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.     

Role of residue Phe225 in the cofactor-mediated, allosteric regulation of the serine protease coagulation factor VIIa

Functional regulation by cofactors is fundamentally important for the highly ordered, consecutive activation of the coagulation cascade. The initiating protease of the coagulation system, factor VIIa (VIIa), retains zymogen-like features after proteolytic cleavage of the activating Arg(15)-Ile(16) peptide bond and requires the binding of the cofactor tissue factor (TF) to stabilize the protease domain in an active enzyme conformation. Structural comparison of TF-bound and free VIIa failed to provide a conclusive mechanism for this catalytic activation. This study provides novel insight into the cofactor-dependent regulation of VIIa by demonstrating that the side chain of Phe(225), an aromatic residue that is common to allosterically regulated serine proteases, is necessary for optimal TF-mediated activation of VIIa's catalytic function. However, mutation of Phe(225) did not abolish the cofactor-induced stabilization of the Ile(16)-Asp(194) salt bridge, previously considered the primary switch mechanism for activating VIIa. Moreover, mutation of other residue side chains in the VIIa protease domain resulted in a reduced level of or no stabilization of the amino-terminal insertion site upon TF binding, with little or no effect on the TF-mediated enhancement of catalysis. This study thus establishes a crucial role for the aromatic Phe(225) residue position in the allosteric network that transmits the activating switch from the cofactor interface to the catalytic cleft, providing insight into the highly specific conformational linkages that regulate serine protease function.     

Thermal effects on an enzymatically latent conformation of coagulation factor VIIa.

Activation of the zymogen factor VII yields an enzyme form, factor VIIa, with only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Factor VIIa activity measured with a chromogenic peptide substrate is characterized by an unusual temperature dependency which indicates that the activated protease exists in an equilibrium between a latent (enzymatically inactive) and an active conformation. As shown by calorimetry and activity measurements the thermal effects on factor VIIa are fully reversible below the denaturation temperature of 58.1 degrees C. A model for factor VIIa has been proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Biol. Chem. 269, 18891-18898] in which the protease is supposed to exist primarily as a latent enzyme form because of the poor incorporation into the protease structure of the N-terminal Ile153 released by proteolytic cleavage during activation of factor VII. Binding of tissue factor to factor VIIa is assumed to shift the equilibrium towards an active conformation in which the N-terminal Ile153 forms a salt bridge with Asp343. We corroborate the validity of this model by: (a) chemical modification of factor VIIa; this suggests that the thermal effect on the equilibrium between the active and inactive conformation is reflected in the relative accessibility of the active site and the N-terminal Ile153; (b) measurements of factor VIIa binding to tissue factor indicating that complex formation is favoured by stabilization of the active conformation; and (c) activity measurements of a cross-linked factor VIIa-tissue factor complex; this showed that cross-linking stabilized the active conformation of factor VIIa and essentially prevented its thermally-induced transformation into the inactive state.     

The factor VII zymogen structure reveals reregistration of beta strands during activation

BACKGROUND: Coagulation factor VIIa (FVIIa) contains a Trypsin-like serine protease domain and initiates the cascade of proteolytic events leading to Thrombin activation and blood clot formation. Vascular injury allows formation of the complex between circulating FVIIa and its cell surface bound obligate cofactor, Tissue Factor (TF). Circulating FVIIa is nominally activated but retains zymogen-like character and requires TF in order to complete the zymogen-to-enzyme transition. The manner in which TF exerts this effect is unclear. The structure of TF/FVIIa is known. Knowledge of the zymogen structure is helpful for understanding the activation transition in this system. RESULTS: The 2 A resolution crystal structure of a zymogen form of FVII comprising the EGF2 and protease domains is revealed in a complex with the exosite binding inhibitory peptide A-183 and a vacant active site. The activation domain, which includes the N terminus, differs in ways beyond those that are expected for zymogens in the Trypsin family. There are large differences in the TF binding region. An unprecedented 3 residue shift in registration between beta strands B2 and A2 in the C-terminal beta barrel and hydrogen bonds involving Glu154 provide new insight into conformational changes accompanying zymogen activation, TF binding, and enzymatic competence. CONCLUSIONS: TF-mediated allosteric control of the activity of FVIIa can be rationalized. The reregistering beta strand connects the TF binding region and the N-terminal region. The zymogen registration allows H bonds that prevent the N terminus from attaining a key salt bridge with the active site. TF binding may influence an equilibrium by selecting the enzymatically competent registration.     

The cofactor function of the N-terminal domain of tissue factor

Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury. TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa. In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex. Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface. It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates. To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF(2-219) in a novel expression/purification vector system in the periplasmic space of bacteria. Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays. The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N- and C-terminal domains of TF are required for high affinity interaction with fVIIa. On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X. However, it was discovered the Arg(74) mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease.     

Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes.

