Role of zymogen and activated factor X as scaffolds for the inhibition of the blood coagulation factor VIIa-tissue factor complex by recombinant nematode anticoagulant protein c2

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa.TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa.TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF(S)), or reconstituted into membranes, containing phosphatidylcholine (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa.TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.fX(a) complex assembled on an appropriate phospholipid surface.     

Identification of a basic region on tissue factor that interacts with the first epidermal growth factor-like domain of factor X

Tissue factor (TF) facilitates the recognition and rapid activation of factor X (fX) by factor VIIa (fVIIa) in the extrinsic Xase pathway. TF makes extensive interactions with both light and heavy chains of fVIIa; however, with the exception of a basic recognition site for the Gla domain of fX, no other interactive site on TF for the substrate has been identified. Structural and modeling data have predicted that a basic region of TF comprised of residues Asn-199, Arg-200, and Lys-201 is located at a proper height on the membrane surface to interact with either the C-terminus of the Gla domain or the EGF-1 domain of fX. To investigate this possibility, we prepared the Ala substitution mutants of these residues and evaluated their ability to function as cofactors for fVIIa in the activation of wild-type fX and its two mutants which lack either the Gla domain (GD-fX) or both the Gla and EGF-1 domains (E2-fX). All three TF mutants exhibited normal cofactor activity in the amidolytic activity assays, but the cofactor activity of Arg-200 and Lys-201 mutants in fVIIa activation of both fX and GD-fX, but not E2-fX, was impaired approximately 3-fold. Further kinetic analysis revealed that kcat values with both TF mutants are impaired with no change in Km. These results suggest that both Arg-200 and Lys-201 of TF interact with EGF-1 of fX to facilitate the optimal docking of the substrate into the catalytic groove of the protease in the activation complex.     

Active and exo-site inhibition of human factor Xa: structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum

Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K(i)=43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 A resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 A) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex.     

Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy.

Factor VIIa (fVIIa) is composed of four discrete domains, a gamma-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca(2+)-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca(2+)-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca(2+)-induced ordering of alpha-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.     

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.     

Sequential coagulation factor VIIa domain binding to tissue factor

Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the γ-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.     

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.     

Surface-dependent coagulation enzymes. Flow kinetics of factor Xa generation on live cell membranes

The initial surface reactions of the extrinsic coagulation pathway on live cell membranes were examined under flow conditions. Generation of activated coagulation factor X (fXa) was measured on spherical monolayers of epithelial cells with a total surface area of 41-47 cm(2) expressing tissue factor (TF) at >25 fmol/cm(2). Concentrations of reactants and product were monitored as a function of time with radiolabeled proteins and a chromogenic substrate at resolutions of 2-8 s. At physiological concentrations of fVIIa and fX, the reaction rate was 3.05 +/- 0.75 fmol fXa/s/cm(2), independent of flux, and 10 times slower than that expected for collision-limited reactions. Rates were also independent of surface fVIIa concentrations within the range 0.6-25 fmol/cm(2). The transit time of fX activated on the reaction chamber was prolonged relative to transit times of nonreacting tracers or preformed fXa. Membrane reactions were modeled using a set of nonlinear kinetic equations and a lagged normal density curve to track the expected surface concentration of reactants for various hypothetical reaction mechanisms. The experimental results were theoretically predicted only when the models used a slow intermediate reaction step, consistent with surface diffusion. These results provide evidence that the transfer of substrate within the membrane is rate-limiting in the kinetic mechanisms leading to initiation of blood coagulation by the TF pathway.     

Role of basic residues of the autolysis loop in the catalytic function of factor Xa

The autolysis loop of factor Xa (fXa) has four basic residues (Arg(143), Lys(147), Arg(150), and Arg(154)) whose contribution to protease specificity of fXa has not been examined. Here, we substituted these basic residues individually with Ala in the fX cDNA and expressed them in mammalian cells using a novel expression/purification vector system. Following purification to homogeneity and activation by the factor X activator from Russell viper venom, the mutants were characterized with respect to their ability to assemble into the prothrombinase complex to activate prothrombin and interact with target plasma fXa inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin. We show that all mutants interacted with factor Va with normal affinities and exhibited wild-type-like prothrombinase activities toward prothrombin. Lys(147) and Arg(154) mutants were inhibited by TFPI approximately 2-fold slower than wild type; however, both Arg(143) and Arg(150) mutants were inhibited normally by the inhibitor. The reactivities of Arg(143) and Lys(147) mutants were improved approximately 2-fold with antithrombin in the absence but not in the presence of heparin cofactors. On the other hand, the pentasaccharide-catalyzed reactivity of antithrombin with the Arg(150) mutant was impaired by an order of magnitude. These results suggest that Arg(150) of the autolysis loop may specifically interact with the activated conformation of antithrombin.     

