Antiangiogenic antithrombin blocks the heparan sulfate-dependent binding of proangiogenic growth factors to their endothelial cell receptors: evidence for differential binding of antiangiogenic and anticoagulant forms of antithrombin to proangiogenic heparan sulfate domains

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.     

Characterization of the conformational alterations, reduced anticoagulant activity, and enhanced antiangiogenic activity of prelatent antithrombin

A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion of native to latent antithrombin (Larsson, H., Akerud, P., Nordling, K., Raub-Segall, E., Claesson-Welsh, L., and Björk, I. (2001) J. Biol. Chem. 276, 11996–12002). Here, we show that the previously characterized prelatent antithrombin is a mixture of native antithrombin and a modified, true prelatent antithrombin that are resolvable by heparin-agarose chromatography. Kinetic analyses revealed that prelatent antithrombin is an intermediate in the conversion of native to latent antithrombin whose formation is favored by stabilizing anions of the Hofmeister series. Purified prelatent antithrombin had reduced anticoagulant function compared with native antithrombin, due to a reduced heparin affinity and consequent impaired ability of heparin to either bridge prelatent antithrombin and coagulation proteases in a ternary complex or to induce full conformational activation of the serpin. Significantly, prelatent antithrombin possessed an antiangiogenic activity more potent than that of latent antithrombin, based on the relative abilities of the two forms to inhibit endothelial cell growth. The prelatent form was conformationally altered from native antithrombin as judged from an attenuation of tryptophan fluorescence changes following heparin activation and a reduced thermal stability. The alterations are consistent with the limited structural changes involving strand 1C observed in a prelatent form of plasminogen activator inhibitor-1 (Dupont, D. M., Blouse, G. E., Hansen, M., Mathiasen, L., Kjelgaard, S., Jensen, J. K., Christensen, A., Gils, A., Declerck, P. J., Andreasen, P. A., and Wind, T. (2006) J. Biol. Chem. 281, 36071–36081), since the ¹H NMR spectrum, electrophoretic mobility, and proteolytic susceptibility of prelatent antithrombin most resemble those of native rather than those of latent antithrombin. Together, these results demonstrate that limited conformational alterations of antithrombin that modestly reduce anticoagulant activity are sufficient to generate antiangiogenic activity.Antithrombin and its glycosaminoglycan activators, heparin and heparan sulfate, are well established anticoagulant regulators of blood clotting proteases (1–3). Antithrombin acts as an anticoagulant by irreversibly inhibiting clotting proteases through a conformational trapping mechanism that is unique to the serpin superfamily of proteins of which antithrombin is a member (4, 5). Heparin and heparan sulfate are required to activate antithrombin to ensure that clotting proteases are inhibited at a physiologically significant rate. This activating effect is the basis for the widespread clinical use of heparin for anticoagulant therapy. The activation results from heparin binding to antithrombin through a specific pentasaccharide sequence and inducing a conformational change in the serpin (6, 7). Conformational activation greatly enhances the affinity of antithrombin for heparin and exposes exosites on the inhibitor that promote its interaction with the target proteases, factor Xa and factor IXa (8–11). Heparin additionally accelerates antithrombin-protease reactions by providing a bridging exosite for the protease to bind next to antithrombin and thereby promote its interaction with the serpin in a ternary complex with heparin (12–14). The latter is the predominant mechanism involved in accelerating antithrombin inhibition of thrombin.Antithrombin has more recently been shown to express a potent antiangiogenic activity after having undergone conformational alterations induced either by limited proteolysis in a reactive protease binding loop or by mild heating (15, 16). Such conformational alterations transform the native metastable protein to a much more stable but inactive form in which the reactive loop has inserted into the major β-sheet, the A-sheet, of the serpin (17, 18). These conformationally altered forms of antithrombin are produced under physiologic conditions (19) and have antiangiogenic activity comparable with that of other naturally produced angiogenesis inhibitors (20). The requirement for conformational change to generate antiangiogenic activity sets antithrombin apart from other serpins, such as pigment epithelium-derived factor, maspin, and kallistatin, which have been shown to also possess antiangiogenic activity but without the need for conformational change (21–23). Interestingly, mild heat treatment was also found to produce a distinct form of antithrombin, termed the prelatent form, which possessed antiangiogenic activity (24). However, unlike the cleaved and latent forms of antithrombin that have lost their ability to inhibit clotting proteases, prelatent antithrombin was found to retain clotting protease inhibitory activity and to have its reaction with these proteases accelerated by heparin. The only reported difference between the native and prelatent forms of antithrombin was a greater susceptibility of the latter to proteolysis by nontarget proteases. Since these findings suggested that an antiangiogenic epitope may be generated by more limited conformational changes than those having occurred in the cleaved and latent forms of antithrombin, it has been of interest to characterize the nature of these conformational differences between then native and prelatent forms of the serpin.In the present report, we show that prelatent antithrombin generated as in past studies is actually a mixture of a novel antiangiogenically active species of antithrombin, the true prelatent form, and the antiangiogenically inactive native serpin. The purified prelatent antithrombin has a more potent antiangiogenic activity than latent or cleaved antithrombins but retains the anticoagulant functions of the native serpin. It is shown to be generated as an intermediate on the pathway to latent antithrombin in the presence of stabilizing anions of the Hofmeister series (25, 26). Significantly, only limited conformational alterations are involved in transforming native to prelatent antithrombin, as judged from the modest changes in heparin affinity, heparin-induced conformational activation, thermal stability, electrophoretic mobility, proteolytic susceptibility, and ¹H NMR spectrum. Overall, our findings suggest that limited conformational changes, comparable with those recently demonstrated in a prelatent form of the serpin, plasminogen activator inhibitor-1 (27), are required to express antiangiogenic activity in antithrombin.     

Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes

The serpin, antithrombin, requires allosteric activation by a sequence-specific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar induced-fit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric activation.     

A novel anti-angiogenic form of antithrombin with retained proteinase binding ability and heparin affinity

Latent antithrombin, an inactive antithrombin form with low heparin affinity, has previously been shown to efficiently inhibit angiogenesis and tumor growth. We now show that heat treatment similar to that used for preparation of latent antithrombin also transforms antithrombin to another form, which we denote prelatent, with potent anti-angiogenic and anti-tumor activity but with retained proteinase- and heparin-binding properties. The ability of prelatent antithrombin to inhibit angiogenesis is presumably due to a limited conformational change, which may partially resemble that in latent antithrombin. Such a change is evidenced by a different cleavage pattern of prelatent than of native antithrombin by nontarget proteinases. Prelatent antithrombin exerts its anti-angiogenic effect by a similar mechanism as latent antithrombin, i.e. by inhibiting focal adhesion formation and focal adhesion kinase activity, thereby leading to decreased proliferation of endothelial cells. The proteinase inhibitory fractions in commercial antithrombin preparations, which have been heat treated during production, also have anti-angiogenic activity, comparable with that of the prelatent antithrombin form.     

The infective polymerization of conformationally unstable antithrombin mutants may play a role in the clinical severity of antithrombin deficiency

Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.     

Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin

The interference of the heparin-neutralizing plasma component S protein (vitronectin) (Mr = 78,000) with heparin-catalyzed inhibition of coagulation factor Xa by antithrombin III was investigated in plasma and in a purified system. In plasma, S protein effectively counteracted the anticoagulant activity of heparin, since factor Xa inhibition was markedly reduced in comparison to heparinized plasma deficient in S protein. Using purified components in the presence of heparin, S protein induced a concentration-dependent reduction of the inhibition rate of factor Xa by antithrombin III. This resulted in a decrease of the apparent pseudo-first order rate constant by more than 10-fold at a physiological ratio of antithrombin III to S protein. S protein not only counteracted the anticoagulant activity of commercial heparin but also of low molecular weight forms of heparin (mean Mr of 4,500). The heparin-neutralizing activity of S protein was found to be mainly expressed in the range 0.2-10 micrograms/ml of high Mr as well as low Mr heparin. S protein and high affinity heparin reacted with apparent 1:1 stoichiometry to form a complex with a dissociation constant KD = 1 X 10(-8) M as determined by a functional assay. As deduced from dot-blot analysis, direct interaction of radiolabeled heparin with S protein revealed a dissociation constant KD = 4 X 10(-8) M. Heparin binding as well as heparin neutralization by S protein increased significantly when reduced/carboxymethylated or guanidine-treated S protein was employed indicating the existence of a partly buried heparin-binding domain in native S protein. Radiolabeled heparin bound to the native protein molecule as well as to a BrCN fragment (Mr = 12,000) containing the heparin-binding domain as demonstrated by direct binding on nitrocellulose replicas of sodium dodecyl sulfate-polyacrylamide gels. Kinetic analysis revealed that the heparin neutralization activity of S protein in the inhibition of factor Xa by antithrombin III could be mimicked by a synthetic tridecapeptide from the amino-terminal portion of the heparin-binding domain. These data provide evidence that the heparin-binding domain of S protein appears to be unique in binding to heparin and thereby neutralizing its anticoagulant activity in the inhibition of coagulation factors by antithrombin III. The induction of heparin binding and neutralization may be considered a possible physiological mechanism initiated by conformational alteration of the S protein molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.     

