Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa

The catalytic activity of the trypsin-like serine protease coagulation factor VIIa is allosterically regulated. In this work, we employed monoclonal antibodies as probes to analyze conformational changes in the VII protease domain that are induced by zymogen activation, cofactor tissue factor (TF) binding, and active site occupancy. The epitopes of three monoclonal antibodies were mapped using a panel of 57 individual alanine replacement mutants in the protease domain. Two of the antibodies had typical "hot spot" epitopes in a basic cluster above the active site cleft and antibody binding to these epitopes was not affected by zymogen activation, TF binding, or active site occupancy. In contrast, the binding kinetics of VII/VIIa to a monoclonal antibody that mapped to an extended epitope overlapping with the macromolecular substrate exosite was affected by each of the conformational transitions of the VIIa protease domain. The changes in antibody affinity are consistent with a transition from zymogen VII to the TF.VIIa complex, with free enzyme VIIa as an intermediate that retains some zymogen-like features responsible for its low catalytic activity. In contrast, active site occupancy resulted in effects that were qualitatively different from the effects of zymogen activation on the antibody epitope. This provides novel insight into the conformational interdependence between the active site, the region for macromolecular substrate recognition, and the cofactor binding exosite of this allosterically regulated serine protease.     

The structural basis of function of the TF. VIIa complex in the cellular initiation of coagulation

Cell surface tissue factor (TF), the major in vivo initiator of coagulation, activates coagulation by binding and allosteric activation of the serine protease factor. VIIa (VIIa). A graphic scheme to account for function of this initial bimolecular activation complex has emerged from the integration of structural with functional analyses. The VIIa light chain, specifically the Gla and EGF-1 domains, form extended hydrophobic contacts with TF which account for most of the free energy of binding. These contacts tether VIIa and facilitate interactions of the protease domain with TF necessary for induction of protease function. Several contact residues in the VIIa protease domain-TF interface are involved in the activation of VIIa by complex allosteric effects. Macromolecular substrate zymogens interact with both the VIIa protease domain and the carboxyl-terminal module of TF. Docking of the VIIa Gla-domain to the latter region of TF appears to contribute to substrate assembly. The current data suggest an extended embrace between TF and VIIa to form the bimolecular enzyme TF.VIIa.     

Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF.VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF. VIIa reversibly inhibited with a potent (Ki=0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 A resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa.5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF.VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.     

A novel soluble tissue factor variant with an altered factor VIIa binding interface

Tissue factor (TF) residues Lys20 and Asp58 form part of a binding epitope previously shown by alanine scanning to be critical for high affinity interactions with factor VIIa (FVIIa). To explore the possibility of enhancing the affinity of a TF-based antagonist for FVIIa, we created libraries in which residues at 20, 58, and adjacent positions were varied in constructs containing the soluble extracellular domain of TF (sTF) fused to the bacteriophage M13 tail coat protein. TF variants monovalently displayed on phage were then sorted on the basis of binding to FVIIa. Sorting of preliminary libraries, in which position 58 and/or 20 and surrounding residues were randomized, led to the selection of TF proteins of essentially wild-type sequence. Therefore, we devised a strategy wherein TF position 20 was held fixed as alanine and 5 specific residues near to, and including, position 58 were randomized to effectively obtain alternative sequences at this interface. The consensus sequence reached with this library included wild-type residues at positions 61, 62, 65, and 66 but exclusively tryptophan at position 58. Analyses of the soluble K20A,D58W (A20W58) TF protein indicated that it binds FVIIa with an affinity comparable with wild-type sTF but is defective as a cofactor for FVIIa-dependent factor X activation. Further experiments designed to elucidate the mechanism of binding suggest that the new binding interactions involve more than the simple addition of hydrophobic surface area.     

Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis

Tissue factor (TF), the initiating cell surface receptor of the coagulation cascade, plays important roles in embryogenesis, angiogenesis, and tumor cell metastasis. It is controversial whether proteolytic function of TF complexed with its serine protease ligand VIIa is required for metastatic tumor dissemination. We show here in a model for TF-dependent experimental hematogenous metastasis, that TF supports metastasis by both proteolytic activity of the TF-VIIa complex and currently undefined functions of the cytoplasmic domain. We demonstrate that ligand binding of VIIa to TF is required for metastasis. Antimetastatic properties of covalently inactivated VIIa provide evidence that ligand binding is insufficient per se to support metastasis, emphasizing that proteolytic activity is necessary for the metastatic process. Ala or Asp mutations of cytoplasmic serine residues were introduced to preclude or mimic phosphorylation. In vivo analysis of these mutants suggests that local protease generation on the tumor cell surface does not serve simply to activate the cytoplasmic domain of TF by serine phosphorylation. Thus, extracellular functions of the catalytically active TF-VIIa complex cooperate with specific functions of the TF cytoplasmic domain to support the complex process of hematogenous tumor cell dissemination.     