The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.     

The tissue factor region that interacts with substrates factor IX and Factor X

The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis.     

The length of the linker between the epidermal growth factor‐like domains in factor VIIa is critical for a productive interaction with tissue factor

Formation of the factor VIIa (FVIIa)‐tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure‐based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)‐like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ‐carboxyglutamic acid (Gla)‐containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly‐Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF‐mediated stabilization of the active conformation of FVIIa and abrogated TF‐induced activity enhancement. In addition, FVIIa variants with short linkers associated 80‐fold slower with soluble TF (sTF) as compared with wild‐type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca²⁺‐free FVIIa; stepwise truncation resulting in gradually higher activity with des(83–86)‐FVIIa reaching the level of Gla‐domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo‐FVIIa activity.     

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.     

Role of the Gla and first epidermal growth factor-like domains of factor X in the prothrombinase and tissue factor-factor VIIa complexes

Factor X (FX) has high structure homology with other proteins of blood coagulation such as factor IX (FIX) and factor VII (FVII). These proteins present at their amino-terminal extremity a gamma-carboxyglutamic acid containing domain (Gla domain), followed by two epidermal growth factor-like (EGF1 and EGF2) domains, an activation peptide, and a serine protease domain. After vascular damage, the tissue factor-FVIIa (TF-FVIIa) complex activates both FX and FIX. FXa interacts stoichiometrically with tissue pathway inhibitor (TFPI), regulating TF-FVIIa activity by forming the TF-FVIIa-TFPI-FXa quaternary complex. Conversely, FXa boosts coagulation by its association with its cofactor, factor Va (FVa). To investigate the contribution of the Gla and EGF1 domains of FX in these complexes, FX chimeras were produced in which FIX Gla and EGF1 domains substituted the corresponding domains of FX. The affinity of the two chimeras, FX/FIX(Gla) and FX/FIX(EGF1), for the TF-FVIIa complex was markedly reduced compared with that of wild-type-FX (wt-FX) independently of the presence of phospholipids. Furthermore, the association rate constants of preformed FX/FIX(Gla)-TFPI and FX/FIX(EGF1)-TFPI complexes with TF-FVIIa were, respectively, 10- and 5-fold slower than that of wt-FXa-TFPI complex. Finally, the apparent affinity of FVa was 2-fold higher for the chimeras than for wt-FX in the presence of phospholipids and equal in their absence. These data demonstrate that FX Gla and EGF1 domains contain residues, which interact with TF-FVIIa exosites contributing to the formation of the TF-FVIIa-FX and TF-FVIIa-TFPI-FXa complexes. On the opposite, FXa Gla and EGF1 domains are not directly involved in FVa binding.     

Substitution of the Gla domain in factor X with that of protein C impairs its interaction with factor VIIa/tissue factor: lack of comparable effect by similar substitution in factor IX

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.     

Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor.

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.     

Identification of factor Xa residues critical for interaction with protein Z-dependent protease inhibitor: both active site and exosite interactions are required for inhibition

Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.     

