Mapping of the catalytic groove preferences of factor Xa reveals an inadequate selectivity for its macromolecule substrates

Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P(3)-P(2)-P(1)-P(1)' residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P(3), P(2), P(1)', P(2)', and P(3)' and determined the k(cat)/K(m) values of cleavage. Contrary to expectation, glycine was not the "best" P(2) residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P(2), the ratio of the k(cat)/K(m) values for the most favorable side chain over the least was 289 (12 with trypsin), but in P(1)', this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P(2) and P(1)'. Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove.     

Determinants of the specificity of protease-activated receptors 1 and 2 signaling by factor Xa and thrombin

Factor Xa (FXa) elicits intracellular signaling responses through the activation of protease-activated receptor 2 (PAR2) and possibly also through PAR1 in endothelial cells. In this study, we investigated FXa signaling in endothelial cells when the protease was either in free form or assembled into the prothrombinase complex. Furthermore, we prepared several wild-type and mutant PAR1 and PAR2 cleavage-reporter constructs in which their exodomains were fused to cDNA encoding for a soluble alkaline phosphatase (ALP). In the mutants, P2 residues were exchanged between PAR1 and PAR2 cleavage-reporter constructs and the hirudin-like binding site (HLBS) of PAR1 was inserted into the homologous site of PAR2. In non-transfected cells, FXa elicited a protective response which could be blocked by a specific anti-PAR2 but not by an anti-PAR1 antibody. A similar protective activity was observed for FXa in the prothrombinase complex. Further studies revealed that neither the Gla- nor EGF1-domain of FXa is required for its signaling activity, however, the N-terminus Arg-86 and Lys-87 of the EGF2-domain were essential. In the cleavage-reporter transfected cells, FXa cleaved the PAR2 construct effectively, however, replacing its P2-Gly with P2-Pro of PAR1 impaired its cleavage by FXa but improved it by thrombin. A PAR2 construct containing both P2-Pro and HLBS of PAR1 was poorly cleaved by FXa, but effectively by thrombin. A PAR1 construct containing P2 and P3 residues of PAR2 was poorly cleaved by thrombin but effectively by FXa. These results provide new insight into mechanisms through which coagulation proteases specifically interact with their target PAR receptors.     

Discovery of sulfonylalkylamides: A new class of orally active factor Xa inhibitors

Factor Xa (FXa) is a trypsin-like serine protease involved in the coagulation cascade and has received great interest as a potential target for the development of new antithrombotic agents. Most of amidine-type FXa inhibitors reported have been found to show extremely poor oral bioavailability. Compound 1 is one of the first reported non-amidine type FXa inhibitors. To discover novel and orally active FXa inhibitors, we investigated flexible linear linkers between the 6-chloronaphthalene ring and the 1-(pyridin-4-yl)piperidine moiety of 1 and found the orally active sulfonylalkylamide 2f with an FXa IC(50) of 0.05 microM, comparable with that of 1. Further modification to reduce the CYP3A4 inhibitory activity of 2f resulted in the potent, selective, and orally active 2-methylpyridine analogue 2s (FXa IC(50) of 0.061 microM), for which the liability of CYP3A4 inhibition was significantly weakened compared to 2f. Compound 2s also showed long lasting anticoagulant activity in cynomolgus monkeys.     

Effects of factor Xa and protein S on the individual activated protein C-mediated cleavages of coagulation factor Va

Activated protein C inhibits the procoagulant function of activated factor V (FVa) through proteolytic cleavages at Arg-306, Arg-506, and Arg-679. The cleavage at Arg-506 is kinetically favored but protected by factor Xa (FXa). Protein S has been suggested to annihilate the inhibitory effect of FXa, a proposal that has been challenged. To elucidate the effects of FXa and protein S on the individual cleavage sites of FVa, we used recombinant FVa:Q306/Q679 and FVa:Q506/Q679 variants, which can only be cleaved at Arg-506 and Arg-306, respectively. In the presence of active site blocked FXa (FXa-1.5-dansyl-Glu-Gly-Arg), the FVa inactivation was followed over time, and apparent second order rate constants were calculated. Consistent with results on record, we observed that FXa-1.5-dansyl-Glu-Gly-Arg decreased the Arg-506 cleavage by 20-fold, with a half-maximum inhibition of approximately 2 nM. Interestingly and in contrast to the inhibitory effect of FXa on the 506 cleavage, FXa stimulated the Arg-306 cleavage. Protein S counteracted the inhibition by FXa of the Arg-506 cleavage, whereas protein S and FXa yielded additive stimulatory effect of the cleavage at Arg-306. This suggests that FXa and protein S interact with distinct sites on FVa, which is consistent with the observed lack of inhibitory effect on FXa binding to FVa by protein S. We propose that the apparent annihilation of the FXa protection of the Arg-506 cleavage by protein S is due to an enhanced rate of Arg-506 cleavage of FVa not bound to FXa, resulting in depletion of free FVa and dissociation of FXa-FVa complexes.     

