Synthesis and X-ray crystal structures of substituted fluorobenzene and benzoquinone inhibitors of the tissue factor VIIa complex

Multistep syntheses of substituted benzenes and benzoquinone inhibitors of tissue Factor VIIa are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa (TF/VIIa) enzyme. The compounds exhibited modest potency on TF/VIIa with selectivity over Factor Xa and thrombin. The X-ray crystal structures of the targeted fluorobenzene 12a and benzoquinone 14 inhibitors bound to TF/VIIa were obtained and will be described.     

Polymer-assisted solution-phase (PASP) parallel synthesis of an alpha-ketothiazole library as tissue factor VIIa inhibitors

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-L-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis affords the desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.     

Rational combinatorial chemistry-based selection,synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca(2+)-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (approximately 1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.     

Factor VIIa/tissue factor generates a form of factor V with unchanged specific activity, resistance to activation by thrombin, and increased sensitivity to activated protein C

Factor VIIa, in complex with tissue factor (TF), is the serine protease responsible for initiating the clotting cascade. This enzyme complex (TF/VIIa) has extremely restricted substrate specificity, recognizing only three previously known macromolecular substrates (serine protease zymogens, factors VII, IX, and X). In this study, we found that TF/VIIa was able to cleave multiple peptide bonds in the coagulation cofactor, factor V. SDS-PAGE analysis and sequencing indicated the factor V was cleaved at Arg679, Arg709, Arg1018, and Arg1192, resulting in a molecule with a truncated heavy chain and an extended light chain. This product (FVTF/VIIa) had essentially unchanged activity in clotting assays when compared to the starting material. TF reconstituted into phosphatidylcholine vesicles was ineffective as a cofactor for the factor VIIa cleavage of factor V. However, incorporation of phosphatidylethanolamine in the vesicles had little effect over the presence of 20% phosphatidylserine. FVTF/VIIa was as sensitive to inactivation by activated protein C (APC) as thrombin activated factor V as measured in clotting assays or by the appearance of the expected heavy chain cleavage products. The FVTF/VIIa could be further cleaved by thrombin to release the normal light chain, albeit at a significantly slower rate than native factor V, to yield a fully functional product. These studies thus reveal an additional substrate for the TF/VIIa complex. They also indicate a new potential regulatory pathway of the coagulation cascade, i.e., the production of a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.     

Elevated function of blood clotting factor VIIa mutants that have enhanced affinity for membranes. Behavior in a diffusion-limited reaction

Blood clotting factor VIIa is involved in the first step of the blood coagulation cascade, as a membrane-associated enzyme in complex with tissue factor (TF). Factor VIIa is also an important therapeutic agent for hemophilia where its function may include TF-independent as well as TF-dependent mechanisms. This study compared the activity of wild type factor VIIa (WT-VIIa) with that of a mutant with elevated affinity for membrane (P10Q/Q32E, QE-VIIa). Phospholipid and cell-based assays showed the mutant to have up to 40-fold higher function than WT-VIIa in both TF-dependent and TF-independent reactions. Tissue factor-dependent reactions displayed the maximum enhancement when binding had reached equilibrium in competition with another TF-binding protein. In liposome-based assays, the association rate of WT-VIIa with TF occurred at a physical maximum and could not be improved by site-directed mutagenesis. A practical consequence was identical function of WT-VIIa and QE-VIIa in assays that depended entirely on assembly kinetics. Thus, factor VIIa mutants provided unique reagents for probing the mechanism of factor VIIa action. They may also offer superior agents for therapy.     

Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation

The activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca2+ (TF.VIIa.PCPS.Ca2+) and factor Xa.PCPS.Ca2+ enzyme complexes was investigated. Reactions were performed in a highly purified system consisting of isolated human plasma proteins and recombinant human tissue factor with synthetic phospholipid vesicles (PCPS: 75% phosphatidylcholine (PC), 25% phosphatidylserine (PS)). Factor IX activation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]factor IX activation peptide assay, colorimetric substrate thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) hydrolysis, and specific incorporation of a fluorescent peptidyl chloromethyl ketone. Factor IX activation by the TF.VIIa.PCPS.Ca2+ enzyme complex was observed to proceed through the obligate non-enzymatic intermediate species factor IX alpha. The simultaneous activation of human coagulation factors IX and X by the TF.VIIa.PCPS.Ca2+ enzyme complex were investigated. When factors IX and X were presented to the TF.VIIa complex, at equal concentrations, it was observed that the rate of factor IX activation remained unchanged while the rate of factor X activation slowed by 45%. When the proteolytic cleavage products of this reaction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was observed that the intermediate species factor IX alpha was generated more rapidly when factor X was present in the reaction mixture. When factor IX was treated with factor Xa.PCPS in the presence of Ca2+, it was observed that factor IX was rapidly converted to factor IX alpha. The activation of factor IX alpha by the TF.VIIa.PCPS.Ca2+ complex was evaluated, and it was observed that factor IX alpha was activated more rapidly by the TF.VIIa.PCPS.Ca2+ complex than was factor IX itself. These data suggest that factors IX and X, when presented to the TF.VIIa.PCPS.Ca2+ enzyme complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IX alpha.     

Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces.

The kinetics of the binding of rVIIa to cell surface tissue factor (TF) and the resultant expression of VIIa/TF activity were studied. Binding of 125I-rVIIa (10 nM) to cell surface TF required 30-60 min for saturation, whereas VIIa/TF activity was fully expressed toward factor X (F X) on intact monolayers after only 1 min of incubation. At the time only 10-20% of the total VIIa TF complexes present at saturation had formed. Freeze-thawing the monolayers before assay increased VIIa/TF activity up to 30-fold, and the time course of its expression was similar to that of TF-specific binding of VIIa to the monolayers. Equilibrium binding revealed a single high affinity binding class of TF sites on intact monolayers for rVIIa with a Kd of 1.6 nM. Experiments with active-site inhibited rVIIa yielded evidence for two populations of VIIa. TF complexes on intact monolayers: (1) a minor population (less than 20%) that formed within 1 min of incubation and accounted for all VIIa/TF activity toward F X present on the intact monolayers, and (2) a major population that was inactive toward F X on intact monolayers but which was fully active after the monolayers were lysed. Tissue factor pathway inhibitor (TFPI).F Xa complexes inhibited the VIIa/TF activity of the first population, i.e. of the complexes active on intact monolayers, half maximally at a concentration of 0.2 nM TFPI. TFPI/Xa also bound to the second population of VIIa.TF complexes on intact monolayers and inhibited their expression of VIIa/TF activity following cell lysis with a half-maximal inhibitory concentration of 2.0 nM. The potential physiologic implications of these findings are discussed.     

Use of an oriented transmembrane protein to probe the assembly of a supported phospholipid bilayer.

Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer.     

Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking.

The upstream coagulation enzymes are homologous trypsin-like serine proteases that typically function in enzyme-cofactor complexes, exemplified by coagulation factor VIIa (VIIa), which is allosterically activated upon binding to its cell surface receptor tissue factor (TF). TF cooperates with VIIa to create a bimolecular recognition surface that serves as an exosite for factor X binding. This study analyzes to what extent scissile bond docking to the catalytic cleft contributes to macromolecular substrate affinity. Mutation of the P1 Arg residue in factor X to Gln prevented activation by the TF.VIIa complex but did not reduce macromolecular substrate affinity for TF.VIIa. Similarly, mutations of the S and S' subsites in the catalytic cleft of the enzyme VIIa failed to reduce affinity for factor X, although the affinity for small chromogenic substrates and the efficiency of factor X scissile bond cleavage were reduced. Thus, docking of the activation peptide bond to the catalytic cleft of this enzyme-cofactor complex does not significantly contribute to affinity for macromolecular substrate. Rather, it appears that the creation of an extended macromolecular substrate recognition surface involving enzyme and cofactor is utilized to generate substrate specificity between the highly homologous, regulatory proteases of the coagulation cascade.     

Structure-based design and synthesis of pyrazinones containing novel P1 'side pocket' moieties as inhibitors of TF/VIIa

We describe the structure-based design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors contain substituents meta to the P(1) amidine designed to explore additional interactions with the VIIa residues in the so-called 'S(1) side pocket'. A crystal structure of the designed inhibitors demonstrates the ability of the P(1) side pocket moiety to engage Lys192 and main chain of Gly216 via hydrogen bond interactions, thus, providing additional possibility for chemical modification to improve selectivity and/or physical properties of inhibitors.     

