Heparin Binding Site Of Antithrombin Research Articles

Molecular Mechanisms of the Impaired Heparin Pentasaccharide Interactions in 10 Antithrombin Heparin Binding Site Mutants Revealed by Enhanced Sampling Molecular Dynamics.

Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments.

Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site.

Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin.

An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: application to neuroserpin reveals regions that differentially regulate inhibitory activity.

The analysis of sequence conservation is commonly used to predict functionally important sites in proteins. We have developed an approach that first identifies highly conserved sites in a set of orthologous sequences using a weighted substitution-matrix-based conservation score and then filters these conserved sites based on the pattern of conservation present in a wider alignment of sequences from the same family and structural information to identify surface-exposed sites. This allows us to detect specific functional sites in the target protein and exclude regions that are likely to be generally important for the structure or function of the wider protein family. We applied our method to two members of the serpin family of serine protease inhibitors. We first confirmed that our method successfully detected the known heparin binding site in antithrombin while excluding residues known to be generally important in the serpin family. We next applied our sequence analysis approach to neuroserpin and used our results to guide site-directed polyalanine mutagenesis experiments. The majority of the mutant neuroserpin proteins were found to fold correctly and could still form inhibitory complexes with tissue plasminogen activator (tPA). Kinetic analysis of tPA inhibition, however, revealed altered inhibitory kinetics in several of the mutant proteins, with some mutants showing decreased association with tPA and others showing more rapid dissociation of the covalent complex. Altogether, these results confirm that our sequence analysis approach is a useful tool that can be used to guide mutagenesis experiments for the detection of specific functional sites in proteins.

Limitations of conventional anticoagulant therapy and the promises of non-heparin based conformational activators of antithrombin

An elevated prothrombotic state is a major risk factor for venous thromboembolism, atrial fibrillation and cardiac strokes. The regulation of various coagulation cascade proteases plays an important role in determining a prothrombotic state. Clinically used anticoagulants are inhibitor of enzymes that are involved in the coagulation pathway, primarily thrombin and factor Xa. The conformational activation of antithrombin by heparin is a critical step in the inhibition of factor Xa by antithrombin. Despite heparin being the most potent physiological activator which enhances the otherwise very lethargic antithrombin inhibition of factor Xa by approximately 1,000-fold, the conventional heparin therapy poses serious complications because of heparin's polyanionic nature and its cross-reactivity. A number of attempts have been carried out in designing alternative non-heparin based conformational activators of antithrombin for factor Xa inhibition. Studies have demonstrated appreciable activation of antithrombin by small organic molecules, but not much is known about the specificity and effects of these molecules on structure and stability. It is assumed that these activators of antithrombin perform their function by binding to heparin binding site. A recently identified cavity which links the heparin binding site to the strand 2A for antithrombin activation also seems to be an ideal target apart the heparin binding site of antithrombin. There are opportunities in discovering more activators from naturally available organic scaffolds and also for modifying such scaffolds for designing better conformational activators with minimum associated complications. This review summarizes the current literature on the mainstay anticoagulants and non-heparin based antithrombin conformation modulators.

Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody.

N-glycosylation occurs in the variable region of about 10% of antibodies but the role of carbohydrate at this location is still poorly understood. We investigated the function of N-glycosylation in the variable region of the heavy chain of a human monoclonal antibody, mAb-LE2E9, that partially inhibits factor VIII (FVIII) activity during coagulation. Enzymatic deglycosylation indicated that the oligosaccharides do not determine the affinity of the antibody but enhance its FVIII neutralizing activity. A mutant antibody lacking the N-glycosylation site in the variable region of the heavy chain inhibited FVIII activity by up to 40%, while inhibition by the native antibody was 80%. To evaluate the physiological effect of such a FVIII inhibition, we investigated the ability of the mutant antibody devoid of N-glycosylation in the variable region to prevent thrombosis in mice with a strong prothombotic phenotype resulting from a type II deficiency mutation in the heparin binding site of antithrombin. Despite its moderate inhibition of FVIII activity, the mutant antibody significantly prevented thrombosis in treated animals. We also carried out glycan analysis of native and mutant antibodies. Modification of glycosylation in the variable region of antibodies contributes to the diversity of FVIII type II inhibition possibly by steric hindrance of the active site of FVIII by glycans, and may provide a novel strategy to modulate the functional activity of therapeutic antibodies.

