

To probe the functional role of tryptophan 49 in human antithrombin III, a mutant antithrombin, W49K, has been expressed in baby hamster kidney cells. The mutation reduces the affinity for heparin pentasaccharide by 1.8 kcal mol-1 but does not alter the heparin enhancement of the rate of factor Xa inhibition. 1H NMR spectra of W49K antithrombin show that the structure of the protein and the mode of heparin binding appear to be unaltered by the mutation, although tryptophan 49 is perturbed by heparin binding. 19F NMR spectra of 6-fluorotryptophan-substituted antithrombin show that tryptophan 49 is in a solvent-exposed environment. The heparin-induced fluorescence enhancement of W49K antithrombin is significantly different from that of wild-type antithrombin. Pentasaccharide induces only a 24% enhancement of antithrombin fluorescence, while high affinity heparin induces an enhancement of 40%. The results indicate that tryptophan 49 is probably a heparin contact residue but can be mutated without altering the remaining heparin-antithrombin interactions or the heparin-induced conformational change and resultant activation toward Factor Xa. Hydrophobic as well as charge interactions are thus probably involved in the specificity of the antithrombin-heparin pentasaccharide interaction. The lower fluorescence enhancements suggest that the heparin-induced 40% fluorescence enhancement used as the hallmark of activating heparin species is not the best indicator of the structural change in antithrombin that results in enhancement of the rate of proteinase inhibition.     

Reactive site peptide structural similarity between heparin cofactor II and antithrombin III

Heparin cofactor II (Mr = 65,600) was purified 1800-fold from human plasma to further characterize the structural and functional properties of the protein as they compare to antithrombin III (Mr = 56,600). Heparin cofactor II and antithrombin III are functionally similar in that both proteins have been shown to inhibit thrombin at accelerated rates in the presence of heparin. There was little evidence for structural homology between heparin cofactor II and antithrombin III when high performance liquid chromatography-tryptic peptide maps and NH2-terminal sequences were compared. A partially degraded form of heparin cofactor II was also obtained in which a significant portion (Mr = 8,000) of the NH2 terminus was missing. The rates of thrombin inhibition (+/- heparin) by native and partially degraded-heparin cofactor II were not significantly different, suggesting that the NH2-terminal region of the protein is not essential either for heparin binding or for thrombin inhibition. A significant degree of similarity was found in the COOH-terminal regions of the proteins when the primary structures of the reactive site peptides, i.e. the peptides which are COOH-terminal to the reactive site peptide bonds cleaved by thrombin, were compared. Of the 36 residues identified, 19 residues in the reactive site peptide sequence of heparin cofactor II could be aligned with residues in the reactive site peptide from antithrombin III. While the similarities in primary structure suggest that heparin cofactor II may be an additional member of the superfamily of proteins consisting of antithrombin III, alpha 1-antitrypsin, alpha 1-antichymotrypsin and ovalbumin, the differences in structure could account for differences in protease specificity and reactivity toward thrombin. In particular, a disulfide bond which links the COOH-terminal (reactive site) region of antithrombin III to the remainder of the molecule and is important for the heparin-induced conformational change in the protein and high affinity binding of heparin does not appear to exist in heparin cofactor II. This observation provides an initial indication that while the reported kinetic mechanisms of action of heparin in accelerating the heparin cofactor II/thrombin and antithrombin III/thrombin reactions are similar, the mechanisms and effects of heparin binding to the two inhibitors may be different.     

A peptide model for the heparin binding site of antithrombin III.

A peptide model for the heparin binding site of antithrombin III (ATIII) was synthesized to elucidate the structural consequences of heparin binding. This peptide [ATIII(123-139)] and a sequence-permuted analogue (ATIII random) showed similar conformational behavior (as analyzed by circular dichroism spectroscopy) in aqueous and organic media. In the presence of heparin, however, the peptide ATIII(123-139) assumed a stable conformation, whereas peptide ATIII random did not. Complex formation was saturable and sensitive to salt. The ATIII(123-139)-heparin complex contained beta-structure, rather than helical structure. This finding is incompatible with current models of heparin binding and suggests that heparin binding may induce nonnative structures at the binding site which could, in turn, lead to activation of ATIII. The peptide ATIII(123-139) was able to inhibit the binding of ATIII by heparin, consistent with the notion that this peptide may be a model for the heparin binding site.     

Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin

Fucoidan, poly(L-fucopyranose) linked primarily alpha 1----2 with either a C3- or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G. F., Wurzel, H. A., McNeal, G. M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc. Soc. Exp. Biol. Med. 94, 404-409). We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin III and heparin cofactor II. Fucoidan enhances the heparin cofactor II-thrombin reaction more than 3500-fold. The apparent second-order rate constant of thrombin inhibition by heparin cofactor II increases from 4 x 10(4) (in the absence of fucoidan) to 1.5 x 10(8) M-1 min-1 as the fucoidan concentration increases from 0.1 to 10 micrograms/ml and then decreases as fucoidan is increased above 10 micrograms/ml. The fucoidan reaction with heparin cofactor II-thrombin is kinetically equivalent to a "template model." Apparent fucoidan-heparin cofactor II and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively. The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor II or of thrombin. The fucoidan-antithrombin III reactions with thrombin and factor Xa are accelerated maximally 285- and 35-fold at fucoidan concentrations of 30 and 500 micrograms/ml, respectively. Using human plasma and 125I-labeled thrombin in an ex vivo system, the heparin cofactor II-thrombin complex is formed preferentially over the antithrombin III-thrombin complex in the presence of 10 micrograms/ml fucoidan. Our results indicate that heparin cofactor II is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor II and not by antithrombin III.     

A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence

A heparin-binding peptide within antithrombin III (ATIII) was identified by digestion of ATIII with Staphylococcus aureus V8 protease followed by purification on reverse-phase high pressure liquid chromatography using a C-4 column matrix. The column fractions were assayed for their ability to bind heparin by ligand blotting with 125I-fluoresceinamine-heparin as previously described (Smith, J. W., and Knauer, D. J. (1987) Anal. Biochem. 160, 105-114). This analysis identified at least three fractions with heparin binding ability of which the peptide eluting at 25.4 min gave the strongest signal. Amino acid sequence analysis of this peptide gave a partially split sequence which was consistent with regions encompassing amino acids 89-96 and 114-156. These amino acids are present in a 1:1 molar ratio which is consistent with a disulfide linkage between Cys-95 and Cys-128. High affinity heparin competed more effectively for the binding of 125I-fluoresceinamine-heparin to this peptide than low affinity heparin. Chondroitin sulfate did not block the binding of 125I-fluoresceinamine-heparin to the peptide. These data strongly suggest that the isolated peptide represents a native heparin-binding region within intact ATIII. Computer generation of a plot of running charge density of ATIII confirms that the region encompassing amino acid residues 123-141 has the highest positive charge density within the molecule. A hydropathy plot of ATIII was generated using a method similar to that of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). This plot indicates that amino acid residues 126-140 are exposed to the exterior surface of the molecule. Based on these data, we suggest that the region corresponding to amino acid residues 114-156 is a likely site for the physiological heparin-binding domain of ATIII. We also conclude that the proposed disulfide bridges within the protein are suspect and should be re-examined (Petersen, T. E., Dudek-Wojiechowska, G., Sottrup-Jensen, L., and Magnussun, S. (1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen, D., Wiman, B., and Verstaeta, M., eds) pp. 43-54, Elsevier Scientific Publishing Co., Amsterdam).     

Effect of heparin modification on its activity in enhancing the inhibition of thrombin by antithrombin III

Studies were conducted to determine the effect of modifying specific functional groups of heparin on its antithrombin III-enhancing activity. The derivatives employed were heparin methyl ester, heparinylglycine and N-desulfated heparin. The carboxyl-modified derivatives increase the rate of inhibition of thrombin by antithrombin III, although not to the same extent as heparin. N-Desulfated heparin is devoid of any activity. Heparin methyl ester is more potent than heparinylglycine in activating antithrombin III, as exhibited by its immediate effect on the thrombin-fibrinogen reaction. However, heparinylglycine is the more effective of the two, in increasing the rate of thrombin deactivation by antithrombin III. The results indicate that although free carboxyl groups of heparin are not crucial for its binding to antithrombin III, they are important for the combination of the latter with thromobin. In contrast, N-sulfates are critical for the interaction of heparin with antithrombin III.     

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.     

Evidence that arginine-129 and arginine-145 are located within the heparin binding site of human antithrombin III

Arginyl residues of human antithrombin III have been implicated to involve in the heparin binding site [Jorgensen, A. M., Borders, C. L., & Fish, W. W. (1985) Biochem, J. 231, 59-63]. We have performed chemical modification of antithrombin with (p-hydroxyphenyl)glyoxal (HPG) in order to determine the locations of these arginine residues. Antithrombin was modified with 12 mM HPG in the absence and presence of heparin (2-fold by weight to antithrombin). In the absence of heparin, about 3-4 mol of arginines/mol of antithrombin were modified within 60 min, and the modification led to the loss of 95% of the inhibitor's heparin cofactor activity as well as heparin-induced fluorescence enhancement and 50% of its progressive inhibitory activity. In the presence of heparin, the extent of modification was diminished by 30% and modified antithrombin retained approximately 70% of its heparin cofactor activity. Peptide mapping and subsequent sequence analysis revealed that selective HPG modification occurred at Arg129 and Arg145 and that their modifications were protected upon binding of heparin to antithrombin. We conclude that Arg129 and Arg145 are situated within the heparin binding site of human antithrombin III.     