Highly efficacious factor Xa inhibitors containing α-substituted phenylcycloalkyl P4 moieties

We previously disclosed a series of highly potent FXa inhibitors bearing α-substituted (CH₂NR¹R²) phenylcyclopropyl P4 moieties in the pyrazolodihydropyridone core system. Herein, we describe our continuous SAR efforts in this series. Effects of the C-3 substitution of the pyrazolodihydropyridone core and the α-substitution (R group) of the cyclopropyl ring on FXa binding affinity (FXa K_i), human plasma anticoagulant activity (PT EC_2×) and permeability are discussed. A set of compounds obtained from optimization of the R group and the C-3 substituent were orally bioavailable in dogs. Furthermore, representative compounds were highly efficacious in the rabbit arterio-venous shunt thrombosis model (EC₅₀s = 29–81 nM).     

Synthesis of human big endothelin-1 by sequence-specific proteolysis of a fusion protein in Escherichia coli.

Three DNA constructs, pETB-40, 41, and 42, encoding human big endothelin-1 (ET-1) preceded by the specific recognition sequence (Ile-Glu-Gly-Arg) for the activated blood coagulation factor Xa (FXa), fused in frame to the N-terminal portion of beta Gal, were expressed in Escherichia coli. The fusion proteins, pETB-40P, 41P, or 42P, consisted of the 55-, 51-, or 42-aa N-terminal peptide of beta Gal and the 38-aa of big ET-1, and had 1, 0, or 0 Cys residues and 5, 5, or 1 Arg residues in the N-terminal peptide of beta Gal, respectively. Enzymatic cleavage of the purified fusion proteins by FXa or trypsin allowed the recovery of authentic human big ET-1. The rates of conversion of pETB-40P, 41P, and 42P to big ET-1 by FXa digestion were 5.6, 11.2, and 30.0%, respectively. pETB-40P with a deletion of one Cys residue and four Arg residues in the N-terminal part was a better substrate than the other two for FXa or trypsin in the production of big ET-1.     

A computational modeling and molecular dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)

Protein Z-dependent protease inhibitor (ZPI) and antithrombin III (AT3) are members of the serpin superfamily of protease inhibitors that inhibit factor Xa (FXa) and other proteases in the coagulation pathway. While experimental structural information is available for the interaction of AT3 with FXa, at present there is no structural data regarding the interaction of ZPI with FXa, and the precise role of this interaction in the blood coagulation pathway is poorly understood. In an effort to gain a structural understanding of this system, we have built a solvent equilibrated three-dimensional structural model of the Michaelis complex of human ZPI/FXa using homology modeling, protein–protein docking and molecular dynamics simulation methods. Preliminary analysis of interactions at the complex interface from our simulations suggests that the interactions of the reactive center loop (RCL) and the exosite surface of ZPI with FXa are similar to those observed from X-ray crystal structure-based simulations of AT3/FXa. However, detailed comparison of our modeled structure of ZPI/FXa with that of AT3/FXa points to differences in interaction specificity at the reactive center and in the stability of the inhibitory complex, due to the presence of a tyrosine residue at the P1 position in ZPI, instead of the P1 arginine residue in AT3. The modeled structure also shows specific structural differences between AT3 and ZPI in the heparin-binding and flexible N-terminal tail regions. Our structural model of ZPI/FXa is also compatible with available experimental information regarding the importance for the inhibitory action of certain basic residues in FXa. Figure Solvent equilibrated models for protein z-dependent protease inhibitor and its initial reactive complex with coagulation factor Xa (show here) are developed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. V.C. and C.J.L. contributed equally to this work. The solvent-equilibrated PDB structure of the ZPI/FXa will be made available upon request. Conflict of interest statement  The authors state that they have no conflict of interest.     

Amino(methyl) pyrrolidines as novel scaffolds for factor Xa inhibitors

The design and synthesis of a novel class of amino(methyl) pyrrolidine-based sulfonamides as potent and selective FXa inhibitors is reported. The amino(methyl) pyrrolidine scaffolds were designed based on the proposed bioisosterism to the piperazine core in known FXa inhibitors. The SAR study led to compound 15 as the most potent FXa inhibitor in this series, with an IC(50) of 5.5 nM and PT EC(2x) of 1.7 microM. The proposed binding models show that the pyrrolidine cores are in van der Waals contact with the enzyme surface, and the flexibility of amino(methyl) pyrrolidines allows the two nitrogen atoms to anchor both the P1 and P4 groups to fit similarly in the S1 and S4 pockets.     

