Low-affinity Heparin Research Articles

Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors.

Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.

PKC (Protein Kinase C)-δ Modulates AT (Antithrombin) Signaling in Vascular Endothelial Cells.

Native and latent conformers of AT (antithrombin) induce anti-inflammatory and proapoptotic signaling activities, respectively, in vascular endothelial cells by unknown mechanisms. Synd-4 (syndecan-4) has been identified as a receptor that is involved in transmitting signaling activities of AT in endothelial cells. Approach and Results: In this study, we used flow cytometry, signaling assays, immunoblotting and confocal immunofluorescence microscopy to investigate the mechanism of the paradoxical signaling activities of high-affinity heparin (native) and low-affinity heparin (latent) conformers of AT in endothelial cells. We discovered that native AT binds to glycosaminoglycans on vascular endothelial cells via its heparin-binding D-helix to induce anti-inflammatory signaling responses by recruiting PKC (protein kinase C)-δ to the plasma membrane and promoting phosphorylation of the Synd-4 cytoplasmic domain at Ser179. By contrast, the binding of latent AT to endothelial cells to a site(s), which is not competed by the native AT, induces a proapoptotic effect by localizing PKC-δ to the perinuclear/nuclear compartment in endothelial cells. Overexpression of a dominant-negative form of PKC-δ resulted in inhibition of anti-inflammatory and proapoptotic signaling activities of both native and latent AT. These results indicate that the native and latent conformers of AT may exert their distinct intracellular signaling effects through differentially modulating the subcellular localization of PKC-δ in endothelial cells.

High levels of latent antithrombin in plasma from patients with antithrombin deficiency.

Antithrombin is an anticoagulant serpin that efficiently inhibits multiple procoagulant proteases. The cost for the structural flexibility required for this function is the vulnerability to mutations that impact its folding pathway. Most conformational mutations identified in serpins cause polymerisation. Only three mutations in SERPINC1 affecting two residues have been found to favour transformation to the latent conformation of antithrombin, another hyperstable non-anticoagulant form with strong antiangiogenic activity that constitutes 3 % of plasma antithrombin in healthy subjects. The analysis of latent antithrombin in 141 unrelated patients with antithrombin deficiency carrying 89 different SERPINC1 mutations identified four cases with higher levels than that of controls: p.Pro439Thr, p.Pro461Ser, p.Met283Val, and p.His401Tyr, the last also with circulating polymers. Heating of plasma at 42ºC exacerbated the transformation to the latent conformation in p.Pro439Thr and p.Pro461Ser. The conformational effect of p.Met283Val, the mutation associated with the highest levels of latent antithrombin detected in four members of a family, was verified in a recombinant model. Antithrombin deficiency in these cases should be classified as pleiotropic based on the impaired reactivity and low heparin affinity of the variant. Despite high levels of latent antithrombin (up to 80 µg/ml in p.Met283Val carriers), no vascular defects were described in carriers of these mutations. In conclusion, our study identifies new residues involved in the structural stability of antithrombin (and potentially of all serpins). High levels of endogenous latent antithrombin seem to play a minor antiangiogenic effect. Finally, pleiotropic deficiencies may be caused by mutations inducing transformation to the latent conformation.

Oxidized antithrombin is a dual inhibitor of coagulation and angiogenesis: Importance of low heparin affinity

Endogenous proteins that promote vascular endothelial cell based inhibition of angiogenesis are an attractive option for antitumor therapy. Inactive cleaved and latent conformations of antithrombin (AT) are antiangiogenic, but not its native form which is an inhibitor of proteases involved in blood coagulation. Unlike native, the cleaved and latent conformations are reactive center loop inserted conformations which binds heparin with very low affinity. We use a sulfoxy modified AT to assess the role of reactive center loop insertion and heparin affinity in antiangiogenic function. Chorioallantoic membrane assay (CAM) shows that antiangiogenic activity of latent and oxidized AT are better than thalidomide, a potent antiangiogenic drug. Wound healing experiments suggest that latent and oxidized conformations can influence endothelial cell migration. Latent and cleaved conformations of AT shows an increase in α-helical content in the presence of unfractionated heparin, but not the oxidized AT. Unlike the loop inserted polymer, cleaved and latent conformations, oxidized AT has factor Xa inhibitory activity indicating that loop insertion is not necessary for antiangiogenic role. The results of our study establish that active conformation of AT can become antiangiogenic while maintaining its anticoagulant activity possibly through chelation of low affinity heparin in the vicinity of endothelial cell.

