Intermolecular interactions and characterization of the novel factor Xa exosite involved in macromolecular recognition and inhibition: crystal structure of human Gla-domainless factor Xa complexed with the anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K(i)=8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.     

The tissue factor/factor VIIa/factor Xa complex: a model built by docking and site-directed mutagenesis

Factor X is activated to factor Xa (fXa) in the extrinsic coagulation pathway by the tissue factor (TF)/factor VIIa (fVIIa) complex. Upon activation, the fXa molecule remains associated with the TF/fVIIa complex, and this ternary complex is known to activate protease-activated receptors (PARs) 1 and 2. Activation of fVII in the TF complex by fXa is also seen at physiologic concentrations. The ternary complexes TF/fVII/fXa, TF/fVIIa/fX, and TF/fVIIa/fXa are therefore all physiologically relevant and of interest as targets for inhibition of both coagulation and cell-signaling pathways that are important in cardiovascular disease and inflammation. We therefore present a model of the TF/fVIIa/fXa complex, built with the use of the available structures of the TF/fVIIa complex and fXa by protein-protein docking calculations with the program Surfdock. The fXa model has an extended conformation, similar to that of fVIIa in the TF/fVIIa complex, with extensive interactions with TF and the protease domain of fVIIa. All four domains of fXa are involved in the interaction. The gamma-carboxyglutamate (Gla) and epithelial growth factor (EGF1 and EGF2) domains of fVIIa are not significantly involved in the interaction. Docking of the Gla domain of fXa to TF/fVIIa has been reported previously. The docking results identify potential interface residues, allowing rational selection of target residues for site-directed mutagenesis. This combination of docking and mutagenesis confirms that residues Glu51 and Asn57 in the EGF1 domain, Asp92 and Asp95 in the EGF2 domain, and Asp 185a, Lys 186, and Lys134 in the protease domain of factor Xa are involved in the interaction with TF/fVIIa. Other fX protease domain residues predicted to be involved in the interaction come from the 160s loop and the N-terminus of the fX protease domain, which is oriented in such a way that activation of both fVII by fXa, and the reciprocal fX activation by fVIIa, is possible.     

Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.     

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.     

Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 A, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 A. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (Cys5T-Cys15T) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 A to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 A with the guanidinium group forming a cation-pi-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 A in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N-terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.     

Inherent flexibility in a potent inhibitor of blood coagulation, recombinant nematode anticoagulant protein c2.

Nematode anticoagulant proteins (NAPs) from the hematophagous nematode Ancylostoma caninum inhibit blood coagulation with picomolar inhibition constants, and have been targeted as novel pharmaceutical agents. NAP5 and NAP6 inhibit factor Xa by binding to its active site, whereas NAPc2 binds to factor Xa at a different, as yet unidentified, site and the resultant binary complex inhibits the tissue factor-factor VIIa complex. We have undertaken NMR studies of NAPc2, including the calculation of a solution structure, and found that the protein is folded, with five disulfide bonds, but is extremely flexible, especially in the acidic loop. The Halpha secondary shifts and 3JHNHalpha coupling constants indicate the presence of some beta structure and a short helix, but the intervening loops are highly conformationally heterogeneous. Heteronuclear NOE measurements showed the presence of large amplitude motions on a subnanosecond timescale at the N-terminus and C-terminus and in the substrate-binding loop, indicating that the conformational heterogeneity observed in the NMR structures is due to flexibility of the polypeptide chain in these regions. Flexibility may well be an important factor in the physiological function of NAPc2, because it must interact with other proteins in the inhibition of blood coagulation. We suggest that this inhibitor is likely to become structured on binding to factor Xa, because the inhibition of the tissue factor-factor VIIa complex requires both NAPc2 and factor Xa.     

Reaction pathway for inhibition of blood coagulation factor Xa by tick anticoagulant peptide.