A 2.8 A resolution structure of 6-phosphogluconate dehydrogenase from the protozoan parasite Trypanosoma brucei: comparison with the sheep enzyme accounts for differences in activity with coenzyme and substrate analogues

The three-dimensional structure of 6-phosphogluconate dehydrogenase (6PGDH) from the parasitic protozoan Trypanosoma brucei has been solved at 2.8 A resolution. This pentose phosphate pathway enzyme is NADP-dependent; NADPH generated in the reaction protects against oxidative stress. The enzyme crystallises in the space-group P3121 with a dimer in the asymmetric unit and cell dimensions a=b=135.13 A, c=116.74 A, alpha=beta=90 degrees, gamma=120 degrees. The structure has refined to R=18.6% (Rfree=27.3%) with good geometry. The amino acid sequence of T. brucei 6PGDH is only 35% identical to that of the sheep liver enzyme and significant activity differences have been observed. The active dimer assembles with the C-terminal tail of one subunit threaded through the other, forming part of the substrate binding site. The tail of T. brucei 6PGDH is shorter than that of the sheep enzyme and its terminal residues associate tightly with the second monomer. The three-dimensional structure shows this generates additional interactions between the subunits close to the active site; the coenzyme binding domain is thereby associated more tightly with the helical domain. Three residues, conserved in all other known sequences, are important in creating a salt bridge between monomers close to the substrate binding site. The differences could explain the 200-fold enhanced affinity observed for the substrate analogue 6-phospho-2-deoxy-D-gluconate and suggest targets for anti-parasite drug design. The coenzyme binding domain of 6PGDH has a beta-alpha-beta fold; while in most species the "fingerprint" sequence is GxAxxG, in the T. brucei enzyme it is GxGxxG. Additional interactions between the enzyme and the coenzyme bis-phosphate are likely in the parasite 6PGDH, accounting for greater inhibition (40-fold) of 2'5'-ADP. While the core of the T. brucei dimer was restrained during refinement, several conformational differences have been found between the monomers; those at the coenzyme binding site suggest the molecule could be asymmetric during the enzyme reaction.     

A study of subunit interface residues of fructose-1,6-bisphosphatase by site-directed mutagenesis: effects on AMP and Mg2+ affinities

The structural transformation of fructose-1,6-bisphosphatase upon binding of the allosteric regulator AMP dramatically changes the interactions across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Met18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose-1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spectrometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in kcat with Met18Arg and Asn9Asp, respectively. The K(m) for fructose 1,6-bisphosphate increased about 2-approximately 4-fold relative to that of the wild-type enzyme in the four mutants. A 50-fold lower Ka value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18Ile with no alteration of the Ki for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fold) without a change in Mg2+ affinity. Increases of 6- and 2-fold in the Ki values for AMP were found with Asn9Asp and Ser87Ala, respectively. There was no difference in the cooperativity for AMP inhibition between the wild-type and the mutant forms of fructose-1,6-bisphosphatase. This study demonstrates that the mutation of residues in the C1-C4 (C2-C3) interface of fructose-1,6-bisphosphatase can significantly affect the affinity for Mg2+, which is presumably bound 30 A away. Moreover the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the corresponding allosteric states of the enzyme. The kinetics and structural modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.     

Crystal structure of a complement factor D mutant expressing enhanced catalytic activity

Complement factor D is a serine protease regulated by a novel mechanism that depends on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumed to be induced by the single natural substrate, C3bB, and to result in reversible reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, and Ser215 of factor D (chymotrypsinogen numbering has been used for comparison purposes) with the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to induce substantially higher catalytic activity associated with a typical serine protease alignment of the catalytic triad residues His57, Asp102, and Ser195. These results provide a partial structural explanation for the low reactivity of "resting-state" factor D toward synthetic substrates.     

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.     

Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.     