Angiostatic and chemotactic activities of the CXC chemokine CXCL4L1 (platelet factor-4 variant) are mediated by CXCR3

AbstractWe investigated possible cellular receptors for the human CXC chemokine platelet factor-4 variant/CXCL4L1, a potent inhibitor of angiogenesis. We found that CXCL4L1 has lower affinity for heparin and chondroitin sulfate-E than platelet factor-4 (CXCL4) and showed that CXCL10 and CXCL4L1 could displace each other on microvascular endothelial cells. Labeled CXCL4L1 also bound to CXCR3A- and CXCR3B-transfectants and was displaced by CXCL4L1, CXCL4, and CXCL10. The CXCL4L1 anti-angiogenic activity was blocked by anti-CXCR3 antibodies (Abs) in the Matrigel and cornea micropocket assays. CXCL4L1 application in CXCR3−/− or in wild-type mice treated with neutralizing anti-CXCR3 Abs, resulted in reduced inhibitory activity of CXCL4L1 on tumor growth and vascularization of Lewis lung carcinoma. Furthermore, CXCL4L1 and CXCL4 chemoattracted activated T cells, human natural killer cells, and human immature dendritic cells (DCs). Migration of DCs toward CXCL4 and CXCL4L1 was desensitized by preincubation with CXCL10 and CXCL11, inhibited by pertussis toxin, and neutralized by anti-CXCR3 Abs. Chemotaxis of T cells, natural killer cells, and DCs is likely to contribute to the antitumoral action. However, the in vivo data indicate that the angiostatic property of CXCL4L1 is equally important in retarding tumor growth. Thus, both CXCR3A and CXCR3B are implicated in the chemotactic and vascular effects of CXCL4L1.

The structure of adeno-associated virus serotype 3B (AAV-3B): Insights into receptor binding and immune evasion

Adeno-associated viruses (AAVs) are leading candidate vectors for human gene therapy. AAV serotypes have broad cellular tropism and use a variety of cellular receptors. AAV serotype 3 binds to heparan sulfate proteoglycan prior to cell entry and is serologically distinct from other serotypes. The capsid features that distinguish AAV-3B from other serotypes are poorly understood. The structure of AAV-3B has been determined to 2.6A resolution from twinned crystals of an infectious virus. The most distinctive structural features are located in regions implicated in receptor and antibody binding, providing insights into the cell entry mechanisms and antigenic nature of AAVs. We show that AAV-3B has a lower affinity for heparin than AAV-2, which can be rationalized by the distinct features of the AAV-3B capsid. The structure of AAV-3B provides an additional foundation for the future engineering of improved gene therapy vectors with modified receptor binding or antigenic characteristics.

Hyperglycaemia impairs antithrombin secretion: Possible contribution to the thrombotic risk of diabetes

