Correlation of factor IXa subsite modulations with effects on substrate discrimination.

BACKGROUND: A key feature of factor IXa is its allosteric transformation from an enzymatically latent form into a potent procoagulant. Although several small molecules have been found to be capable of partially affecting FIXa function (i.e. ethylene glycol, Ca(2+), and low molecular weight heparin [LMWH]), the resulting modest changes in peptidolytic activity have made the study of their mechanisms of action challenging. As these effects provide hints about potential regulatory forces that may be operational in the full expression of FIXa coagulant activity, their description remains of great interest. Studies of crystal structures have yielded insights into the structural changes induced by these effectors, but there remains a paucity of information to correlate any given structural change with specific consequences for FIXa function. OBJECTIVES: To correlate structural changes induced by these modulators with defined consequences for FIXa substrate discrimination and function. METHODS: A peptidomics-based mass spectrometry (MS) approach was used to examine the patterns of hydrolysis of four combinatorial chemistry-derived pentapeptide libraries by FIXa under various conditions in a soluble, active enzyme system. RESULTS: Ethylene glycol specifically altered the S3 subsite of FIXa to render it more tolerant to side chains at the P3 substrate position, whereas Ca(2+) enhanced tolerance at the S2 subsite. In contrast, LMWH altered both the S2 and S1' subsites. CONCLUSIONS: These results demonstrate the role of plasticity in regulating FIXa function with respect to discrimination of extended substrate sequences, as well as providing crucial insights into active site modulations that may be capitalized on by various physiologic cofactors of FIXa and in future drug design.

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function.

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

Proteolysis of blood coagulation factor VIII by the factor VIIa-tissue factor complex: generation of an inactive factor VIII cofactor.

Activation of factor VIII by thrombin occurs via limited proteolysis at R372, R740, and R1689. The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R336 and R562 by activated protein C subsequently inactivates this cofactor. We now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the trigger of blood coagulation with restricted substrate specificity, can also catalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognized by an anti-factor VIII A2 domain antibody. Time courses indicated the slow conversion of the large fragment to 45 kDa, followed by further degradation into at least two smaller fragments. N-Terminal sequencing along with time courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII first at R740, followed by concomitant cleavage at R336 and R372. Although cleavage of the light chain at R1689 was observed, the majority remained uncleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage of factor VIII by VIIa-TF/PL produces an inactive factor VIII cofactor no longer capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor VIII. We hypothesize that these proteolyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences recognized by this enzyme-cofactor complex.

Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain. A fluorescence resonance energy transfer study.

Coagulation factor VIIa (fVIIa), a soluble serine protease, exhibits full proteolytic activity only when bound to its cofactor, tissue factor (TF). Both proteins interact with membranes; TF is an integral membrane protein, while fVIIa binds reversibly to phospholipid surfaces via its Gla domain. In this study, we examine the extent to which the location of the fVIIa active site in the fVIIa.TF complex is determined by the fVIIa Gla domain. A fluorescein dye was covalently attached to the active site of fVIIa lacking the Gla domain (Gla domainless fVIIa, GD-fVIIa) via a tripeptide tether to yield fluorescein-D-Phe-Pro-Arg-GD-fVIIa (Fl-FPR-GD-fVIIa). The location of the active site of GD-fVIIa relative to the membrane surface was determined using fluorescence resonance energy transfer between the fluorescein dye in the active site of GD-fVIIa and octadecylrhodamine (OR) at the surface of phospholipid vesicles. As expected, no energy transfer was observed between Fl-FPR-GD-fVIIa and OR in vesicles composed of phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) because the Gla domain is required for the binding of fVIIa to phospholipid. However, when Fl-FPR-GD-fVIIa was titrated with PC or PC/PS vesicles into which purified TF had been reconstituted, energy transfer was observed. Based on the dependence of fluorescence resonance energy transfer on OR density, the average distance of closest approach between fluorescein in the active site of Fl-FPR-GD-fVIIa.TF and OR at the vesicle surface was determined to be 78 A (kappa2 = (2)/(3)). Since this value is nearly the same as that obtained with intact Fl-FPR-fVIIa bound to TF, the presence or absence of the fVIIa Gla domain has only a small effect on the location of the active site in the fVIIa.TF complex. The extracellular domain of tissue factor therefore must be fairly rigid and fixed relative to the surface to position and maintain the fVIIa active site far above the membrane even in the absence of the fVIIa Gla domain.