The reaction pathway for inhibition of human factor Xa (fXa) by recombinant tick anticoagulant peptide (rTAP) was studied by stopped-flow fluorometry. In the presence of the fluorogenic substrate N-tert-butyloxycarbonyl-L-isoleucyl-L-glutamylglycyl-L-arginyl-7-amido-4 - methylcoumarin (B-IEGR-AMC) and under pseudo-first-order conditions, inhibition appears to occur via a two-step process. Initially, a weak enzyme-inhibitor complex forms with a dissociation constant (Ki) of 68 +/- 6 microM. The initial complex then rearranges to a more stable fXa-rTAP complex with a rate constant (k2) of 123 +/- 5 s-1. The apparent second-order rate constant (k2/Ki) describing formation of the stable complex is (1.8 +/- 0.2) x 10(6) M-1 s-1. Studies of the reaction of rTAP with fXa in the presence of the fluorescent active-site probe p-amino-benzamidine (P) revealed a reaction pathway wherein rTAP initially binds to the fXa-P complex in a two-step process prior to displacing P from the active site. These results indicate that rTAP can bind fXa via a site distinct from the active site (an exosite). The subsequent displacement of P from the active site of fXa by rTAP exhibits a dependence on the concentration of P, indicating that rTAP is locked into the active site in a third step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative molecular modeling analysis of­5‐amidinoindole and benzamidine binding to thrombin and trypsin: specific H‐bond formation contributes to high 5‐amidinoindole potency and selectivity for thrombin and factor Xa

The coagulation cascade enzymes thrombin and factor Xa are known to have specificity pockets very similar to those of trypsin and plasmin. However, comparative molecular modeling analysis of the crystal structures of benzamidine–thrombin and benzamidine–trypsin, in conjunction with a docking analysis of 5‐amidinoindole and related inhibitors in both enzymes reveals subtle differences between the specificity sites of the two types of enzymes. Specifically, thrombin and factor Xa, which have an alanine residue at position 190, exhibit increased activities for the rigid and more bulky bicyclic derivatives of benzamidine (e.g. amidinobenzofuran, amidinothiophene and amidinoindole), because of additional hydrophobic and H‐bond interactions between the inhibitors and the specificity sites, whereas enzymes with a serine residue at position 190, like trypsin and plasmin, exhibit little difference in activity among the same set of compounds because of the orientational restriction imposed on the inhibitors by Ser190, which forms an additional H‐bond with the amidino group of the inhibitors. Enzymes of both groups show similar responses to the flexible aminobenzamidine since the smaller size and the rotatable anilino group of the inhibitor would allow the inhibitor to achieve favorable electrostatic interactions with both groups of enzymes. 5‐Amidinoindole is the most dramatic example of the rigid bicyclic type inhibitor. Based on our docking analysis, we propose that a selective H‐bond with the hydroxyl group of the catalytic Ser195 and the subtle differences in steric fit imposed by Ala/Ser at position 190 explain the high potency and selectivity of 5‐amidinoindole for thrombin and factor Xa. Copyright © 1999 John Wiley & Sons, Ltd.     

Role of zymogen and activated factor X as scaffolds for the inhibition of the blood coagulation factor VIIa-tissue factor complex by recombinant nematode anticoagulant protein c2

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa.TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa.TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF(S)), or reconstituted into membranes, containing phosphatidylcholine (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa.TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.fX(a) complex assembled on an appropriate phospholipid surface.     

Cloning, nucleotide sequence and expression of the gene encoding factor Xa inhibitor from the salivary glands of the tick, Ornithodoros savignyi