Conformation of the pentasaccharide corresponding to the binding site of heparin for antithrombin III

The conformation in solution of the pentasaccharide methyl glycoside (As-G-A*-Is-AM; 1), which represents the binding site of heparin for Antithrombin III, has been investigated using molecular mechanics and 1H-n.m.r. spectroscopy. The pentasaccharide has a rather rigid (As-G-A*) and a more flexible (Is-AM) region. A simplified model of 1, comprising two conformations, corresponding to the 1C4 and the 2S0 forms of the iduronate residue, and modified at the G-A* glycosidic linkage with respect to the energy minimum, reproduces most of the observed 3J values and n.O.e. enhancements. The possible role in the binding to Antithrombin III of a low-energy conformer, not observed in solution, is discussed.     

Thermodynamic analysis of heparin binding to human antithrombin.

The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.     

Refolding properties of antithrombin III. Mechanism of binding to heparin

The presence of two unfolding domains in antithrombin III during its denaturation in guanidinium chloride has previously been reported (Villanueva, G. B., and Allen, N. (1983) J. Biol. Chem. 258, 11010-11013). In the present work, we report the results of refolding studies on antithrombin III. Circular dichroism and intrinsic fluorescence studies have demonstrated that the first unfolding domain of low stability (midpoint at 0.7 M guanidinium chloride) is irreversible upon renaturation, whereas the second unfolding domain (midpoint at 2.3 M guanidinium chloride) is reversible. The intermediate form of antithrombin III, termed AT-IIIR, which has lost the structural features of the first domain was investigated. Clotting assays and electrophoretic analyses showed that AT-IIIR had lost 60% of heparin cofactor activity but was still capable of forming sodium dodecyl sulfate-stable complexes with thrombin. Although certain regions of this molecule do not refold to the conformation of native antithrombin III, the tryptophan residues refold to a conformation identical with the native state. This was demonstrated by fluorescence quenching, solvent perturbation, and chemical modification studies. However, the tryptophan-ascribed fluorescence enhancement and absorption difference spectrum which occur when heparin binds to antithrombin III are reduced by 70%. On the basis of these data, the binding of heparin to antithrombin III is interpreted in terms of a two-step mechanism. The primary binding occurs in the region without tryptophan and is followed by a secondary conformational rearrangement which affects the tryptophan environment. The mechanism of the binding of heparin and antithrombin III has been previously studied by kinetic methods, and the data also support a two-step mechanism. The agreement of these two studies employing entirely different approaches to the same problem lends support to the validity of this postulated mechanism.     

Importance of lysine 125 for heparin binding and activation of antithrombin

The anticoagulant sulfated polysaccharide, heparin, binds to the plasma coagulation proteinase inhibitor, antithrombin, and activates it by a conformational change that results in a greatly increased rate of inhibition of target proteinases. Lys125 of antithrombin has previously been implicated in this binding by chemical modification and site-directed mutagenesis and by the crystal structure of a complex between antithrombin and a pentasaccharide constituting the antithrombin-binding region of heparin. Replacement of Lys125 with Met or Gln in this work reduced the affinity of antithrombin for full-length heparin or the pentasaccharide by 150-600-fold at I = 0.15, corresponding to a loss of 25-33% of the total binding energy. The affinity decrease was due both to disruption of approximately three ionic interactions, indicating that Lys125 and two other basic residues of antithrombin act cooperatively in binding to heparin, and to weakened nonionic interactions. The mutations caused a 10-17-fold decrease in the affinity of the initial, weak binding step of the two-step mechanism of heparin binding to antithrombin. They also increased the reverse rate constant of the second, conformational change step by 10-50-fold. Lys125 is thus a major heparin-binding residue of antithrombin, contributing an amount of binding energy comparable to that of Arg129, but less energy than Lys114. It is the first residue identified so far that has a critical role in the initial recognition of heparin by antithrombin, but also appreciably stabilizes the heparin-induced activated state of the inhibitor. These effects are exerted by interactions of Lys125 with the nonreducing end of the heparin pentasaccharide.     

Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity

Antithrombin III Basel is a hereditary abnormal antithrombin with normal progressive inhibition activity (normal reactive site) and reduced heparin cofactor activity (impaired heparin binding site). Structures of antithrombin III Basel and normal antithrombin III isolated from the same patient were compared by peptide mapping using the dimethylaminoazobenzene isothiocyanate precolumn derivatization technique. Of the approximately 50 tryptic peptides of normal and abnormal antithrombin III, one peptide comprising residues 40-46 had a different retention time in reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide from antithrombin III Basel had a single substitution of Pro (normal) by Leu (abnormal) at position 41. This substitution is close to an Arg (residue 47) and a Trp (residue 49) which have previously been shown to be critical for heparin binding by antithrombin III. Although additional amino acid substitutions in antithrombin III Basel cannot be ruled out, this Pro-Leu replacement could cause a conformational change by increasing both the helical structure and the hydrophobicity around residue 41. These data suggest that: (i) the heparin binding site of antithrombin III encompasses the region containing residues 41, 47, and 49; and (ii) the impaired heparin cofactor activity of antithrombin III Basel is likely due to a conformational change of the heparin binding site induced by the Pro-Leu substitution at position 41.     

The heparin binding site of human extracellular-superoxide dismutase.

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the results. A previous report showed that both lysine and arginine residues probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a "cluster" of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.     

Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III.

The relationship between thrombomodulin-associated O-linked glycosammoglycans (GAGs) and the exogenous GAGs heparin or dermatan sulfate was studied in the inhibition of thrombin by antithrombin III (AT III) or heparin cofactor II (HC II). Both rabbit thrombomodulin (TM) and two glycoforms (a high-Mr form containing GAGs and a low-Mr form lacking the majority of O-linked GAGs) of a recombinant human TM deletion mutant (rec-TM) were used. The rapid inactivation of thrombin by HC II in the presence of dermatan sulfate was prevented by both the high-Mr rec-TM and the rabbit TM. In contrast, both rabbit TM treated with chondroitin ABC lyase to remove O-linked GAGs and the low-Mr form of rec-TM had only weak protecting effects. In the absence of exogeneous dermatan sulfate, thrombin inhibition by a high concentration of HC II was slightly accelerated by the high-Mr form of rec-TM but protected by rabbit TM. When thrombin inhibition by AT III in the presence of heparin was studied, both high-Mr rec-TM and rabbit TM again invoked a similar reduction of inactivation rates, whereas in the absence of exogenous heparin, both high-Mr forms accelerated thrombin inhibition by AT III. The diverse reactivities of various forms of TM towards HC II and AT III were also observed during protein C activation by the thrombin-TM complex. These results suggest that thrombin activity at the vessel wall or in fluid phase may undergo major kinetic modulations depending on the type of protease inhibitor, the presence or absence of exogenous GAGs and the glycosylation phenotype of TM. The dependence of TM anticoagulant function on the presence of an intrinsic GAG moiety suggests that variant glycoforms of this endothelial cell cofactor may be expressed differently in a species-, organ-, or tissue-specific manner as a means to regulate TM function in diverse vasculatures.     

Glycation of apolipoprotein E impairs its binding to heparin: identification of the major glycation site.

The increased glycation of plasma apolipoproteins represents a possible major factor for lipid disturbances and accelerated atherogenesis in diabetic patients. The glycation of apolipoprotein E (apoE), a key lipid-transport protein in plasma, was studied both in vivo and in vitro. ApoE was shown to be glycated in plasma very low density lipoproteins of both normal subjects and hyperglycemic, diabetic patients. However, diabetic patients with hyperglycemia showed a 2-3-fold increased level of apoE glycation. ApoE from diabetic plasma showed decreased binding to heparin compared to normal plasma apoE. The rate of Amadori product formation in apoE in vitro was similar to that for albumin and apolipoproteins A-I and A-II. The glycation of apoE in vitro significantly decreased its ability to bind to heparin, a critical process in the sequestration and uptake of apoE-containing lipoproteins by cells. Diethylenetriaminepentaacetic acid, a transition metal chelator, had no effect on the loss of apoE heparin-binding activity, suggesting that glycation rather than glycoxidation is responsible for this effect. In contrast, glycation had no effect on the interaction of apoE with amyloid beta-peptide. ApoE glycation was demonstrated to be isoform-specific. ApoE(2) showed a higher glycation rate and the following order was observed: apoE(2)>apoE(4)>apoE(3). The major glycated site of apoE was found to be Lys-75. These findings suggest that apoE is glycated in an isoform-specific manner and that the glycation, in turn, significantly decreases apoE heparin-binding activity. We propose that apoE glycation impairs lipoprotein-cell interactions, which are mediated via heparan sulfate proteoglycans and may result in the enhancement of lipid abnormalities in hyperglycemic, diabetic patients.