Factor VIIa-tissue factor: functional importance of protein-membrane interactions.

The first enzyme in the blood clotting cascade consists of two distinct protein subunits: a catalytic subunit (factor VIIa; FVIIa) and an essential regulatory subunit (tissue factor; TF). FVIIa is a soluble plasma protease, while TF is a cell-surface, integral-membrane protein. The recently reported X-ray crystal structure of the complex of FVIIa and the isolated extracellular domain of TF has provided important insights into the protein-protein interactions that bind these two subunits together (1). Equally important in the functioning of the TF-FVIIa complex, but much less well understood, are a series of protein-phospholipid interactions involving TF, FVIIa, and the natural substrates of this enzyme, as well as protein-protein interactions important in substrate recognition by TF-FVIIa. Here we review recent studies on the membrane organization and role of protein-phospholipid interactions in the function of TF-FVIIa, the enzyme that triggers blood clotting in hemostasis and thrombosis.

The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor. A fluorescence energy transfer study.

The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of fVIIa via a tripeptide tether to yield fluorescein-D-phenylalanyl-L-prolyl-L-arginyl-fVIIa (Fl-FPR-fVIIa). The location of the active-site probe relative to the membrane surface was determined, both in the presence and absence of tissue factor (TF), using fluorescence energy transfer between the fluorescein dye and octadecylrhodamine (OR) at the phospholipid vesicle surface. When Fl-FPR-fVIIa was titrated with phospholipid vesicles containing OR, the magnitude of OR-, calcium ion-, and phosphatidylserine-dependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the active site of fVIIa and OR at the vesicle surface is 82 A assuming a random orientation of donor and acceptor dyes (kappa2 = 2/3; the orientational uncertainty totals approximately 10%). The active site of fVIIa is therefore located far above the membrane surface, and the elongated fVIIa molecule must bind at one end to the membrane and project approximately perpendicularly out of the membrane. When Fl-FPR-fVIIa was titrated with vesicles that contained TF, the efficiency of energy transfer was increased by a TF-dependent translational and/or rotational movement of the fVIIa protease domain relative to the membrane surface. If this movement was solely translational, the height of the active site of fVIIa was lowered by an average of 6 A after binding to TF. The association of fVIIa with TF on the membrane surface therefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the active-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.

Substrate recognition by tissue factor-factor VIIa. Evidence for interaction of residues Lys165 and Lys166 of tissue factor with the 4-carboxyglutamate-rich domain of factor X.