The most active factor VIIa (FVIIa) variants identified to date carry concurrent substitutions at positions 158, 296 and 298 with the intention of generating a thrombin-mimicking motif, optionally combined with additional replacements within the protease domain [Persson, E., Kjalke, M. & Olsen, O. H. (2001) Proc. Natl Acad. Sci. USA98, 13583-13588]. Here we have characterized variants of FVIIa mutated at one or two of these positions to assess the relative importance of the individual replacements. The E296V and M298Q mutations gave an increased intrinsic amidolytic activity (about two- and 3.5-fold, respectively) compared with wild-type FVIIa. An additive effect was observed upon their combination, resulting in the amidolytic activity of E296V/M298Q-FVIIa being close to that of the triple mutant. The level of amidolytic activity of a variant was correlated with the rate of inhibition by antithrombin (AT). Compared with wild-type FVIIa, the Ca2+ dependence of the intrinsic amidolytic activity was significantly attenuated upon introduction of the E296V mutation, but the effect was most pronounced in the triple mutant. Enhancement of the proteolytic activity requires substitution of Gln for Met298. The simultaneous presence of the V158D, E296V and M298Q mutations gives the highest intrinsic activity and is essential to achieve a dramatically higher relative increase in the proteolytic activity than that in the amidolytic activity. The N-terminal Ile153 is most efficiently buried in V158D/E296V/M298Q-FVIIa, but is less available for chemical modification also in the presence of the E296V or M298Q mutation alone. In summary, E296V and M298Q enhance the amidolytic activity and facilitate salt bridge formation between the N-terminus and Asp343, E296V reduces the Ca2+ dependence, M298Q is required for increased factor X (FX) activation, and the simultaneous presence of the V158D, E296V and M298Q mutations gives the most profound effect on all these parameters.     

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Prothrombinase assembly and S1 site occupation restore the catalytic activity of FXa impaired by mutation at the sodium-binding site

Two loop segments (183-189 and 221-225) in the protease domain of factor Xa contribute to the formation of a Na(+)-binding site. Studies with factor Xa indicate that binding of a single Na(+) ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr(225) with Pro diminishes sensitivity to Na(+). Using full-length factor Xa(Y225P), the allosteric relationship between the Na(+) site and other structural determinants in factor Xa and prothrombinase was investigated. Direct binding and kinetic measurements with probes that target the S1 specificity pocket indicate that assembly of the mutant in prothrombinase corrected the impaired binding of these probes observed with free factor Xa(Y225P). This appears to result from the apparent allosteric linkage between the factor Va, S1, and Na(+)-binding sites, since binding of the cofactor to membrane-bound factor Xa(Y225P) enhances binding at the S1 site and vice versa. Additional studies revealed that the internal salt bridge (Ile(16)-Asp(194)) of factor Xa(Y225P) is partially destabilized, a process that is reversible upon occupation of the S1 site. The data establish that alterations at the factor Xa Na(+)-binding site shift the zymogen-protease equilibrium to a more zymogen-like state, and as a consequence binding of S1-directed probes and factor Va are adversely affected. Therefore, the zymogen-like characteristics of factor Xa(Y225P) have allowed for the apparent allosteric linkage between the S1, factor Va, and Na(+) sites to become evident and has provided insight into the structural transitions which accompany the conversion of factor X to factor Xa.     

Disulfide locked variants of factor VIIa with a restricted beta-strand conformation have enhanced enzymatic activity

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of beta-strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF*FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V(max)/K(m) values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.     

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.     

Similar molecular interactions of factor VII and factor VIIa with the tissue factor region that allosterically regulates enzyme activity

Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factor VII with high affinity. The structure determined for the sTF-VIIa complex [Banner, D. W., et al. (1996) Nature 380, 41-46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contact with TF. Although a structure is not available for the TF-VII complex, the structure determined for free VII [Eigenbrot, C., et al. (2001) Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K(D)) measured for binding to wild-type sTF are 7.5 +/- 2.4 nM for VII and 5.1 +/- 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.     

Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor

The enzyme factor VIIa (FVIIa) triggers the blood coagulation cascade upon association with tissue factor (TF). The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex, and Met-306 in the serine protease domain of FVIIa participates in this event. We have characterized FVIIa variants mutated in position 306 with respect to their ability to be stimulated by TF. The amidolytic activity of FVIIa mutants with Ser, Thr, and Asn in position 306 was stimulated 9-, 12-, and 7-fold, respectively, by soluble TF as compared to 22-fold for wild-type FVIIa. In contrast, the activity of Met306Asp-FVIIa only increased about 2-fold and that of Met306Asp/Asp309Ser-FVIIa increased about 1.5-fold. Modeling suggests that Asp in position 306 prevents the TF-induced stimulation of FVIIa by disrupting essential intermolecular hydrogen bonds. The ability of the FVIIa variants to catalyze factor X activation and the amidolytic activity were enhanced to a similar extent by soluble TF. This indicates that factor X does not promote its own activation through interactions with exosites on FVIIa made accessible upon FVIIa-TF assembly. Met306Asp-FVIIa binds soluble TF with a dissociation constant of 13 nM (about 3-fold higher than that of FVIIa), and, in sharp contrast to FVIIa, its binding kinetics are unaltered after inactivation with D-Phe-Phe-Arg chloromethyl ketone. We conclude that a single specific amino acid replacement, substitution of Asp for Met-306, virtually prevents the TF-induced allosteric changes which normally result in dramatically increased FVIIa activity and eliminates the effect of the active site inhibitor on TF affinity.     