Antithrombin Nagasaki (Ser 116 to Pro): a rare antithrombin variant with abnormal heparin binding presenting during pregnancy

Quantitative antithrombin deficiency constitutes an important risk factor for venous thromboembolism, stillbirth, and other complications of pregnancy. Studies suggest, however, that individuals heterozygous for missense mutations involving the heparin-binding site of antithrombin do not have a significantly increased thrombotic risk. Owing to the rarity of such mutations, it remains unclear whether any specific heparin-binding site defects might be associated with thrombotic potential. We report here the case of a pregnant woman with an exceptionally rare Type II heparin-binding site antithrombin variant. This case highlights the difficult issues that are associated with the management of Type II antithrombin deficiency during pregnancy.

The heparin-binding site of antithrombin is crucial for antiangiogenic activity

AbstractThe heparin-binding site of antithrombin is shown here to play a crucial role in mediating the antiangiogenic activity of conformationally altered cleaved and latent forms of the serpin. Blocking the heparin-binding site of cleaved or latent antithrombin by complexation with a high-affinity heparin pentasaccharide abolished the serpin's ability to inhibit proliferation, migration, capillary-like tube formation, basic fibroblast growth factor (bFGF) signaling, and perlecan gene expression in bFGF-stimulated human umbilical vein endothelial cells. Mutation of key heparin binding residues, when combined with modifications of Asn-linked carbohydrate chains near the heparin-binding site, also could abrogate the anti-proliferative activity of the cleaved serpin. Surprisingly, mutation of Lys114, which blocks anticoagulant activation of antithrombin by heparin, caused the native protein to acquire antiproliferative activity without the need for conformational change. Together, these results indicate that the heparin-binding site of antithrombin is of crucial importance for mediating the serpin's antiangiogenic activity and that heparin activation of native antithrombin constitutes an antiangiogenic switch that is responsible for turning off the antiangiogenic activity of the native serpin.

Treatment of symptomatic venous thromboembolism: improving outcomes.

Antithrombotic therapy with unfractionated heparin (UFH) and warfarin for the treatment of deep venous thrombosis (DVT) and pulmonary embolism (PE) became widely accepted in the early 1960s. Subsequent clinical trials have focused on the importance of early intravenous UFH in the treatment of venous thromboembolic events, the method of heparin administration, the importance of rapid anticoagulation to prevent recurrent venous thromboembolism (VTE), and the optimal duration of UFH administration. Low-molecular-weight heparin (LMWH) represents a significant advance over UFH therapy for VTE. Developed in the late 1980s, LMWH has become an excellent alternative to UFH because it offers superior efficacy and safety, improved pharmacokinetics, longer half-life, and once- or twice-daily subcutaneous administration without the need for laboratory monitoring. Fondaparinux is the first of a new class of antithrombotic agents that targets factor Xa. Based on studies of the heparin binding site of antithrombin, this novel pentasaccharide has superior pharmacokinetics and bioavailability when compared with LMWH and can be administered once daily. Studies in patients undergoing major orthopedic surgery and in those with proximal DVT indicate that the efficacy and safety of fondaparinux may represent an improvement over those of LMWH.

Inhibition of proliferation and induction of differentiation of endothelial cells by antithrombin

Anti-angiogenic and anti-tumor activities of latent and cleaved antithrombin has been described, and in vitro, the serpin inhibited proliferation of endothelial cells. We have recently observed that direct cellular effects of antithrombin are mediated by syndecan-4, a heparan sulfate proteoglycan known to bind to the heparin-binding site of antithrombin. Syndecan-4 is known to affect proliferation and differentiation of a variety of cell types. We have, therefore, studied direct effects of intact antithrombin on endothelial cells. Human umbilical vein and calf pulmonary artery endothelial cells were studied in the presence or absence of antithrombin concentrate or monoclonal antibody purified antithrombin with and without concomitant presence of synthetic pentasaccharide. Proliferation was assessed in BrDU incorporation and MTT assays. For testing endothelial cell differentiation, capillary tube formation was investigated in matrigel assays. Proliferation of the two types of endothelial was significantly inhibited by 1–10 U/ml of both antithrombin concentrate and antibody-purified antithrombin. Capillary tube formation induced by matrigel was augmented by the presence of 1–10 U/ml of antithrombin concentrate which was partly reversed with pentasaccharide. Results show that in vitro effects of antithrombin on angiogenesis-related endothelial cell functions may be directly exerted by the intact serpine and can be antagonised by pentasaccharide

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.