Tissue factor (TF) is the protein cofactor for factor VIIa (FVIIa), the first serine protease of the clotting cascade. Previous studies using alanine mutagenesis have identified TF residues Lys165 and Lys166 as important for factor X (FX) activation, hypothesizing either that these residues interact with phospholipid head groups or that they directly or indirectly promote macromolecular substrate binding. In the recently reported x-ray crystal structure of the isolated extracellular domain of TF, both Lys165 and Lys166 are solvent-exposed and predicted to be near the phospholipid surface in intact TF. We hypothesized that these residues may in fact be ideally positioned to interact with the 4-carboxyglutamate-rich domain (Gla domain) of FX. We therefore predicted that mutations at Lys165 and Lys166 should have no effect on the activation of Gla domainless FX. To test this hypothesis, we mutated both residues Lys165 and Lys166 of TF to Ala, Glu, or Gln and examined the ability of these double mutants to support FVIIa-mediated activation of FX, Gla domainless FX, and factor IX (FIX). Each TF mutant was equivalent to wild-type TF in both FVIIa binding and promotion of FVIIa amidolytic activity. However, all three mutants were markedly deficient in supporting FIX and FX activation, with FX activation rates decreased more than FIX activation rates. In both reactions, the TF mutants exhibited different extents of activity: Gln165-Gln166 > Ala165-Ala166 > Glu165-Glu166. In sharp contrast, all three TF mutants were equivalent to wild-type TF in supporting activation of Gla domainless FX by FVIIa. Interestingly, the deficiency of the mutants in FX activation was less pronounced when Gla domainless FVIIa was used in place of native FVIIa. Together, these findings suggest that TF residues Lys165 and Lys166 contribute to a binding site for the Gla domain of FX (and perhaps other substrates) and that this interaction may be facilitated by the presence of the Gla domain of FVIIa.

Effect of soluble tissue factor on the kinetic mechanism of factor VIIa: enhancement of p-guanidinobenzoate substrate hydrolysis.

The mechanism by which the protein cofactor, tissue factor, enhances the activity of its cognate serine protease, coagulation factor VIIa (FVIIa), has been studied using the fluorogenic ester substrate 4-methylumbelliferyl p'-guanidinobenzoate (MUGB). Kinetic data were collected at pH 8.4 and pH 7.6 in the presence and absence of soluble tissue factor (sTF; recombinant human tissue factor containing only the extracellular domain). Pre-steady-state techniques allowed the determination of the individual rate constants for acylation (k2) and deacylation (k3) of the sTF.FVIIa complex as well as the dissociation constant for the noncovalent Michaelis complex with MUGB. Alternative methods were required for determination of these parameters for free FVIIa due to extremely slow hydrolysis of MUGB in the absence of sTF. Under all experimental conditions, deacylation was found to be rate-limiting. The major effect of sTF was to raise the affinity of FVIIa for MUGB (31-fold at pH 8.4 and 36-fold at pH 7.6); only minor changes in k2 and k3 were observed. Thus, we conclude that for the ester substrate MUGB, sTF exerts greater allosteric effects on substrate binding than on the later steps involved in the catalytic pathway.

Phosphatidylethanolamine augments factor VIIa-tissue factor activity: enhancement of sensitivity to phosphatidylserine.

The effect of phosphatidylethanolamine (PE) on the activity of the factor VIIa-tissue factor complex (fVIIa-TF) has been examined with respect to plasma clotting activity and activation of factor X (fX) in a purified system. Vesicles prepared by relipidating membrane-anchored TF (dcTF; TF1-244, lacking the C-terminal cytoplasmic tail) into phospholipid vesicles containing 6 mol % phosphatidylserine (PS) and increasing levels of PE up to 40 mol % (the balance consisting of phosphatidylcholine) were found to progressively shorten TF-initiated clotting in normal human plasma to levels comparable to those observed using dcTF relipidated with cephalin. The shortened clotting times were at least in part due to the ability of PE-containing membranes to better support the activation of fX by the fVIIa.TF complex, as vesicles with increased PE content yielded progressively higher initial rates of fX activation. Surprisingly, PE substantially altered the sensitivity of fX activation to low levels of PS, yielding near-maximal rates of activation at only 3 mol % PS compared to 15-20 mol % PS required in the absence of PE. The effect of PE was not synergistic with that of PS since PE did not increase fX activation rates at high levels of PS (20 mol %). Examination of the kinetic parameters for fX activation revealed that the majority of the effect of PE was in decreasing the apparent Km for fX.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of the substrate and inhibitor specificities of blood coagulation factor VIIa: importance of amino acid residue K192.