Targeted deletion of the cytosolic domain of tissue factor in mice does not affect development

The role of the cytosolic domain of tissue factor (TF) in signal transduction and gene regulation was studied in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids. This deletion was introduced in exon 6 along with a floxed neo(R) selection cassette in intron 5 using homologous recombination in embryonic stem cells. Removal of the floxed neo(R) cassette by in vivo Cre-mediated loxP recombination yielded TF(+/deltaCT) and TF(deltaCT/deltaCT) mice. In contrast to TF(-/-) mice, TF(+/deltaCT) and TF(deltaCT/deltaCT) mice displayed normal embryonic development, survival, fertility, and blood coagulation. Factor VIIa or factor Xa stimulation produced similar p44/42 MAPK activation in TF(+/+) and TF(deltaCT/deltaCT) fibroblasts. These data, based on expression of a TF(deltaCT) molecule from the endogenous TF locus, provide conclusive proof that the cytosolic domain of TF is not essential for signal transduction in embryogenesis and in physiological postnatal processes.     

The interaction of human factor VIIa with tissue factor.

The interaction of factor VIIa with tissue factor (TF) results in an increase in the catalytic efficiency for the hydrolysis of several synthetic peptidyl p-nitroanilide substrates by factor VIIa. The binding of human recombinant factor VIIa to recombinant human TF incorporated into vesicles containing phosphatidylcholine (TF/PC) or phosphatidylcholine/phosphatidylserine (TF/PCPS) was studied using the increased rate of H-D-phenylalanyl L-pipecoyl L-arginine p-nitroanilide (S2238) hydrolysis as a signal for the interaction. The saturable dependence of rate on increasing concentrations of factor VIIa or TF/PCPS yielded no obvious evidence for cooperativity and could be analyzed according to the interaction of factor VIIa with independent noninteracting sites (Kd = 259 +/- 60 pM, n = 1.05 +/- 0.12 mol of factor VIIa/mol of TF at saturation). Identical titration curves and equilibrium parameters were derived from titrations using TF/PC or TF in the absence of phospholipids, indicating that possible protein-membrane interactions do not further stabilize the extrinsic Xase complex. The dissociation constant for the interaction of factor VIIa with TF/PCPS inferred from measurements of factor X activation (Kd = 197 +/- 38 pM) was comparable with the values obtained from measurements of S2238 hydrolysis. In contrast to the membrane-independent nature of the enzyme-cofactor interaction, the rate of factor X activation was reduced by approximately 50-fold when the enzyme complex was assembled using solution-phase TF. Collectively, the result indicate that the membrane dependence of extrinsic Xase function primarily results from an influence of the membrane surface on factor X utilization.     

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.     

Intrinsic versus extrinsic coagulation. Kinetic considerations.

A study to compare the kinetics of activation of factor IX by Factor XIa/Ca2+ and by Factor VIIa/tissue factor/Ca2+ has been undertaken. When purified human proteins, detergent-extracted brain tissue factor and tritiated-activation-peptide-release assays were utilized, the kinetic constants obtained were: Km = 310 nM, kcat. = 25 min-1 for Factor XIa and Km = 210 nM, kcat. = 15 min-1 for Factor VIIa. The kinetic constants for the activation of Factor X by Factor VIIa/brain tissue factor were: Km = 205 nM, kcat. = 70 min-1. Predicted rates for the generation of Factor IXa and Factor Xa were obtained when human monocytic tumour U937 cells (source of tissue factor) and Factor VIIa were used to form the activator. In other experiments, inclusion of high-Mr kininogen did not increase the activation rates of Factor IX by Factor XIa in the presence or absence of platelets and/or denuded rabbit aorta. These kinetic data strongly indicate that both Factor XIa and Factor VIIa play physiologically significant roles in the activation of Factor IX.     

Inhibitors of the tissue factor/factor VIIa-induced coagulation: synthesis and in vitro evaluation of novel specific 2-aryl substituted 4H-3,1-benzoxazin-4-ones

The synthesis of a series of novel 2-aryl substituted 4H-3,1-benzoxazin-4-ones and their evaluation as specific inhibitors of the Tissue Factor (TF)/Factor VIIa (FVIIa)-induced pathway of coagulation is reported. Inhibitory activities (IC50 values) in the range 0.17 to > 40 microM on the activation of Factor X (FX) by the TF/FVIIa complex were found for compounds having one or two electronegative substituents such as F, Cl and NO2 in the 2-aryl substituent. Different substitutions both electron-attracting and donating groups were allowed in the 5, 6, 7 and 8 positions. Several of the compounds showed a selectivity ratio towards FX and thrombin of > 50, thus being the first small molecules described as potential drugs for oral antithrombotic treatment without side effects such as bleeding which is observed especially with thrombin inhibitors. The best substituent pattern being the 2-aryl group substituted with: 2-F; 2,6-F2; or 2-FX; 6-Cl; together with electronegative substitution in the 5, 6, 7, or 8 positions. 2-Heteroaryl substituents like thienyl and furanyl were of low activity while some 2-(2-chloro-3-pyridyl) derivatives had inhibitory activity < 10 microM and a good selectivity.     

