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PubMed1997

Factor Xa as an interface between coagulation and inflammation. Molecular mimicry of factor Xa association with effector cell protease receptor-1 induces acute inflammation in vivo.

Coagulation proteases were tested in a rat model of acute inflammation. Subplantar injection of Factor Xa (10-30 microg) produced a time- and dose-dependent edema in the rat paw, and potentiated carrageenin-induced edema. In contrast, the homologous protease Factor IXa was ineffective. This inflammatory response was recapitulated by the Factor Xa sequence L83FTRKL88(G), which mediates ligand binding to effector cell protease receptor-1 (EPR-1), while a control scrambled peptide did not induce edema in vivo. Conversely, injection of the EPR-1-derived peptide S123PGKPGNQNSKNEPP137 (corresponding to the receptor binding site for Factor Xa) inhibited carrageenin-induced rat paw edema, while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was without effect. EPR-1-Factor Xa-induced inflammation was characterized by fast onset and prominent perivascular accumulation of activated and degranulated mast cells, was inhibited by the histamine/serotonin antagonists cyproheptadine and methysergide, but was unaffected by the thrombin-specific inhibitor, Hirulog. These findings suggest that through its interaction with EPR-1, Factor Xa may function as a mediator of acute inflammation in vivo. This pathway may amplify both coagulation and inflammatory cascades, thus contributing to the pathogenesis of tissue injury in vivo.

Amino Acid Sequence↗
PubMed1988

Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation.

We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.

Algorithms↗
PubMed2001

The use of 3D structural data in the design of specific factor Xa inhibitors.

Factor Xa (fXa) is a serine protease that plays a critical role in the blood coagulation process and qualifies as an attractive target for finding new antithrombotics. In the case of fXa several structure based drug design strategies have been followed because of the difficulty in growing fXa co-crystals routinely. This has led to the use of surrogate proteins such as trypsin. Factor Xa inhibitors for which the binding mode has been determined experimentally or modeled are described in this review. The inhibitors are divided into three fragments: a P1 group, a central scaffold and a P4 group. In this review, interactions in each sub-site of fXa with various inhibitor fragments have been examined at the molecular level and were shown to bind, in most cases, independently of the rest of the molecule. Knowledge of the 3D structure of the binding mode of ligands to target proteins has been successfully applied in designing fXa inhibitors with enhanced specificity, affinity and has provided hints to modulate the physico-chemical properties of the small molecule ligand.

Animals↗
PubMed2001

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin.

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.

Allosteric Regulation↗
PubMed1976

Mechanism of activation of bovine factor VII. Products of cleavage by factor Xa.

Coagulation Factor VII from bovine plasma is a glycoprotein containing a single peptide chain. The NH2-terminal sequence of Ala-Asx-Gly-Phe-Leu- is homologous with the NH2 termini of prothrombin, Factor IX, and the light chain of Factor X. Factor Xa in the presence of calcium ions and phospholipid cleaves Factor VII at an Arg-Ile bond in the sequence Arg-Ile-Val-Gly-Gly-, producing a two-chain molecule with at least 85 times the coagulant activity of single-chain Factor VII and a new NH2-terminal sequence homologous with the corresponding chains of thrombin, Factor IXa and Factor Xa. A second slower cleavage at an Arg-Gly bond destroys Factor VII activity. Bovine Factor VII, unlike prothrombin, Factor IX, and Factor X, is rapidly inhibited by diisopropylphosphorofluoridate (iPr2PF). [3H]iPr2PF is readily incorporated into one-chain, two-chain, and three-chain forms of Factor VII up to ratios of approximately 0.9 moles of [3H]diisopropylphosphate per mole of protein. The radioactive peptides generated from each form of [32P]iPr2PF-inhibited Factor VII by tryptic and thermolytic digestion were found to migrate together on paper electrophoresis. This indicates that the iPr2PF is incorporated stoichiometrically into the same specific site in each form.

Amino Acid Sequence↗
PubMed1999

Synthesis, characterization, and structure-activity relationships of amidine-substituted (bis)benzylidene-cycloketone olefin isomers as potent and selective factor Xa inhibitors.

Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in blood coagulation linking the intrinsic and extrinsic pathways to the final common pathway of the coagulation cascade. During our initial studies, we observed facile photochemical conversion of the known FXa/tPA inhibitor, BABCH ¿(E,E)-2, 7-bis(4-amidinobenzylidene)cycloheptan-1-one, 1a, to the corresponding (Z,Z) olefin isomer, 1c (FXa K(i) = 0.66 nM), which was over 25,000 times more potent than the corresponding (E,E) isomer (1a, FXa K(i) = 17 000 nM). In order to determine the scope of this observation, we expanded on our initial investigation through the preparation of the olefin isomers in a homologous series of cycloalkanone rings, 4-substituted cyclohexanone analogues, and modified amidine derivatives. In most cases the order of potency of the olefin isomers was (Z,Z) > (E,Z) > (E,E) with the cycloheptanone analogue (1c) showing the most potent factor Xa inhibitory activity. In addition, we found that selectivity versus thrombin (FIIa) can be dramatically improved by the addition of a carboxylic acid group to the cycloalkanone ring as seen with 8c (FXa K(i) = 6.9 nM, FIIa K(i) > 50,000 nM). Compounds with one or both of the amidine groups substituted with N-alkyl substituents or replaced with amide groups led to a significant loss of activity. In this report we have demonstrated the importance of the two amidine groups, the cycloheptanone ring, and the (Z,Z) olefin configuration for maximum inhibition of FXa within the BABCH template. The results from this study provided the foundation for the discovery of potent, selective, and orally active FXa inhibitors.

Benzylidene Compounds↗
PubMed1992

Bacterial expression, purification, and partial characterization of amino acids 94-155 of human tissue factor pathway inhibitor (TFPI) as an inhibitor of blood coagulation factor Xa.

Tissue factor pathway inhibitor (TFPI) is a plasma-derived protein which inhibits two of the active serine proteases present during normal blood coagulation. Inhibition of both of these proteases, factors VIIa and Xa, is thought to require a factor Xa-TFPI complex. To begin to investigate the interactions between factor Xa and TFPI, amino acids 94-155, which encode for the second Kunitz domain (K2) of TFPI, were expressed, purified, and partially characterized. Expression of the recombinant peptide was accomplished using an E. coli expression system which produced the peptide at an expression level of approximately 2-5% of total cell protein. The peptide was localized to disulfide-linked refractile bodies which were solubilized by reduction in the presence of denaturant and the soluble protein refolded. Oxidized K2 was purified from the refold mixture using a two-step procedure employing gel filtration chromatography and reverse-phase HPLC. The unprocessed form of the recombinant peptide, Met-Ala-K2 (rMA-K2), was characterized. This peptide was purified to apparent homogeneity as determined by SDS-PAGE, quantitative amino acid, Edman degradation, and electrospray mass spectrometry analyses (> 95% pure). The product bound to factor Xa covalently coupled to a solid support in the presence of 2M sodium chloride demonstrating its affinity for this enzyme. Preincubation of rMA-K2 peptide with factor Xa neutralized, with 1.1:1 stoichiometry, the ability of factor Xa to hydrolyze a small chromogenic substrate. Additionally, rMA-K2 prolonged the time to clot formation in a plasma-based assay dependent on factor Xa concentration. Finally, this peptide mildly prolonged the prothrombin and modified prothrombin times of normal pooled plasma. Taken together this data demonstrates that this region of TFPI inhibits factor Xa activity and allows for further characterization of this enzyme-inhibitor complex.

Amino Acid Sequence↗
PubMed1994

Effect of gamma carboxylation on prothrombinase inhibitory activity of catalytically inactive factor XA.

Recombinant catalytically inactive factor Xa (factor rXai) is capable of assembly into inactive prothrombinase complexes, thus serving as a competitive inhibitor (Ki = 0.3nM) of active factor Xa. In order to study the role of gamma carboxylation in prothrombinase complex assembly, we have prepared differentially gamma carboxylated factor rXai and have measured the activities of these proteins in prothrombinase complex inhibition and in extension of plasma clotting. A factor rXai preparation containing 8 out of a possible maximum of 11 g carboxyglutamic acid (GLA) residues was found to be as active as chemically inactivated plasma factor Xa which was fully gamma carboxylated. Loss of a single additional g carboxyglutamic acid in the recombinant protein, however lead to a marked loss in activity. Factor rXai preparation with 8 GLA residues is also detected by a monoclonal antibody specific for a GLA dependent epitope. Thus assembly of the factor Va/Xa complex on phospholipid membranes does not require the presence of all of the g carboxyglutamic acid residues present in the plasma protein.

1-Carboxyglutamic Acid↗
PubMed2001

Residues Y179 and H101 of a hydrophobic patch of factor VII are involved in activation by factor Xa.