Dynamic character of the complex of human blood coagulation factor VIIa with the extracellular domain of human tissue factor: a normal mode analysis

As an attempt to investigate the dynamic interactions between plasma serine protease, coagulation factor VIIa (VIIa) and its cofactor, tissue factor (TF), we performed normal mode analysis (NMA) of the complex of VIIa with soluble TF (the extracellular part of TF; sTF). We compared fluctuations of Calpha atoms of VIIa or sTF derived from NMA in the VIIa-sTF complex with those of VIIa or sTF in an uncomplexed condition. The atomic fluctuations of the Calpha atoms of sTF complexed with VIIa did not significantly differ from those of sTF without VIIa. In contrast, the atomic fluctuations of VIIa complexed with sTF were much smaller than those of VIIa without sTF. These results suggest that domain motions of VIIa molecule alone are markedly dampened in the VIIa-sTF complex and that the sTF molecule is relatively more rigid than the VIIa molecule. This may indicate functions of TF as a cofactor.     

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.     

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.     

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.     

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.     

Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity

Factor VII requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active factor VIIa (FVIIa) conformation. The former event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. We have designed a number of FVIIa mutants with the aim of mimicking the effect of TF, that is, creating molecules with increased intrinsic (TF-independent) enzymatic activity. Based on a possible structural difference between free and TF-bound FVIIa (Pike, A. C. W., Brzozowski, A. M., Roberts, S. M., Olsen, O. H., and Persson, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8925--8930), we focused on the helical region encompassing residues 307-312 and residues in its spatial vicinity. For instance, FVIIa contains Phe-374 and Leu-305, whereas a Phe/Tyr residue in the position corresponding to 374 in homologous coagulation serine proteases is accompanied by Val in the position corresponding to 305. This conceivably results in a unique orientation of this helix in FVIIa. Substitution of Val for Leu-305 in FVIIa resulted in a 3--4-fold increase in the intrinsic amidolytic and proteolytic activity as compared with wild-type FVIIa, whereas the activity in complex with soluble TF remained the same. In accordance with this, L305V-FVIIa exhibited an increased rate of inhibition as compared with wild-type FVIIa, both by d-Phe-Phe-Arg-chloromethyl ketone and antithrombin III in the presence of heparin. The increased FVIIa activity upon replacement of Leu-305 by Val may be mediated by a movement of the 307--312 helix into an orientation resembling that found in factors IXa and Xa and thrombin. The corresponding shortening of the side chain of residue 374 (Phe --> Pro) had a smaller effect (about 1.5-fold increase) on the intrinsic activity of FVIIa. Attempts to increase FVIIa activity by introducing single or multiple mutations at positions 306, 309, and 312 to stabilize the 307-312 helix failed.     

Selective attenuation of the extrinsic limb of the tissue factor-driven coagulation protease cascade by occupancy of a novel peptidyl docking site on tissue factor

Tissue factor (TF), the receptor and cofactor for factor VIIa (VIIa) for cellular initiation of the coagulation protease cascade, drives thrombogenesis, inflammation, tumor cell metastasis, and the lethality of severe sepsis. To identify TF surface loci that can selectively inhibit substrate zymogen association and activation, TF(1-218), the extracellular domain, was used as the target for the phage display search. This resulted in selection of 59 clones from a phage gpVIII surface protein-expressed library of constrained combinatorial peptides. Of these, one encoding the peptide Glu-Cys-Leu-Arg-Ser-Val-Val-Thr-Cys on gpVIII most avidly bound TF(1-218), as did the synthetic peptide. Inhibition of binding was selective with an IC(50) of 30 nM for proteolytic activation of factor X by the TF(1-218)-VIIa complex. In contrast, there was no inhibition of factor IX activation. The selective inhibition of only factor X association with TF(1-218) will spare the intrinsic hemostatic pathway while attenuating the extrinsic thrombogenic pathway. This and related peptidyl structures provide the potential for the more precise identification of TF surface loci that mediate selective functional properties of the protein as well as a structural basis for the design of novel molecules for selectively attenuating initiation of the extrinsic limb of the coagulation protease cascade and other functions of TF.