Properties of thrombin- and elastase-modified human antithrombin III

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydropathic interaction analyses of small organic activators binding to antithrombin

Recently we designed the first small organic ligands, sulfated flavanoids and flavonoids, that act as activators of antithrombin for accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade [Gunnarsson and Desai, Bioorg. Med. Chem. Lett. (2003) 13:579]. To better understand the binding properties of these activators at a molecular level, we have utilized computerized hydropathic interaction (HINT) analyses of the sulfated molecules interacting in two plausible electropositive regions, the pentasaccharide- and extended heparin-binding sites, of antithrombin in its native and activated forms. HINT analyses indicate favorable multi-point interactions of the activators in both binding sites of the two forms of antithrombin. Yet, HINT predicts better interaction of most activators, except for (-)-catechin sulfate, with the activated form of antithrombin than with the native form supporting the observation in solution that these molecules function as activators of the inhibitor. Further, whereas (+)-catechin sulfate recognized the activated form of antithrombin better in both the pentasaccharide- and extended heparin- binding sites, the native form was better recognized by (-)-catechin sulfate, thus explaining its weaker binding and activation potential in solution. A reasonable linear correlation between the overall HINT score and the solution free energy of binding of the sulfated activators was evident. This investigation indicates that HINT is a useful tool in understanding interactions of antithrombin with small sulfated organic ligands at a molecular level, has some good predictive properties, and is likely to be useful for rational design purposes.     

Preparative conversion of native human antithrombin to the latent form

Human native antithrombin (AT) can be converted to a partially denaturated form of AT, known as latent AT (L-AT). This latent form of AT has been shown to exhibit strong antiangiogenic activity and also to suppress tumor growth in mice models. In the present work, a method is presented which induces the conversion of native AT to L-AT, using incubation at 60 degrees C, for 16 h, with 0.9 M ammonium sulfate, in 5mM Hepes buffer, pH 7.4, giving a recovery of more than 70%. L-AT was determined by integration of the low heparin affinity peak when analyzed by the affinity chromatography method. Native polyacrylamide gel electrophoresis was used to show that the preparation contained no aggregates. Hydrophobic interaction chromatography was also used for the separation of AT and L-AT.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

Two latent and two hyperstable polymeric forms of human neuroserpin

Human neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin β-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of quasi-native protein molecules. Although hNS polymerization has been intensely studied, a general agreement about serpin polymer organization is still lacking. Here we report a biophysical characterization of native hNS that is shown to undergo two distinct conformational transitions, at 55°C and 85°C, both leading to distinct latent and polymeric species. The latent and polymer hNS forms obtained at 45°C and 85°C differ in their chemical and thermal stabilities; furthermore, the hNS polymers also differ in size and morphology. Finally, the 85°C polymer shows a higher content of intermolecular β-sheet interactions than the 45°C polymer. Together, these results suggest a more complex conformational scenario than was previously envisioned, and, in a general context, may help reconcile the current contrasting views on serpin polymerization.     

Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States

Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations'' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.     

Effect of polyanetholesulphonic acid and xylan sulphate on antithrombin III activity.

Both polyanetholesulphonic acid and xylan sulphate prolonged the partial thromboplastin clotting time of plasma. The anticoagulant effect of both compounds was reduced following pre-incubation of plasma with antiserum specific for antithrombin III. Polyanetholesulphonic acid was more effective than xylan sulphate in inhibiting thrombin-initiated clotting of plasma, and potentiated antithrombin III inhibition of both thrombin and Xa. Xylan sulphate was more effective in potentiating antithrombin III inhibition of Xa than of thrombin. These differential effects of xylan sulphate on different blood serine proteases are discussed in terms of the antithrombin III-mediated anticoagulant activity of heparin.     

Antithrombin-mediated anticoagulant activity of sulfated polysaccharides: different mechanisms for heparin and sulfated galactans

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.     