We studied factor Xa activation of human factor VII in hopes of identifying factor VII residues, not adjacent to the cleavage site, involved in this interaction. We made eight factor VIIs with single mutations (N100A, H101A, D102Q, L144A, R147A, Y179A, D186A, and F256A) and two factor VIIs with multiple mutations [MM3 (L144A/R147A/D186A) and MM4 (N100A/H101A/Y179A/F256A)]. Residues in MM3 have previously been identified as affecting factor X activation, and the residues of MM4 are located at a hydrophobic patch of factor VII on the opposite side of the catalytic domain from those in MM3. Only H101A, Y179A, and MM4 were activated significantly more slowly than the wild type. Results of our kinetic analyses showed that the catalytic efficiency of factor Xa for activation of factor VII was 176- and 234-fold higher than that for H101A andY179A, respectively. All the mutants with measurable activity had affinities for tissue factor similar to those of the wild type. The activated hydrophobic patch residues, except N100A, which is adjacent to one of the catalytic residues, had normal activities toward both a small peptide substrate and factor X. The rest of the activated mutants (except D102Q with no activity) had reduced activities toward the small substrate (except R147A) and factor X. We conclude that factor VII activation by factor Xa and factor VIIa's catalytic interaction with factor X involve different regions in the catalytic domain, and residues H101 and Y179, part of an aromatic hydrophobic patch, are specifically involved in factor Xa activation of factor VII.

Amino Acid Sequence↗
PubMed2001

The tissue factor region that interacts with factor Xa in the activation of factor VII.

Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.

1-Carboxyglutamic Acid↗
PubMed1996

Kinetics of factor VIII light-chain cleavage by thrombin and factor Xa. A regulatory role of the factor VIII heavy-chain region Lys713-Arg740.

Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region Lys713-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3 +/- 0.3 x 10(6) M-1 s-1, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713-Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713-1637)-factor VIII. This suggests that the region Lys713-Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713-1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713-1637)-factor VIII is clearly reduced. Furthermore, the secondorder rate constant (0.2 +/- 0.1 x 10(6) M-1 s-1) of des-(713-1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713-1637)-factor VIII light-chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des-(713-1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes both activation and light-chain proteolysis.

Antibodies, Monoclonal↗
PubMed2006

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.

Ancylostoma↗
PubMed1994

Protein S binds to and inhibits factor Xa.

Although human protein S binds to human factor Va and inhibits prothrombinase activity, this inhibition is not totally dependent on factor Va. Hence, we investigated possible interaction of protein S with human factor Xa. Factor Xa, diisopropylphospho-factor Xa and their biotin derivatives ligand blotted specifically to protein S and protein S ligand blotted specifically to factor X and factor Xa. Biotinylated factors X and Xa bound to immobilized protein S and, reciprocally, protein S bound to immobilized factor Xa with a Kd of approximately 19 nM. In fluid phase, protein S bound to factor Xa with a Kd of approximately 18 nM. Protein S at 33 nM reversibly inhibited 50% of factor Xa amidolytic activity. Protein S inhibition of prothrombin conversion to thrombin by factor Xa was phospholipid-independent and was 1.6 times stimulated by Ca2+ ions. Inhibition of prothrombinase activity by protein S was 2.3-fold more potent in the presence of factor Va, with 50% inhibition at approximately 8 nM protein S. Protein S prolonged the factor Xa one-stage clotting time of protein S-depleted plasma in a dose-dependent manner. These data demonstrate mechanisms of anticoagulant action for protein S that are independent of activated protein C and that involve direct binding to factors Xa and Va and direct inhibition of factor Xa.

Biotin↗
PubMed1998

Binding site for blood coagulation factor Xa involving residues 311-325 in factor Va.

Factor Va inactivation by activated protein C is associated with cleavages at Arg306, Arg506, and Arg679 with Arg306 cleavage causing the major activity loss. To study functional roles of the Arg306 region, overlapping 15-mer peptides representing the sequence of factor Va residues 271-345 were synthesized and screened for anticoagulant activities. The peptide containing residues 311-325 (VP311) noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution with a Kd of 70 microM. Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa or prothrombin bound to immobilized VP311 with relatively high affinity. These results support the hypothesis that residues 311-325, which are positioned between the A1 and A2 domains of factor Va and likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by activated protein C at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311-325, thereby down-regulating the binding of factor Xa to factor Va.

Amino Acid Sequence↗
PubMed1993

The influence of factor Va on the active site of factor Xa.