Initiation of blood coagulation occurs when the plasma serine protease factor VIIa (fVIIa) binds to its cell-surface receptor/cofactor, tissue factor (TF). This binding interaction mediates a large enhancement in both the proteolytic activity and the amidolytic activity (hydrolysis of small peptidylamide substrates) of fVIIa. This necessitates local changes in the catalytic center of fVIIa of which little is understood. Studies with thrombin and activated protein C have demonstrated that residue E192 (chymotrypsinogen numbering system) near the active site of these proteases is an important determinant for substrate and inhibitor specificity. By homology, residue 192 in fVIIa is K, bringing into question the potential role of this residue in fVIIa. We have prepared two mutants of fVIIa in which K192 has been replaced by either Q (as in factors IX and X) or E (as in thrombin). Both mutants were found to be defective in clotting: fVIIK 192Q was 44% active, while fVIIK192E was completely ineffective. This defect was attributable to proportional decreases in specificity constants for activation of factor X. Although both mutant enzymes were catalytically competent with respect to amidolytic activity, the selectivity of fVIIaK192E was greatly altered. Inhibition of both mutants by the TF pathway inhibitor (TFPI) and bovine pancreatic trypsin inhibitor (BPTI) was also drastically altered. Neither mutant was inhibited by TFPI, while fVIIaK192Q was inhibited by BPTI better than wild-type fVIIa. In contrast, fVIIaK192E was poorly inhibited by BPTI and made more refractory to inhibition when bound to TF. These results suggest a potential role for K192 in governing the substrate and inhibitor specificities of fVIIa.

Roles of the membrane-interactive regions of factor VIIa and tissue factor. The factor VIIa Gla domain is dispensable for binding to tissue factor but important for activation of factor X.

The roles of the putative membrane-interactive regions of factor VIIa (fVIIa) and tissue factor (TF) have been examined. Enzymatic removal of the 4-carboxyglutamic acid (Gla) domain of fVIIa had no effect on hydrolysis of a tripeptidyl chromogenic substrate in the absence or presence of TF. Additionally, Gla-domainless fVIIa (GdVIIa) was similar to native fVIIa in activating factor X in the absence of TF and phospholipid. However, GdVIIa in complex with recombinant soluble TF (sTF) was 76-fold less efficient in factor X activation than was fVIIa.sTF. The difference increased to 740-fold using TF relipidated in vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine (TF/PCPS). While Gla domain deletion produced a 10(3)-fold increase in the Kd for binding to TF/PCPS, the Kd for binding to TF/PC increased only 20-fold, and that for sTF in the absence of phospholipid increased 10-fold. Kd values for GdVIIa binding to TF/PCPS, TF/PC, or sTF were nearly identical. Thus, most of the binding energy required for formation of the fVIIa.TF complex was present even after Gla domain deletion. Both fVIIa and GdVIIa were capable of binding sTF in the presence of excess divalent metal-ion chelator, suggesting Ca(2+)-independent binding or the presence of a novel very high affinity Ca2+ binding site in fVIIa. The results demonstrate that the effect of the Gla domain on the Kd is apparent only in the presence of PS, and that interactions involving the fVIIa Gla domain and phospholipid are critical for efficient proteolysis of factor X on a membrane surface.

The biochemical basis for the apparent defect of soluble mutant tissue factor in enhancing the proteolytic activities of factor VIIa.

Tissue factor (TF), an integral membrane protein, is the cofactor for the serine protease, coagulation factor VIIa (FVIIa). Previous studies of the isolated extracellular domain of TF (sTF) reported a kcat for factor X (FX) activation by the sTF.VIIa complex only 4% that of wild-type TF.VIIa and furthermore, a complete inability of sTF to support FVII autoactivation. We now report that in the presence of poly(L-lysine), sTF promoted both FX activation and FVII autoactivation, the latter with an apparent second-order rate constant higher than reported previously for wild-type TF in phospholipid vesicles. This led us to reexamine the cofactor ability of sTF, using high concentrations of phospholipid to promote nearly quantitative binding of sTF.VII(a) complexes to the phospholipid surface. Rate constants for the activation of FX or FVII by sTF.VIIa were similar to those of wild-type TF.VIIa, indicating that the apparent deficiency of sTF is largely attributable to kinetic consequences of relatively weak affinity of FVIIa (and sTF.VIIa) for phospholipid surfaces in these surface-dependent reactions, compared with TF being embedded in the membrane. This supports the notion that sTF recapitulates the protein-protein interactions of wild-type TF with high, and possibly full, catalytic activity. It also provides a biochemical explanation for the specificity of our recently described, sTF-based clotting assay for plasma FVIIa.