Achieving structural diversity using the perpendicular conformation of alpha-substituted phenylcyclopropanes to mimic the bioactive conformation of ortho-substituted biphenyl P4 moieties: discovery of novel, highly potent inhibitors of Factor Xa

Ortho-substituted biphenyl moieties are widely used in drug design. We herein report a successful use of the perpendicular conformation of the alpha-substituted phenylcyclopropyl groups to mimic the aplanar, biologically active conformation of the ortho-substituted biphenyl moieties to achieve structural diversity. This is exemplified by the design and synthesis of a series of highly potent pyrazole bicyclic-based Factor Xa (FXa) inhibitors bearing alpha-substituted phenylcyclopropyl P4 moieties. The designed perpendicular conformation was confirmed by the X-ray structure of FXa-bound compound 2r. The potential structural basis for the high FXa potency in the phenylcyclopropyl P4 analogs and their improved FXa inhibitory activities compared with the biphenyl P4 counterparts are discussed.     

Kinetics of factor Xa inhibition by recombinant tick anticoagulant peptide: both active site and exosite interactions are required for a slow- and tight-binding inhibition mechanism

Recombinant tick anticoagulant peptide (rTAP) is a competitive slow- and tight-binding inhibitor of factor Xa (FXa) with a reported equilibrium dissociation constant (K(I)) of approximately 0.2 nM. The inhibitory characteristics and the high selectivity of rTAP for FXa are believed to arise from the ability of the inhibitor to specifically interact with the residues of both the active site as well as those remote from the active site pocket of the protease. To localize the rTAP-interactive sites on FXa, the kinetics of inhibition of wild-type and 18 different mutants of recombinant FXa by the inhibitor were studied by either a discontinuous assay method employing the tight-binding quadratic equation or a continuous assay method employing the slow-binding kinetic approach. It was discovered that K(I) values for the interaction of rTAP with four FXa mutants (Tyr(99) --> Thr, Phe(174) --> Asn, Arg(143) --> Ala, and a Na(+)-binding loop mutant in which residues 220-225 of FXa were replaced with the corresponding residues of thrombin) were elevated by 2-3 orders of magnitude for each mutant. Further studies revealed that the characteristic slow type of inhibition by rTAP was also eliminated for the mutants. These findings suggest that the interaction of rTAP with the P2-binding pocket, the autolysis loop, and the Na(+)-binding loop is primarily responsible for its high specificity of FXa inhibition by a slow- and tight-binding mechanism.     

Design,synthesis, and structure–activity relationship of novel and effective apixaban derivatives as FXa inhibitors containing 1,2,4-triazole/pyrrole derivatives as P2 binding element

Four series of novel and potent FXa inhibitors possessing the 1,2,4-triazole moiety and pyrrole moiety as P2 binding element and dihydroimidazole/tetrahydropyrimidine groups as P4 binding element were designed, synthesized, and evaluated for their anticoagulant activity in human and rabbit plasma in vitro. Most compounds showed moderate to excellent activity. Compounds 14a, 16, 18c, 26c, 35a, and 35b were further examined for their inhibition activity against human FXa in vitro and rat venous thrombosis in vivo. The most promising compound 14a, with an IC₅₀ (FXa) value of 0.15 μM and 99% inhibition rate, was identified for further evaluation as an FXa inhibitor.     

Design, synthesis, and in vitro biological activity of benzimidazole based factor Xa inhibitors

Inhibitors based on the benzimidazole scaffold showed subnanomolar potency against Factor Xa with 500-1000-fold selectivity against thrombin and 50-100-fold selectivity against trypsin. The 2-substituent on the benzimidazole ring had a strong impact on the FXa inhibitory activity. Crystallography studies suggest that the 2-substituent may have a conformational effect favoring the extended binding conformation.     

The extended interactions and Gla domain of blood coagulation factor Xa

The serine protease factor Xa (FXa) is inhibited by ecotin with picomolar affinity. The structure of the tetrameric complex of ecotin variant M84R (M84R) with FXa has been determined to 2.8 A. Substrate directed induced fit of the binding interactions at the S2 and S4 pockets modulates the discrimination of the protease. Specifically, the Tyr at position 99 of FXa changes its conformation with respect to incoming ligand, changing the size of the S2 and S4 pockets. The role of residue 192 in substrate and inhibitor recognition is also examined. Gln 192 from FXa forms a hydrogen bond with the P2 carbonyl group of ecotin. This confirms previous biochemical and structural analyses on thrombin and activated protein C, which suggested that residue 192 may play a more general role in mediating the interactions between coagulation proteases and their inhibitors. The structure of ecotin M84R-FXa (M84R-FXa) also reveals the structure of the Gla domain in the presence of Mg(2+). The first 11 residues of the domain assume a novel conformation and likely represent an intermediate folding state of the domain.     