The role of IL-4, IL-10, and TNF-alpha in the immune suppression induced by ultraviolet radiation

The two main catalytic residues Cys25 and His159 of the monomeric cysteine protease papain are located on different walls of a cleft formed by two domains. This topology suggests a possible relationship between relative domain organization and catalytic mechanism. The effect on enzymatic parameters of structural modifications at various locations of the two-domain interface of papain was examined by individual or double replacements by Ala of pairs of interacting residues. Most modifications had no effect on enzyme activity. However, the enzyme's substrate turnover (kcat) decreased following simultaneous alteration of the two most conserved residues, forming an apolar contact located 15 A away from the active site. The pH activity profile of the double mutant was unchanged, indicating a conserved ionization state of the active site thiolate-imidazolium ion pair. This state is strongly dependent on the distance separating the two residues, thus suggesting that the active site geometry has not been significantly altered. Efficient enzymatic activity in papain requires more than a correct active site geometry and is influenced by domain packing properties in a region remote from the active site.     

Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases

Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.     

Pro region C-terminus:protease active site interactions are critical in catalyzing the folding of alpha-lytic protease

alpha-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is required transiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L. , and Agard, D. A. (1989) Nature 341, 462-464; Baker, D., et al. (1992) Nature 356, 263-265]. The pro region also acts as a potent inhibitor of the mature enzyme [Baker, D., et al. (1992) Proteins: Struct.,Funct., Genet. 12, 339-344]. This inhibition is mediated through direct steric occlusion of the active site by the C-terminal residues of the pro region [Sohl, J. L., et al. (1997) Biochemistry 36, 3894-3904]. Through mutagenesis and structure-function analyses we have begun to investigate the mechanism by which the pro region acts as a single turnover catalyst to facilitate folding of the mature protease. Of central interest has been mapping the interface between the pro region and the protease and identifying interactions critical for stabilizing the rate-limiting folding transition state. Progressive C-terminal deletions of the pro region were found to have drastic effects on the rate at which the pro region folds the protease but surprisingly little effect on inhibition of protease activity. The observed kinetic data strongly support a model in which the detailed interactions between the pro region C-terminus and the protease are remarkably similar to those of known substrate/inhibitor complexes. Further, mutation of two protease residues near the active site have significant effects on stabilization of the folding transition state (kcat) or in binding to the folding intermediate (KM). Our results suggest a model for the alpha-lytic protease pro region-mediated folding reaction that may be generally applicable to other pro region-dependent folding reactions.     

An induced proximity model for caspase-8 activation

The assembly of the CD-95 (Fas/Apo-1) receptor death-inducing signaling complex occurs in a hierarchical manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8 (FLICE/Mach-1) by a homophilic interaction involving the death effector domains. Immediately after recruitment, the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large and small catalytic subunits. Since all caspases cleave their substrates after Asp residues and are themselves processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it follows that an upstream caspase can process a downstream zymogen. However, since FLICE represents the most apical caspase in the Fas pathway, its mode of activation has been enigmatic. We hypothesized that the FLICE zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active protease. Support for this was provided by (i) the synthesis of chimeric Fpk3FLICE molecules that can be oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2. Cells transfected with Fpk3FLICE underwent apoptosis after exposure to FK1012H2; (ii) the creation of a nonprocessable zymogen form of FLICE that retained low but detectable protease activity.     

Structural role of the 30's loop in determining the ligand specificity of the human immunodeficiency virus protease

The structural basis of ligand specificity in human immunodeficiency virus (HIV) protease has been investigated by determining the crystal structures of three chimeric HIV proteases complexed with SB203386, a tripeptide analogue inhibitor. The chimeras are constructed by substituting amino acid residues in the HIV type 1 (HIV-1) protease sequence with the corresponding residues from HIV type 2 (HIV-2) in the region spanning residues 31-37 and in the active site cavity. SB203386 is a potent inhibitor of HIV-1 protease (Ki = 18 nM) but has a decreased affinity for HIV-2 protease (Ki = 1280 nM). Crystallographic analysis reveals that substitution of residues 31-37 (30's loop) with those of HIV-2 protease renders the chimera similar to HIV-2 protease in both the inhibitor binding affinity and mode of binding (two inhibitor molecules per protease dimer). However, further substitution of active site residues 47 and 82 has a compensatory effect which restores the HIV-1-like inhibitor binding mode (one inhibitor molecule in the center of the protease active site) and partially restores the affinity. Comparison of the three chimeric protease structures with those of HIV-1 and SIV proteases complexed with the same inhibitor reveals structural changes in the flap regions and the 80's loops, as well as changes in the dimensions of the active site cavity. The study provides structural evidence of the role of the 30's loop in conferring inhibitor specificity in HIV proteases.     