The N-terminal sequence of the competitive and slow tight-binding factor Xa inhibitor (fXaI; Ki = 0.83 ± 0.10 nM) isolated from the salivary glands of Ornithodoros savignyi ticks (Acari: Argasidae) was employed to design a degenerate gene-specific primer (GSP) for 3-rapid amplification of cDNA ends (3-RACE). The primer consisted of a sequence encoding for amino acid residues 5-11. A full-length gene was next constructed from the 3-RACE product in a two-step PCR procedure and successfully expressed by the BAC-TO-BAC baculovirus expression system. The deduced amino acid sequence of the gene showed 46% identity and 78% homology to an fXaI (TAP) from Ornithodoros moubata. Recombinant fXaI (rfXaI) consists of 60 amino acid residues, has a molecular mass of ~7 kDa and inhibited fXa by ~91%. The availability of the rfXaI will aid further investigations of its potential for therapeutic applications and as vaccine against tick infestation. The authentic nucleotide sequence of the gene encoding tick fXaI furthermore enables studies at the genetic level and probing of other tick species for similar and related genes.     

Activation of antithrombin as a factor IXa and Xa inhibitor involves mitigation of repression rather than positive enhancement

Allosteric activation of antithrombin as a rapid inhibitor of factors IXa and Xa requires binding of a high-affinity heparin pentasaccharide. The currently-accepted mechanism involves removal of a constraint on the antithrombin reactive center loop (RCL) so that the proteinase can simultaneously engage both the P1 arginine and an exosite at Y253. Recent results suggest that this mechanism is incorrect in that activation can be achieved without loop expulsion, while the exosite can be engaged in both low and high activity states. We propose a quite different mechanism in which heparin activates antithrombin by mitigating an unfavorable surface interaction, by altering its nature, and by moving the attached proteinase away from the site of the unfavorable interaction through RCL expulsion.     

Identification of factor Xa residues critical for interaction with protein Z-dependent protease inhibitor: both active site and exosite interactions are required for inhibition

Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.     

Design, synthesis and SAR of novel ethylenediamine and phenylenediamine derivatives as factor Xa inhibitors

We previously reported on a series of cyclohexanediamine derivatives as highly potent factor Xa inhibitors. Herein, we describe the modification of the spacer moiety to discover an alternative scaffold. Ethylenediamine derivatives possessing a substituent at the C1 position in S configuration and phenylenediamine derivatives possessing a substituent at the C5 position demonstrated moderate to strong anti-fXa activity. Further SAR studies led to the identification of compound 30h which showed both good in vitro activity (fXa IC₅₀ = 2.2 nM, PTCT2 = 3.9 μM) and in vivo antithrombotic efficacy.     

Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants.

The coagulation factors IXa (fIXa) and Xa (fXa) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface. However, the amidolytic activity of fIXa is 10(4)-fold lower than that of fXa. To identify determinants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and fXa (rf10a) in Escherichia coli. The crystal structures of fIXa and fXa revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a. Exchanging Glu219 by Gly or exchanging the 148 loop did not increase activity of rf9a, whereas corresponding mutations abolished reactivity of rf10a. Exchanging Ile213 by Val only moderately increased reactivity of rf9a. Exchanging the 99 loop, however, dramatically increased reactivity. Furthermore, combining all four mutations essentially introduced fXa properties into rf9a: the amidolytic activity was increased 130-fold with fXa substrate selectivity. The results suggest a 2-fold origin of fIXa's poor reactivity. A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond. Both defects could be repaired by introducing fXa residues. Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.     

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.     

Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite

Ixolaris is a two-Kunitz tick salivary gland tissue factor pathway inhibitor (TFPI). In contrast to human TFPI, Ixolaris specifically binds to factor Xa (FXa) heparin-binding exosite (HBE). In addition, Ixolaris interacts with zymogen FX. In the present work we characterized the interaction of Ixolaris with human FX quantitatively, and identified a precursor state of the heparin-binding exosite (proexosite, HBPE) as the Ixolaris-binding site on the zymogen. Gel-filtration chromatography demonstrated 1:1 complex formation between fluorescein-labeled Ixolaris and FX. Isothermal titration calorimetry confirmed that the binding of Ixolaris to FX occurs at stoichiometric concentrations in a reaction which is characteristically exothermic, with a favorable enthalpy (DeltaH) of -10.78 kcal/mol. ELISA and plasmon resonance experiments also indicate that Ixolaris binds to plasma FX and FXa, or to recombinant Gla domain-containing FX/FXa with comparable affinities ( approximately 1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction-Arg-93 > Arg-165 > or = Lys-169 > Lys-236 > Arg-125-which was identical to that observed for FXa/Ixolaris interaction. Remarkably, Ixolaris strongly inhibited FX activation by factor IXa in the presence but not in the absence of factor VIIIa, suggesting a specific interference in the cofactor activity. Further, solid phase assays demonstrated that Ixolaris inhibits FX interaction with immobilized FVIIIa. Altogether, Ixolaris is the first inhibitor characterized to date that specifically binds to FX HBPE. Ixolaris may be a useful tool to study the physiological role of the FX HBPE and to evaluate this domain as a target for anticoagulant drugs.     