Orally bioavailable factor Xa inhibitors containing alpha-substituted gem-dimethyl P4 moieties

In an effort to identify a potential back-up to apixaban (Eliquis®), we explored a series of diversified P4 moieties. Several analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent FXa binding affinity (FXa K_i), good human plasma anticoagulant activity (PT EC_2x), cell permeability, and oral bioavailability.     

Inhibition of tissue factor-factor VIIa-catalyzed factor X activation by factor Xa-tissue factor pathway inhibitor. A rotating disc study on the effect of phospholipid membrane composition.

The physiological inhibitor of tissue factor (TF).factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPI(FL)) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF.FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF.PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF.PSPC) was measured in the presence of preformed complexes of FXa.TFPI(FL) or FXa.TFPI(1-161) (TFPI lacking the third Kunitz domain and C terminus). At TF.PC, FXa.TFPI(FL) and FXa.TFPI(1-161) showed similar rate constants of inhibition (0.07 x 10(8) M(-1) s(-1) and 0.1 x 10(8) M(-1) s(-1), respectively). With phosphatidylserine present, the rate constant of inhibition for FXa.TFPI(FL) increased 3-fold compared with a 9-fold increase in the rate constant for FXa. TFPI(1-161). Incubation of TF.PSPC with FXa.TFPI(FL) in the absence of FVIIa followed by depletion of solution FXa.TFPI(FL) showed that FXa.TFPI(FL) remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa.TFPI(1-161) or at TF.PC membranes. These data suggest that the membrane-bound pool of FXa.TFPI(FL) may be of physiological importance in an on-site regulation of TF.FVIIa activity.     

Factor Xa Binding to Phosphatidylserine-Containing Membranes Produces an Inactive Membrane-Bound Dimer

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.     

A Computer Modeling Study of the Interaction Between Tissue Factor Pathway Inhibitor and Blood Coagulation Factor Xa

Activation of blood coagulation factor X to factor Xa (FXa) is inhibited by tissue factor pathway inhibitor (TFPI). The second Kunitz-type inhibitory domain (K2) of TFPI binds a catalytic domain of FXa, whereas the first domain (K1) does not. We analyzed computer models of complexes of FXa with K1 or K2, which were made using a crystal structure of FXa. Favorable hydrophobic interaction was observed in the complex of FXa with K2. Furthermore, we constructed a tertiary structure of FXa using CHIMERA to assess the accuracy of a homology modeling method. The isolated model structure of FXa agreed well with the crystal structure, but analyses of complexes of this structure with K1 or K2 revealed that the models of complexes could not provide clear evidence of greater binding ability to K2 because of the positional difference of a few side chains interacting with the inhibitor.     

抗凝蛋白EH体外活性检测条件的建立

EH蛋白是一种水蛭素衍生物,它是在水蛭素的N端引入了一小段寡肽,该寡肽可被凝血因子XIa(factor XIa,FXIa)和Xa(factor Xa,FXa)裂解释放水蛭素的抗凝血酶活性.比较在不同条件下FXa裂解EH蛋白的效果,包括裂解时间(2h,4h,6h,8h,10h,20h)、裂解温度(25℃,37℃,40℃)...     

Conformation of a bound inhibitor of blood coagulant factor Xa

13C[(15)N] and (13)C[(19)F] rotational-echo double-resonance NMR have been used to characterize the enzyme-bound structure of ZK-816042, an amidine-imidazoline inhibitor of human factor Xa (FXa). The NMR experiments were performed on a lyophilized FXa-inhibitor complex. The complex was formed in solution in the presence of stabilizing excipients and frozen after gradual supercooling prior to lyophilization. The results indicate that the inhibitor binds with a distribution of orientations of the imidazoline ring.     

Discovery of piperazinylimidazo[1,2-a]pyridines as novel S4 binding elements for orally active factor Xa inhibitors

We have recently reported the discovery of orally active sulfonylalkylamide Factor Xa (FXa) inhibitors, as typified by compound 1 (FXa IC(50)=0.061 microM). Since the pyridylpiperidine moiety was not investigated in our previous study, we conducted detailed structure-activity relationship studies on this S4 binding element. This investigation led to the discovery of piperazinylimidazo[1,2-a]pyridine 2b as a novel and potent FXa inhibitor (FXa IC(50)=0.021 microM). Further modification resulted in the discovery of 2-hydroxymethylimidazo[1,2-a]pyridine 2e (FXa IC(50)=0.0090 microM), which was found to be a selective and orally bioavailable FXa inhibitor with reduced CYP3A4 inhibition.