Inactivation of human antithrombin by neutrophil elastase. Kinetics of the heparin-dependent reaction

Human neutrophil elastase catalyzes the inactivation of antithrombin by a specific and limited proteinolytic cleavage. This inactivation reaction is greatly accelerated by an active anticoagulant heparin subfraction with high binding affinity for antithrombin. A potentially complex reaction mechanism is suggested by the binding of both neutrophil elastase and antithrombin to heparin. The in vitro kinetic behavior of this system was examined under two different conditions: 1) at a constant antithrombin concentration in which the active anticoagulant heparin was varied from catalytic to saturating levels; and 2) at a fixed, saturating heparin concentration and variable antithrombin levels. Under conditions of excess heparin, the inactivation could be continuously monitored by a decrease in the ultraviolet fluorescence emission of the inhibitor. A Km of approximately 1 microM for the heparin-antithrombin complex and a turnover number of approximately 200/min was estimated from these analyses. Maximum acceleratory effects of heparin on the inactivation of antithrombin occur at heparin concentrations significantly lower than those required to saturate antithrombin. The divergence in acceleratory effect and antithrombin binding contrasts with the anticoagulant functioning of heparin in promoting the formation of covalent antithrombin-enzyme complexes and is likely to derive from the fact that neutrophil elastase is not consumed in the inactivation reaction. A size dependence was observed for the heparin effect since an anticoagulantly active octasaccharide fragment of heparin, with avid antithrombin binding activity, was without effect on the inactivation of antithrombin by neutrophil elastase. Despite the completely nonfunctional nature of elastase-cleaved antithrombin and the altered physical properties of the inhibitor as indicated by fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the inactivated inhibitor exhibited a circulating half-life in rabbits that was indistinguishable from native antithrombin. These results point to an unexpected and apparently contradictory function for heparin which may relate to the properties of the vascular endothelium in pathological situations.     

Heterogeneity of heparin: characterization of one hundred components with different anticoagulant activities by a combination of electrophoretic and affinity chromatography methods

A comparative study of heparin fractions obtained by affinity chromatography, electrofocusing, selective barium precipitation, polyacrylamide and agarose gel electrophoresis is reported. It is concluded that commercial heparin preparations are heterogeneous, containing at least 120 components which differ in molecular weight, in degree of affinity for antithrombin, and in their distribution in monomeric and dimeric forms. High anticoagulant activity for some heparin fractions was obtained by most of the methods used.     

Serpin crystal structure and serpin polymer structure

Serpins are a family of structurally homologous proteins having metastable native structures. As a result, a serpin variant destabilized by mutation(s) has a tendency to undergo conformational changes leading to inactive forms, e.g., the latent form and polymer. Serpin polymers are involved in a number of conformational diseases. Although several models for polymer structure have been proposed, the actual structure remains unknown. Here, we provide a comprehensive list of serpins, both free and in complexes, deposited in the Protein Data Bank. Our discussion focuses on structures that potentially can contribute to a better understanding of polymer structure.     

Recombinant human elastin polypeptides self-assemble into biomaterials with elastin-like properties

Processes involving self-assembly of monomeric units into organized polymeric arrays are currently the subject of much attention, particularly in the areas of nanotechnology and biomaterials. One biological example of a protein polymer with potential for self-organization is elastin. Elastin is the extracellular matrix protein that imparts the properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Tropoelastin, the approximately 70 kDa soluble monomeric form of elastin, is highly nonpolar in character, consisting essentially of 34 alternating hydrophobic and crosslinking domains. Crosslinking domains contain the lysine residues destined to form the covalent intermolecular crosslinks that stabilize the polymer. We and others have suggested that the hydrophobic domains are sites of interactions that contribute to juxtaposition of lysine residues in preparation for crosslink formation. Here, using recombinant polypeptides based on sequences in human elastin, we demonstrate that as few as three hydrophobic domains flanking two crosslinking domains are sufficient to support a self-assembly process that aligns lysines for zero-length crosslinking, resulting in formation of the crosslinks of native elastin. This process allows fabrication of a polymeric matrix with solubility and mechanical properties similar to those of native elastin.     

Plasma antithrombin III activity in patients with pulmonary thromboembolism]

A decreased plasma antithrombin III activity has been noted in 12 out of 20 patients. In 2 patients it was most probably congenital defect, whereas in the remaining 10 patients--acquired. The observed disorders in the activity of antithrombin III with particular reference to anticoagulant therapy have been discussed.