Effect of Individual Carbohydrate Chains of Recombinant Antithrombin on Heparin Affinity and on the Generation of Glycoforms Differing in Heparin Affinity

Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin, were previously shown to be expressed in BHK or CHO mammalian cell lines (I. Björk,et al.,1992,Biochem. J.286, 793–800; B. Fanet al.,1993,J. Biol. Chem.268, 17588–17596). To determine the source of the glycosylation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 96, 135, 155, and 192, was mutated to Gln to block glycosylation at these sites. Heparin-agarose chromatography of the four antithrombin variants revealed that Gln 96, Gln 135, and Gln 192 variants still displayed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gln 155 variant showed only a single functional high heparin affinity form. These results demonstrate that heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform. Analysis of heparin binding to the higher heparin affinity forms of the four variants showed that all exhibited increased heparin affinities of two- to sevenfold compared to wild-type higher heparin affinity form or to plasma antithrombin, with the Gln 135 variant showing the largest effect on this affinity. The extent of heparin-affinity enhancement was correlated with the distance of the mutated glycosylation site to the putative heparin-binding site in the X-ray structure of antithrombin. All variants displayed normal kinetics of thrombin inhibition in the absence and presence of saturating heparin, indicating that the carbohydrate chains solely affected heparin binding and not heparin-activation or proteinase-binding functions. These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect heparin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin.

Role of Arginine 132 and Lysine 133 in Heparin Binding to and Activation of Antithrombin

The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The Kd values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the Kd values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.

Heparin binding affinity of normal and genetically modified antithrombin III measured using a monoclonal antibody to the heparin binding site of antithrombin III.

The inhibitory activity of the plasma serine proteinase inhibitor antithrombin III (AT III) is enhanced about 1000-fold upon binding to heparin. We have determined the dissociation constants, Kd, of 48.8 nM for the heparin-AT III interaction, of 175 nM for the specific pentasaccharide-AT III interaction, and of 13 microM for the low-affinity heparin-AT III interaction, using a binding assay based on a monoclonal antibody (MAb) that recognizes an epitope at or close to the heparin binding site of AT III. The heparin binding affinities and proportions of normal and variant AT III in plasma from patients with mutations of AT III have been quantitated for the first time using the binding assay. Substitution mutations in three regions of AT III have been investigated: (i) mutations in the reactive site loop affecting Ala382, Arg393, and Ser394 have no discernible effect on heparin binding; (ii) mutations in the previously identified N-terminal heparin binding region, affecting Arg47, Leu99, and Arg129, produce variant AT III molecules with heparin affinities reduced 11-924-fold, the largest reduction being observed for the substitution mutation Arg47-Cys in Padua 2, which has an affinity of 65.6 microM; (iii) mutations in the hydrophobic regions around strand 1C of the C terminus, affecting Phe402, Ala404, Asn405, Pro407, and Pro429, have pleiotropic effects that include the production of reduced amounts of low-affinity AT III with dissociation constants from 6 to 43 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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).

On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR spectroscopy.

The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.

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.

Predictions of the secondary structure of antithrombin III and the location of the heparin-binding site.

The secondary structure of antithrombin III was investigated using the predictive scheme based on the amino acid sequence (Fasman, G. D., Chou, P. Y., and Adler, A. J. (1976) Biophys. J. 16, 1201-1238. The relative composition of the conformational regions is: 31% alpha-helix, 16% beta-sheet, 9% beta-turns, and the rest of the molecule in unordered conformation. When the alpha-helical regions are carefully delineated, 44% are predicted to be unstable helices because of unfavorable electrostatic interactions. Circular dichroism studies showed that the disruption of the first unfolding domain in antithrombin III by low concentration of guanidinium chloride (Villanueva, G. B., and Allen, N. (1983) J. Biol. Chem. 258, 14048-14053) was accompanied by a 42% decrease in the alpha-helical content. This suggested that the first unfolding domain demonstrated to be essential for heparin binding (Villanueva, G. B., and Allen, N. (1983) J. Biol. Chem. 258, 11010-11013) corresponds to the predicted unstable helical region. Model-binding studies of the unstable helical segment which contain tryptophan and lysine residues reveal that Lys-282, Lys-286, and Lys-289 are aligned on the same side of the alpha-helix. A model of the antithrombin III-binding octasaccharide (Lindahl, U., Backstrom, G., Thunberg, L., and Leder, I. G. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555) was also constructed based on available NMR and x-ray data of the repeating units in heparin. These two models can be positioned such that the three lysine residues in the alpha-helix can be matched for maximal interaction with the three sulfate groups in the octasaccharide demonstrated as essential for binding antithrombin III. Based on these models, it is proposed that the unstable helical segment composed of residues 281-292 is the heparin-binding site in antithrombin III.

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.