Recent data support that diabetes might be a conformational disease. Certainly, hyperglycaemia causes a broad range of deleterious effects that might facilitate protein aggregation. We have evaluated the effects of hyperglycaemia on antithrombin, a conformationally sensitive serpin with a potent anticoagulant role. Moreover, these studies might also help to understand the thrombotic risk associated to diabetes. We incubated in vitro plasma and purified antithrombin and human hepatoma cells (HepG2) with methyl-glyoxal and glucose. Moreover, a mouse model of acute diabetes was generated with streptozotocin. Antigen, anti-FXa activity, heparin affinity and conformational features of antithrombin were analysed. Histological and intracellular features and distribution of antithrombin in HepG2 and livers of mice were also evaluated. Hyperglycaemia i n vitro induced a transition of antithrombin to a form with low heparin affinity that explained the loss of anticoagulant activity, without generation of abnormal conformers (polymers or latent antithrombin). However, these effects were not observed on circulating antithrombin from diabetic mice. In contrast, hyperglycaemia in vivo had significant effects on intracellular antithrombin, which was retained, forming microaggregates within the lumen of dilated cisterns of the endoplasmic reticulum. These effects explained the moderate type I deficiency observed in diabetic mice. Similar intracellular consequences were observed for another hepatic serpin, α1-antitrypsin. Our data further support that diabetes has conformational effects on structurally sensitive proteins. These effects on antithrombin, the main natural anticoagulant, might contribute to the hypercoagulable status of diabetic patients.

Release rate controls biological activity of nerve growth factor released from fibrin matrices containing affinity‐based delivery systems

Previously, combinatorial techniques were used to identify peptide sequences exhibiting high, medium, and low affinity for heparin. Bidomain peptides were synthesized containing a transglutaminase sequence for one domain and one of the heparin-affinity sequences for the other domain. A delivery system was made consisting of bi-domain peptides, heparin, and nerve growth factor (NGF), which binds to heparin with moderate affinity. The goal of this research was to determine whether peptide affinity for heparin and the molar ratio of peptide to heparin affected the release rate of NGF from the delivery system and the biological activity of NGF released. This study also explored whether peptide affinity modulated biological activity independent of release rate. Mathematically modeling the delivery system confirmed that release could be controlled by both peptide affinity and molar ratio of peptide to heparin. Experimentally, the rate of NGF release from the delivery system was found to be affected by peptide affinity and molar ratio. The delivery system presented biologically active NGF as assayed by embryonic chick dorsal root ganglia (DRGs) neurite extension, where extension was similar to or increased for DRGs grown in fibrin matrices containing the delivery system compared to DRGs grown with NGF in the culture media. Furthermore, by modulating the molar ratio of peptide to heparin in the delivery system, similar release rates of NGF were obtained for different affinity peptides and these conditions promoted similar levels of neurite extension, demonstrating that release rate appears to be the main mechanism controlling the biological activity of released NGF.

Mycobacterium smegmatis produces an HBHA homologue which is not involved in epithelial adherence

Mycobacterium tuberculosis produces heparin-binding hemagglutinin (TB-HBHA), an adhesin involved in binding to non-professional phagocytes and in extrapulmonary dissemination. TB-HBHA binds sulphated glycoconjugates through its C-terminal lysine-rich domain and can be purified by heparin-Sepharose chromatography. Homologues of HBHA are found in other pathogenic mycobacteria, but previous investigations failed to demonstrate them in non-pathogenic Mycobacterium smegmatis. We identified a gene encoding a HBHA-like protein, named MS-HBHA, from the complete M. smegmatis genome. The deduced MS-HBHA amino acid sequence revealed 68% identity with that of TB-HBHA and contains lysine-rich repeats in its C-terminal domain. However, in contrast to TB-HBHA, the lysine-rich domain of MS-HBHA is preceded by a stretch of acidic residues. This difference likely explains the low affinity for heparin displayed by MS-HBHA compared to TB-HBHA. Isolation by heparin-Sepharose chromatography procedure and mass spectrometry analysis indicated that MS-HBHA, similar to TB-HBHA contains several methylated lysine residues in its C-terminal domain. Although MS-HBHA is associated with M. smegmatis cell wall fractions, it does not seem to play a role in epithelial adherence and its function remains unknown. We therefore conclude that TB-HBHA may have evolved as an adhesin in pathogenic mycobacteria from a homolog that serves a different function in a saprophytic mycobacterium.