Role of zymogenicity-determining residues of coagulation factor VII/VIIa in cofactor interaction and macromolecular substrate recognition

Factor VIIa (VIIa) remains in a zymogen-like state following proteolytic activation and depends on interactions with the cofactor tissue factor (TF) for function. Val(21), Glu(154), and Met(156) are residues that are spatially close in available zymogen and enzyme structures, despite major conformational differences in the corresponding loop segments. This residue triad displays unusual side chain properties in comparison to the properties of other coagulation serine proteases. By mutagenesis, we demonstrate that these residues cooperate to stabilize the enzyme conformation and to enhance the affinity for TF. In zymogen VII, however, substitution of the triad did not change the cofactor affinity, further emphasizing the crucial role of the activation pocket in specifically stabilizing the active enzyme conformation. In comparison to VIIa(Q156), the triple mutant VIIa(N21I154Q156) had a stabilized amino-terminal Ile(16)-Asp(194) salt bridge and enhanced catalytic function. However, proteolytic and amidolytic activities of free VIIa variants were not concordantly increased. Rather, a negatively charged Asp at position 21 was the critical factor that determined whether an amidolytically more active VIIa variant also more efficiently activated the macromolecular substrate. These data thus demonstrate an unexpected complexity by which the zymogenicity-determining triad in the activation pocket of VIIa controls the active enzyme conformation and contributes to exosite interactions with the macromolecular substrate.     

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.     

Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions

Protein-phospholipid as well as protein-protein interactions may be critical for tight binding of the serine protease factor VIIa (VIIa) to its receptor cofactor tissue factor (TF). To elucidate the role of protein-protein interactions, we analyzed the interaction of VII/VIIa with TF in the absence of phospholipid. Binding of VII occurred with similar affinity to solubilized and phospholipid-reconstituted TF. Lack of the gamma-carboxyglutamic acid (Gla)-domain (des-(1-38)-VIIa) resulted in a 10- to 30-fold increase of the Kd for the interaction, as did blocking the Gla-domain by Fab fragments of a specific monoclonal antibody. These results suggest that the VII Gla-domain can participate in protein-protein interaction with the TF molecule per se rather than only in interactions with the charged phospholipid surface. Gla-domain-independent, low affinity binding of VII to TF required micromolar Ca2+, indicating involvement of high affinity calcium ion binding sites suggested to be localized in VII rather than TF. Interference with Gla-domain-dependent interactions with TF did not alter the TF. VIIa-dependent cleavage of a small peptidyl substrate, whereas the proteolytic activation of the protein substrate factor X was markedly decreased, suggesting that the VIIa Gla-domain not only participates in the formation of a more stable TF. VIIa complex but contributes to extended substrate recognition.     

Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function

Membrane anchoring of tissue factor (TF), the cell receptor for coagulation factor VIIa (VIIa), exemplifies an effective mechanism to localize proteolysis at the cell surface. A recombinant TF mutant (TF1-219), deleted of membrane spanning and intracellular domains, was used to evaluate the role of phospholipid interactions for assembly of substrate with the catalytic TF.VIIa complex. TF1-219 was secreted by cells rather than expressed as a cell membrane protein. Unlike free VIIa, TF1-219 as well as the TF1-219.VIIa complex demonstrated no stable association with phospholipid. In the absence of lipid, kinetic evaluation of substrate factor X cleavage by free VIIa, TF.VIIa, and TF1-219.VIIa suggests that the catalytic function of VIIa rather than substrate recognition is enhanced by complex formation. Furthermore, compared with free factor X, factor X on phospholipid was preferentially cleaved as a substrate by TF1-219.VIIa. TF-dependent initiation of the coagulation protease cascades thus involves an enhancement of the activation of factor X on the cell surface by a crucial role of the TF transmembrane domain to membrane anchor the reaction, by the TF extracellular domain to provide protein-protein interactions with VIIa to enhance the activity of the catalytic domain of VIIa, and the preferential presentation of factor X as a substrate when associated with phospholipid surfaces.