The interaction of factor Xa with factor Va on the membrane surface results in a 3,000-fold increase in the kcat for the activation of prothrombin catalyzed by factor Xa. The reaction between the transition state irreversible inhibitor dansylglutamyl-glycyl-arginyl chloromethyl ketone (DEGRck) and factor Xa was characterized and employed to evaluate changes in the active site of factor Xa resulting from its interaction with factor Va, which may account for the increased catalytic efficiency of the enzyme complex. Inhibition studies indicated a two-step inhibition reaction involving a reversible binding step (Ki = 1.13 microM) and an irreversible alkylation step (ki = 0.65 s-1). The interaction between factors Va and Xa in solution or on membranes resulted in a small decrease in the overall second-order rate constant (ki/Ki) for the inhibition reaction. The incorporation of DEGRck into the active site of factor Xa results in a large change in the fluorescence intensity of the dansyl moiety. The fluorescence change was employed to study the reaction between enzyme and inhibitor directly by stopped-flow fluorescence measurements. The fluorescence traces were biphasic, indicating that the association of DEGRck with factor Xa and the subsequent covalent modification of the active site could be resolved because of differences in fluorescence intensities of the intermediate and product. This interpretation was verified by rapid chemical quench experiments. The reaction between DEGRck and factor Xa was characterized by a second-order association rate constant of 8.38 +/- 0.28 x 10(5) M-1.s-1 and an intrinsic rate constant for the alkylation step of 1.67 +/- 0.25 s-1. The rate constant for the alkylation step was unchanged in the presence of factor Va and membranes, whereas the association rate constant was modestly decreased by approximately 50%. The decrease in the association rate constant did not result from the partitioning of factor Xa to the membrane surface and could therefore be ascribed to an effect of factor Va on the protease. The data suggest that the interaction between factors Va and Xa on the membrane surface does not detectably alter the catalytic residues but may result in changes in the binding or accessibility of substrate to extended macromolecular recognition sites on the protease.

Amino Acid Chloromethyl Ketones↗
PubMed1994

Kinetics of the inhibition of factor Xa and the tissue factor-factor VIIa complex by the tissue factor pathway inhibitor in the presence and absence of heparin.

The inhibition by tissue factor pathway inhibitor (TFPI) of its two target enzymes--factor Xa and the tissue factor-factor VIIa complex (TF:VIIa)--has been studied under near-physiological reactant concentrations and conditions. Over a TFPI range of 0-1 nM, the rate of inhibition of factor Xa, in the presence of Ca2+ and anionic phospholipid vesicles at 37 degrees C, was proportional to TFPI concentration, giving an association rate, k1, of 0.96 x 10(9) M-1 min-1. Factor Xa inhibition did not proceed to completion, the reaction attaining a near-equilibrium that was dependent on the TFPI concentration. The estimated dissociation rate of the TFPI:Xa complex, k-1, was independent of TFPI concentration, with a mean value of 0.02 min-1. The resulting calculated value of K1, the apparent dissociation constant for the initial step, is 21 pM. Slow decay of the remaining factor Xa in such incubations, detectable after attainment of the rapid initial near-equilibrium, confirmed the two-step mechanism proposed by Huang et al. (1993) [J. Biol. Chem. 268, 26950-26955], but did not permit determination of a rate constant for the second step. Omission of anionic phospholipid had no significant effect on either k1 or k-1. A high-molecular-weight fraction of heparin, at saturating levels (> or = 0.05 unit/mL, congruent to 25 nM), increased k1 2-fold, with no detectable effect on k-1. The second stage of TFPI action was studied by preformation of the TFPI:Xa complex, and its incubation with the TF:VIIa complex in the presence of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Discovery of 1-(2-aminomethylphenyl)-3-trifluoromethyl-N- [3-fluoro-2'-(aminosulfonyl)[1,1'-biphenyl)]-4-yl]-1H-pyrazole-5-carboxyamide (DPC602), a potent, selective, and orally bioavailable factor Xa inhibitor(1).

Factor Xa, a serine protease, is at the critical juncture between the intrinsic and extrinsic pathways of the coagulation cascade. Inhibition of factor Xa has the potential to provide effective treatment for both venous and arterial thrombosis. We recently described a series of meta-substituted phenylpyrazoles that are highly potent, selective, and orally bioavailable factor Xa inhibitors. In this paper we report our efforts to further optimize the selectivity profile of our factor Xa inhibitors with a series of ortho- and/or para-substituted phenylpyrazole derivatives. The most potent compounds display sub-nanomolar inhibition constants for factor Xa and show greater than 1000-fold selectivity against other serine proteases. These compounds are also effective in a rabbit model of arteriovenous shunt thrombosis. Optimization of this series led to the preclinical development of DPC602, a 2-(aminomethyl)phenylpyrazole analogue, as a highly potent, selective, and orally bioavailable factor Xa inhibitor.

Administration, Oral↗
PubMed1995

Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C.

Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively. We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation. Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg306 (20-fold). Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg506. Inactivation of factor VaR506Q, which was isolated from the plasma of a homozygous APC-resistant patient and which lacks the Arg506 cleavage site, was also stimulated by protein S but was not affected by factor Xa. This confirms that the target sites of protein S and factor Xa involve Arg306 and Arg506, respectively. Factor Xa completely blocked APC-catalyzed cleavage at Arg506 in normal factor Va (1 nM) with a half-maximal effect (K1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va. In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor VaR506Q were almost annihilated in the presence of factor Xa and protein S. This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition.

Amino Acid Sequence↗