Mechanism of Poly(acrylic acid) Acceleration of Antithrombin Inhibition of Thrombin: Implications for the Design of Novel Heparin Mimics

The bridging mechanism of antithrombin inhibition of thrombin is a dominant mechanism contributing a massive approximately 2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antithrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in thrombin inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269). To better understand this interesting phenomenon, we have studied the mechanism of PAA-dependent acceleration in antithrombin inhibition of thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antithrombin in both the pentasaccharide- and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the K(D) of the PAA-antithrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic forces is miniscule, resulting in an interaction that is significantly weaker than that observed for heparins. A bell-shaped profile of the observed rate constant for antithrombin inhibition of thrombin as a function of PAA concentration was observed, suggesting that inhibition proceeds through the "bridging" mechanism. The knowledge gained in this mechanistic study highlights important rules for the rational design of orally available heparin mimics.

Effect of antithrombin III on neutrophil deformability

As the spherical diameter of pulmonary capillaries is smaller than that of neutrophils, increased neutrophil stiffness or conversely, decreased neutrophil deformability is a key step in the initial sequestration of neutrophils within the lungs during inflammatory processes. Antithrombin III (AT) is known to exert a therapeutic effect against disseminated intravascular coagulation, and accumulating evidence suggests that AT also has anti-inflammatory properties. The mechanisms of its anti-inflammatory effects remain unclear, but in a rat endotoxin model, AT apparently inhibited neutrophil sequestration in the lung. In the present in vitro study, therefore, we examined the effect of AT on the deformability of human neutrophils and correlated those findings with their F-actin content. Isolated human neutrophils were stimulated with formyl-Met-Leu-Phe (1 muM, 2 min) in the presence or absence of the alpha, beta, or low heparin-affinity isoforms of AT (1 IU/ml, 20 min), and deformability was evaluated using a filter assay system. Neutrophils were also stained with fluorescein isothiocyanate-phalloidin and subjected to a fluorescein-activated cell sorter scan to assess F-actin content. The results showed that pretreatment with any of the three AT isoforms similarly inhibited the decreased neutrophil deformability and increased F-actin content of stimulated cells. Notably, heparinase had no effect on deformability or F-actin content in the presence or absence of AT, which was somewhat unexpected, as heparin sulfate proteoglycans likely function as AT receptors. These findings suggested that AT inhibits the increase in neutrophil stiffness seen during inflammatory processes by inhibiting actin polymerization via a heparin-independent pathway.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin -Like Activation Mechanism.

Heparin cofactor II(HCII) is a serpin which specifically inhibits thrombin. Like antithrombin(AT) it requires glycosaminoglycan(GAG) binding before full activation. However unlike antithrombin no specific activators have been identified. What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of GAG binding specificity. We have investigated the heparin binding properties of HCII using size fractionated heparins. The affinity of heparins for HCII, ranging in size from 8 to 26 saccharide units, were determined by following change in fluorescence of the extrinsic probe TNS. The effect of heparin concentration(from 1 to 100nM) on the rate of thrombin inhibition was determined for the same heparins under psuedo first-order conditions. Based on these studies we can conclude that binding is non specific with a minimal heparin length of 13 monosaccaride units required. The observed dependence of catalytic activity on heparin length is caused entirely by the size dependence of heparin affinity for HCII. By studying affinity at different ionic strengths it is clear that binding is critically dependent on ionic strength with a very weak non-ionic contribution to binding. Rapid binding experiments were conducted under psuedo first-order conditions in heparin using a stopped-flow fluorometer. These studies of heparin binding indicate an induced- fit mechanism which involves a conformational change in HCII. The low affinity of HCII can now be understood as a failure of interactions to reduce the rate of the reverse conformational, in a manner analogous to the interaction of AT with low affinity heparin. HCII is activated by a fully allosteric mechanism which produces a 2000 fold increase in activation on heparin binding. We conclude that the heparin binding mechanism of HCII is closely analogous to that of AT, and that the design or discovery of high affinity, HCII-specific GAGs is thus feasible.