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.     

1H nuclear magnetic resonance studies of hen lysozyme-N-acetylglucosamine oligosaccharide complexes in solution. Application of chemical shifts for the comparison of conformational changes in solution and in the crystal

Two-dimensional 1H nuclear magnetic resonance spectroscopy has been used to examine the complexes formed in solution between hen egg-white lysozyme and N-acetylglucosamine (GlcNAc) oligosaccharides. Changes in chemical shift have been measured for resonances of the majority of residues of lysozyme on binding the monomer, dimer and trimer of GlcNAc. The three inhibitors induce very similar changes in chemical shift, and these increase slightly with the length of the oligosaccharide. The largest changes are confined principally to the vicinity of site C in the active site cleft of the enzyme. These changes in chemical shift have been compared with differences in the ring current chemical shifts calculated from the crystal structures of unbound and GlcNAc3 bound lysozyme. This comparison suggests that the major conformational changes of residues in the vicinity of site C of the enzyme, that are caused by the binding of GlcNAc3, observed in the diffraction studies are at least consistent with the changes that occur in solution. Small changes in chemical shift are observed in the enzyme in regions remote from the active site, which indicate that the effects of inhibitor binding are felt throughout the enzyme. These changes in chemical shift correlate to a lesser extent than those near site C with the changes in chemical shift predicted from changes in conformation observed in the crystal structures. The results illustrate that chemical shifts are useful in assessing the significance of small conformational changes in proteins, although the usefulness of this approach will be limited by the resolution of the crystallographic structures, as well as the uncertainties in the origins of the chemical shift. Although conformational changes in site C account for many of the changes in the NMR spectrum of lysozyme, evidence is, however, presented for multiple binding sites for the GlcNAc oligosaccharides in solution, perhaps involving partial occupancy of site D.     

Tissue factor pathway inhibitor inhibits aortic smooth muscle cell migration induced by tissue factor/factor VIIa complex

Tissue factor (TF), a transmembrane glycoprotein, forms a high affinity complex with factor VII/VIIa (FVIIa) and thereby initiates blood coagulation. Tissue factor pathway inhibitor (TFPI) is an endogenous protease inhibitor of TF/FVIIa-initiated coagulation. We previously reported that TF was a strong chemotactic factor for cultured vascular smooth muscle cells (SMCs). In this study, we examined the contribution of FVIIa and the effect of TFPI to TF-induced cultured SMC migration. TF/FVIIa complex showed a strong migration ability, however, neither TF alone nor FVIIa induced SMC migration. TF/FVIIa treated by a serine protease inhibitor and the complex of TF and inactivated FVIIa (DEGR-FVIIa) did not stimulate SMC migration. Pretreatment with hirudin and the antibodies to alpha-thrombin and factor X had no effect on TF/FVIIa-induced SMC migration, although alpha-thrombin and factor Xa also induced SMC migration respectively. TFPI markedly inhibited TF/FVIIa-induced SMC migration in a concentration-dependent manner, but did not affect the SMC migration induced by platelet-derived growth factor (PDGF)-BB, basic fibroblast-growth factor (bFGF), or alpha-thrombin. These results indicate that the catalytic activity of TF/FVIIa complex is important on SMC migration, and TFPI can reduce SMC migration as well as thrombosis.     

Potent bifunctional anticoagulants: Kunitz domain-tissue factor fusion proteins

A strategy to design potent antagonists of human coagulation factor VIIa (FVIIa) by linking two proteins that independently inhibit activity and bind at separate, nonoverlapping sites is presented. A bifunctional inhibitor (KDTF5), comprising a Kunitz-type domain engineered to inhibit the FVIIa active site and a soluble tissue factor (TF) variant that is defective as a cofactor for factor X (FX) activation, was developed from structure-based modeling of a ternary FVIIa-Kunitz domain-TF complex. KDTF5 inhibited FVIIa-dependent FX activation with a Ki* of 235 +/- 45 pM, a 193-fold and 398-fold increase in potency compared to the TF variant and Kunitz domain individually. Similarly, KDTF5 was a more potent anticoagulant in vitro compared to either inhibitory domain alone. The results demonstrate the harnessing of a macromolecular chelate effect by fusing two inhibitory ligands that bind a target at spatially distinct sites.