Importance of substrate composition, pH and other variables on tissue factor enhancement of factor VIIa activity.

Tissue factor (TF) markedly enhances the ability of factor VIIa (FVIIa) to cleave both macromolecular and small peptidyl substrates. Using soluble mutant TF (sTF) to investigate TF-enhanced FVIIa amidolytic activity in solution, we screened thirty-four commercially available peptidyl-p-nitroanilide substrates and found that substrate hydrolysis rates were influenced by both the peptide sequence and the N-terminal blocking group (MeSO2 > MeO-CO or free N-terminus >> benzoyl). Two substrates (Chromozym t-PA: MeSO2-D-Phe-Gly-Arg-pNA; and CBS 34.47: H-D-cyclohexylglycyl-alpha-aminobutyryl-Arg-pNA) were cleaved at rates higher than those of previously reported chromogenic substrates for FVIIa. The pH range of FVIIa amidolytic activity toward Chromozym t-PA was 6.5 to 10 with an optimum at pH 7.8, while sTF.VIIa had a higher pH optimum (pH 8.4 to 8.5). The degree of enhancement of FVIIa activity by sTF varied from 12-fold at pH 7.5 to 73-fold at pH 9.9. The effect of a variety of agents on FVIIa amidolytic activity was surveyed: most decreased activity, while glycerol and ethylene glycol enhanced the activity of FVIIa but not sTF.VIIa. These results indicate that the effect of sTF on the catalytic center of FVIIa is pH-dependent, and that certain polyalcohols can partially substitute for TF.

Factor VII autoactivation proceeds via interaction of distinct protease-cofactor and zymogen-cofactor complexes. Implications of a two-dimensional enzyme kinetic mechanism.

Tissue factor (TF), an integral membrane protein, enhances the feedback activation of factor VII by factor VIIa (factor VII autoactivation). We found that, in contrast to the other known membrane-dependent coagulation activation reactions, TF-dependent factor VII autoactivation occurred preferentially on neutral phospholipid vesicles relative to negatively charged vesicles containing phosphatidylserine. This reaction was best described by a novel mechanism in which the enzyme and substrate are each bound to separate cofactor (TF) molecules. This unusual mechanism of substrate presentation to a membrane-bound protease predicts that the reaction rate will be directly dependent on the surface density, and hence lateral diffusion, of factor VII.TF and factor VIIa.TF complexes, obeying obligatorily two-dimensional enzyme kinetics. This prediction was confirmed, yielding a two-dimensional second-order rate constant (k2D) of 4.9 (+/- 0.8) x 10(6) m2 mol-1 s-1. Since intact cells normally sequester acidic phospholipids away from the outer leaflet of the plasma membrane, this reaction mechanism should permit factor VII autoactivation to predominate on unactivated/undamaged cell surfaces when other clotting reactions are dormant.

Analysis of the functions of the first epidermal growth factor-like domain of factor X.