The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin-like Activation Mechanism

The serpin heparin cofactor II (HCII) is a glycosaminoglycan-activated inhibitor of thrombin that circulates at a high concentration in the blood. The antithrombotic effect of heparin, however, is due primarily to the specific interaction of a fraction of heparin chains with the related serpin antithrombin (AT). What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of glycosaminoglycan binding specificity. In this report we investigate the heparin binding properties of HCII and conclude that binding is nonspecific with a minimal heparin length of 13 monosaccharide units required and affinity critically dependent on ionic strength. Rapid kinetics of heparin binding indicate an induced fit mechanism that involves a conformational change in HCII. Thus, HCII binds to heparin in a manner analogous to the interaction of AT with low affinity heparin. A fully allosteric 2000-fold heparin activation of thrombin inhibition by HCII is demonstrated for heparin chains up to 26 monosaccharide units in length. We conclude that the heparin-binding mechanism of HCII is closely analogous to that of AT and that the induced fit mechanism suggests the potential design or discovery of specific HCII agonists.

037 Fibroblast growth factor – binding protein cDNA and truncated variants are active in diabetic wound healing

Fibroblast growth factor – binding protein (FGF‐BP) is a secreted protein that appears to function as a low affinity heparin –binding protein. FGF‐BP binds to FGF‐1 and ‐2 in a non‐covalent, reversible manner to mobilize and solubilize these growth factors from their storage sites in the extracellular matrix. FGF‐BP is involved in both developmental and adult tissue homeostasis as well as in angiogenesis and tumorogenesis involving FGF‐1/2. FGF‐BP is overexpressed in several tumor types: head and neck, skin, cervical, and lung cancer, squamous cell carcinoma, and colon and breast adenocarcinoma. To establish the effect of FGF‐BP on wound healing, several forms of FGF‐BP cDNA were administered by particle‐mediated gene transfer into various animal wound models using the gene gun (Bio‐Rad). In a rat incisional wound model, gene gun cDNA delivery of full length FGF‐BP at the time of surgery produced a 117% increase of wound strength in diabetic rats at 10d, although the relative increase did not reach statistical significance (P < 0.08). Two truncated variants of FGF‐BP (pFGFbp10 and 17) were also administered in the rat incisional wound model by gene gun technique. pFGFbp17 increased the wound strength in diabetic rat 129%(p < 0.03), and the relative increase reach statistical significance (P < 0.008). In the rabbit ear ulcer model, particle‐mediated transduction of full length FGF‐BP increased collagen content by 195% and wound closure rate 38% at 10d post‐surgery. These findings show that FGF‐BP gene overexpression has a greater relative effect on wound healing in the diabetic rat model. The cDNA also had a significant effect in a rabbit excisional wound model that depended on granulation tissue formation. Truncated forms of the molecule may have higher therapeutic potency. FGF‐BP has an important role in FGF‐1/2 mobilization and macrophage functions, and FGF‐BP gene therapy for wound healing can improve the process by stimulating angiogenesis, epithelization and collagen synthesis in target tissue.

Detection of conformational transformation of antithrombin in blood with crossed immunoelectrophoresis: new application for a classical method

The structural flexibility of antithrombin is essential for its molecular trapping mechanism but also makes it vulnerable to even minor changes affecting its conformational stability, which influences hemostasis significantly. The conformational transformation of this serpin has been poorly investigated in biologic samples because available immunologic methods hardly differentiate between different conformations of this protein. Crossed immunoelectrophoresis (CIE) in presence of heparin has been classically used to identify mutant antithrombins with low heparin affinity. We demonstrate that this method also separates native and relaxed antithrombin, permitting the analysis of conformational variations of this potent anticoagulant with just a few microliters of plasma. However, CIE does not distinguish between antithrombin conformations with reduced heparin affinity: latent, cleaved, thrombin-antithrombin complexes, or heparin-binding mutants. Therefore, clinical interpretation of CIE results should be examined with caution. Using this and other methods, and evaluating the functional activity of antithrombin, we analyzed the conformational transformation of antithrombin in biologic samples. We confirmed its transformation to the latent configuration by incubating it at 50°C. This conformational change also occurs at 37°C, supporting the idea that this process is involved in the senescence of antithrombin. However, fresh plasma contains only traces of latent antithrombin, suggesting that this conformation is rapidly cleared in vivo. Finally, small increases in temperature (to 40°C) resulted in a faster conformational transformation of antithrombin. Fever has been suggested to have key structural, functional, and clinical consequences in patients with conformational mutations in antithrombin. Our results support a role for small changes in temperature in nonmutated antithrombin, suggesting that fever is a general risk factor for thrombosis.