Lipid-bound factor Xa regulates tissue factor activity

The activation of coagulation factor X by tissue factor (TF) and coagulation factor VIIa (VIIa) on a phospholipid surface is thought to be the key step in the initiation of blood coagulation. In this reaction, the product, fXa, is transiently and reversibly bound to the TF-VIIa enzyme complex. This in effect leads to a probabilistic inhibition of subsequent fX activations; a new fX substrate molecule cannot be activated until the old fXa molecule leaves. In this study, we demonstrate that benzamidine and soybean trypsin inhibitor-conjugated Sepharose beads, which bind fXa and sequester it away from the reaction, serve to enhance fX activation by the TF-VIIa complex. Thus, removal of fXa from the reactive zone, by either flow, fXa sequestration, or binding to distant lipid surfaces, can serve to enhance the levels of TF-VIIa activity. Using resonance energy transfer, we found the dissociation constants of fX and fXa for 100 nm diameter phospholipid vesicles to be on the order of 30-60 nM, consistent with previous measurements employing planar lipid surfaces. On the basis of the measurements of binding of fXa to phospholipid surfaces, we demonstrate that the rates of fX activation by the TF-VIIa complex under a variety of experimental conditions depend inversely on the amount of product (fXa) bound to the TF-phospholipid surface. These data support an inhibitory role for the reaction product, fXa, and indicate that models previously employed in understanding this initial coagulation reaction must now be re-evaluated to account for both the product occupancy of the phospholipid surface and the binding of the product to the enzyme. Moreover, the inhibitory properties of fXa can be described on the basis of the estimated surface density of fXa molecules on the TF-phospholipid surface.     

Directed glycosylation of human coagulation factor X at residue 333. Insight into factor Va-dependent prothrombin catalysis

Based on homology, amino acids 326-336 (143-154 in chymotrypsin numbering) of factor X (fX) comprise a flexible surface loop, which is susceptible to self-proteolysis and influences substrate catalysis. To investigate the role of this autolysis loop in fX function, a recombinant variant with a new site for asparagine-linked glycosylation has been produced by changing glutamine 333 to asparagine. Q333N fX is activated normally by factor VIIa and tissue factor, factors IXa and VIIIa, and Russell's viper venom. Proteolysis of the loop is prevented by the mutation. Reactivity of the free enzyme toward substrates and inhibitors is attenuated 4-20-fold; relative to wild type fXa, Spectrozyme Xa(TM) hydrolysis is 25%, inhibition by antithrombin III and the tissue factor pathway inhibitor is approximately 20%, and prothrombin activation in the absence of the cofactor Va is only 5%. Surprisingly, activities of the variant and wild type enzymes are equivalent when part of the prothrombinase complex. N-Glycanase cleaves the new oligosaccharide from Q333N fXa leaving aspartic acid. Q333D fXa is approximately 1.6-fold more reactive with Spectrozyme Xa(TM), antithrombin III and tissue factor pathway inhibitor, and prothrombin than its glycosylated counterpart, Q333N fXa, but still quite abnormal relative to wild type fXa. Like Q333N fXa, Q333D fXa is fully functional as part of the prothrombinase complex. We conclude that Gln-333 is geographically close to a site of proteolytic degradation but not to activator, cofactor, or membrane binding sites. Mutation of Gln-333 impairs catalytic function, but given normal prothrombin activation by the complexed enzyme, the importance of Gln-333 for catalysis is not manifest in the prothrombinase assembly, suggesting a conformational change in complexed fXa.     

The gamma-carboxyglutamic acid and epidermal growth factor-like domains of factor X. Effect of isolated domains on prothrombin activation and endothelial cell binding of factor X

Factor Xa is the enzymatically active constituent of the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin. We have isolated fragments, from tryptic digests of factor X, that consists of the gamma-carboxyglutamic acid (Gla) region linked to one or two epidermal growth factor (EGF)-like domains. Calcium ion binding measurements indicated that these fragments have a native conformation. The factor X-GlaEGF fragments inhibit factor Xa-induced blood clotting in a manner suggesting that they compete with factor Xa for phospholipid binding sites. The same conclusion was reached when thrombin generation was studied in a system of purified components (factor Xa, factor Va, prothrombin, phospholipid, and Ca2+). There was no evidence for a strong interaction between the EGF-like domains of factor Xa and factor Va in either system. However, experiments in the purified system without phospholipid indicated a direct, albeit weak, interaction between the Gla region of factor Xa and factor Va and between the COOH-terminal EGF-like domain of factor Xa and factor Va. Using domain-specific Fab fragments, we have confirmed that the conformation of the serine protease region alters dramatically upon activation of factor X. Furthermore, we have demonstrated that the conformation of the Gla region is affected by the activation, whereas the EGF-like domains appear to be unaltered. The association constant for factor X binding to endothelial cells was two orders of magnitude lower than that for binding of factor IX to these cells. Binding of the Gla and GlaEGF fragments suggested Gla-mediated binding to phospholipid rather than binding to a specific receptor.