Upon activation, factor X participates in the prothrombin activation complex. Similar to 4-carboxyglutamic acid (Gla)-domainless protein C, the Gla-domainless factor X (GDFX) contains a high affinity Ca(2+)-binding site critical for the function of these molecules. In the case of protein C, we recently demonstrated that the high affinity Ca(2+)-binding site critical for activation is outside the first epidermal growth factor (EGF) homology domain. To examine if this is also true for factor X, we have expressed in human 293 cells a deletion mutant of factor X (E2FX) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of factor X. Direct binding studies by equilibrium dialysis indicate that E2FX contains a single Ca(2+)-binding site with a dissociation constant (Kd) of 154 +/- 48 microM. The functional properties of E2FX are equivalent or improved over those of GDFX. For instance, the factor X coagulant protein of Russell's viper venom activates E2FX three times faster than recombinant GDFX. Kinetic analysis of prothrombin activation in the absence of membranes indicates that activated GDFX and E2FX bind to factor Va with equal affinity (Kd = 4.1 microM). The Ca2+ concentration required for half-maximal prothrombin activation rates in the above activation system shifted from 721 +/- 113 microM for activated GDFX to 193 +/- 64 microM for activated E2FX. GDFX and E2FX activation rates with the soluble tissue factor-factor VIIa complex were identical as was the Ca2+ dependence of the reaction. We conclude that E2FX retains a high affinity Ca(2+)-binding site and that the first EGF homology domain does not appear to have a positive functional role in the GDFX molecule. However, Ca2+ occupancy of the Ca(2+)-binding site in the first EGF domain of intact factor X may be essential for optimal prothrombin activation.

Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation.

Although the majority of factor VII (FVII) circulates in the zymogen form, low levels of activated factor VII (FVIIa) have been postulated to exist in plasma and to serve a priming function for triggering of the clotting cascade. However, direct measurement of plasma FVIIa has not previously been possible. We have quantified plasma FVIIa levels using a novel, highly sensitive assay that is free from interference by FVII. Specificity of this clot-based assay results from the use of a mutant tissue factor that is selectively deficient in promoting FVII activation, but retains FVIIa cofactor function. In normal adults, FVIIa was found to be present in plasma (mean: 3.6 ng/mL) with considerable variation between individuals (range: 0.5 to 8.4 ng/mL). FVIIa levels were only loosely correlated with FVII coagulant activity, but were elevated in pregnancy and reduced with oral anticoagulant therapy. Incubation of plasma on ice in glass containers (cold activation) resulted in substantial FVIIa generation. Measurement of plasma forms of factor VII is of potential clinical importance because elevated FVII coagulant activity has been implicated as a significant risk predictor for ischemic heart disease. Clinically, this new assay will now permit direct assessment of the role of plasma FVIIa in thrombotic disorders.

An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates.

Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8-11D12 and TF9-9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9-9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8-11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8-11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8-11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.

Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.

The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.

Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate.

The decay of human coagulation factor VIIIa has been studied by kinetic methods that ensure no interference through proteolytic feedback. The rate of decay of factor VIIIa activity was found to vary with the activator used to activate factor VIII. Thrombin-activated factor VIII-von Willebrand factor complex (fVIII-vWf) decayed at a rate of 0.31 min-1, whereas factor Xa-activated fVIII-vWf decayed at 0.11 min-1 under the same conditions. Factor VIII free of von Willebrand factor (factor VIII: C), although decaying at a generally slower rate after activation, still showed a dependence of decay rate on activator: thrombin-activated factor VIII:C decaying at a rate of 0.06 min-1, and factor Xa-activated factor VIII: C at 0.01 min-1. Readdition of von Willebrand factor (18 micrograms/ml) to factor VIII:C did not alter the observed activity or decay rate. The decay of the two species of factor VIIIa was studied, using the fVIIIa-vWf complex, in the presence of varying levels of factor IXa. Plots of reciprocal decay rates vs factor IXa concentration were linear, and nearly parallel for the two factor VIIIa species, with a mean slope of 0.56 min.nM-1. In addition to these studies, we have confirmed previous studies showing that the two forms of factor VIIIa differ in cofactor activity, but they do so in the same ratio as in their decay rates. We suggest that this difference and that observed in decay rate have a common cause, and incorporate this into a potential kinetic model of factor VIII activation and decay.