Physical polymer matrices based on affinity interactions between peptides and polysaccharides.

A rapidly forming polymer matrix with affinity-based controlled release properties was developed based upon interactions between heparin-binding peptides and heparin. Dynamic mechanical testing of 10% (w/v) compositions consisting of a 3:1 molar ratio of poly(ethylene glycol)-co-peptide (approximately 18,000 g/mol) to heparin (approximately 18,000 g/mol) revealed a viscoelastic profile similar to that of concentrated, large molecular weight polymer solutions and melts. In addition, the biopolymer mixtures recovered quickly following thermal denaturation and mechanical insult. These gel-like materials were able to sequester exogenous heparin-binding peptides and could release these peptides over several days at rates dependent on relative heparin affinity. The initial release rates ranged from 3.3% per hour for a peptide with low heparin affinity to 0.025% per hour for a peptide with strong heparin affinity. By altering the affinity of peptides to heparin, a series of peptides can be developed to yield a range of release profiles useful for controlled in vivo delivery of therapeutics.

Molecular Mechanism of Host Specificity in Plasmodium falciparum Infection: ROLE OF CIRCUMSPOROZOITE PROTEIN

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.

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 °C, for 16 h, with 0.9 M ammonium sulfate, in 5 mM 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.

Interaction of designed sulfated flavanoids with antithrombin: lessons on the design of organic activators.

Recently, we designed (-)-epicatechin sulfate (ECS), the first small nonsaccharide molecule, as an activator of antithrombin for the accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002, 45, 1233-1243). Although sulfated flavanoid ECS was found to bind antithrombin with an affinity ( approximately 10.7 microM) comparable to the reference trisaccharide DEF ( approximately 4.5 microM), it accelerated the inhibition of factor Xa only 10-fold as compared to the approximately 300-fold observed with DEF. To determine whether this conformational activation of the inhibitor is dependent on the structure of the organic activator and to probe the basis for the deficiency in activation, we studied the interaction of similar sulfated flavanoids with antithrombin. (+)-Catechin sulfate (CS), a chiral stereoisomer of ECS, bound plasma antithrombin with a 3-fold higher affinity (K(D) = 3.5 microM) and a 2-fold higher second-order rate constant for factor Xa inhibition (k(ACT) = 6750 M(-1) s(-1)). On the contrary, the K(D) and k(ACT) were found to be lower approximately 7.4- and approximately 2.4-fold, respectively, for its racemic counterpart, (+/-)-catechin sulfate. Dependence of the equilibrium dissociation constant on the ionic strength of the medium at pH 6.0 and 7.4 suggests that nonionic interactions contribute a major proportion ( approximately 55-73%) of the total binding energy, and only 1-2 ion pairs, in comparison to the expected approximately 4 ion pairs for the reference trisaccharide, are formed in the interaction. Competitive binding experiments indicate that activator CS does not compete with a saccharide ligand that binds antithrombin in the pentasaccharide binding site, while it competes with full-length low-affinity heparin. A molecular docking study suggests plausible binding of CS in the extended heparin binding site, which is adjacent to the binding domain for the reference trisaccharide DEF. In combination, the results demonstrate that although conformational activation of antithrombin with small sulfated flavanoids is dependent on the structure of the activator, the designed activators do not bind in the pentasaccharide binding site in antithrombin resulting in weak activation. The mechanistic investigation highlights plausible directions to take in the rational design of specific high-affinity organic antithrombin activators.