Molecular determinants of the mechanism underlying acceleration of the interaction between antithrombin and factor Xa by heparin pentasaccharide

The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5. The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.     

Synthesis and biological activity of novel 1,4-diazepane derivatives as factor Xa inhibitor with potent anticoagulant and antithrombotic activity

Factor Xa (fXa) is a serine protease involved in the coagulation cascade, which has received great interest as a potential target for the development of new antithrombotic drugs. Herein we report a novel series of fXa inhibitors in which the 1,4-diazepane moiety was designed to interact with the S4 aryl-binding domain of the fXa active site. Compound 13 (YM-96765) showed potent fXa inhibitory activity (IC(50) = 6.8 nM) and effective antithrombotic activity without prolonging bleeding time.     

Tick anticoagulant peptide: kinetic analysis of the recombinant inhibitor with blood coagulation factor Xa

Tick anticoagulant peptide (TAP) is a 60 amino acid protein which is a highly specific inhibitor of human blood coagulation factor Xa (fXa) isolated from the tick Ornithodoros moubata [Waxman, L., Smith, D. E., Arcuri, K. E., & Vlasuk, G. P. (1990) Science 248, 593-596]. Due to the limited quantities of native TAP, a recombinant version of TAP produced in Saccharomyces cerevisiae was used for a detailed kinetic analysis of the inhibition interaction with human fXa. rTAP was determined to be a reversible, slow, tight-binding inhibitor of fXa, displaying a competitive type of inhibition. The binding of rTAP to fXa is stoichiometric with a dissociation constant of (1.8 +/- 0.02) x 10(-10) M, a calculated association rate constant of (2.85 +/- 0.07) x 10(6) M-1 s-1, and a dissociation rate constant of (0.554 +/- 0.178) x 10(-3) s-1. Binding studies show that 35S-rTAP binds only to fXa and not to DFP-treated fXa or zymogen factor X, which suggests the active site of fXa is required for rTAP inhibition. That rTAP is a unique serine proteinase inhibitor is suggested both by its high specificity for its target enzyme, fXa, and also by its unique structure.     

Design,synthesis and biological evaluation of anthranilamide derivatives as potential factor Xa (fXa) inhibitors

Factor Xa (fXa) is a crucial player in various thromboembolic disorders. Inhibition of fXa can provide safe and effective antithrombotic effects. In this study, a series of anthranilamide compounds were designed by utilizing structure-based design strategies. Optimization at P1 and P4 groups led to the discovery of compound 16g: a highly potent, selective fXa inhibitor with pronounced in vitro anticoagulant activity. Moreover, 16g also displayed excellent in vivo antithrombotic activity in the rat venous thrombosis (VT) and arteriovenous shunt (AV-SHUNT) models. The bleeding risk evaluation showed that 16g had a safer profile than that of betrixaban at 1?mg/kg and 5?mg/kg dose. Additionally, 16g also exhibited satisfactory PK profiles. Eventually, 16g was selected to investigate its effect on hypoxia-reoxygenation- induced H9C2 cell viability. MTT results showed that H9C2 cell viability can be remarkably alleviated by 16g.