©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Contribution of Residue 192 in Factor Xa to Enzyme Specificity and Function (*)

Alireza R. Rezaie (1)(§), Charles T. Esmon (1) (2) (3) (4)(¶)

From the (1)Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, the Departments of (2)Pathology and (3)Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, and the (4)Howard Hughes Medical Institute, Oklahoma City, Oklahoma 73104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mutation of residue 192 (chymotrypsin numbering) from Glu to Gln in thrombin and activated protein C has been shown to dramatically alter substrate and inhibitor specificity, in large part by allowing these enzymes to accept substrates with acidic residues in the P3 and/or P3` positions. In factor Xa, residue 192 is already a Gln. We now compare the properties of a Q192E mutant of Gla-domainless factor X (GDFX). Kinetic analysis of prothrombin activation indicates similar affinity of factor Va for GDFXa and GDFXa Q192E (K = 3.6 and 3.7 µM, respectively). Prothrombin activation rates are similar for both enzymes with factor Va, but are 10-fold slower for the Q192E mutant without factor Va. This defect is in the activation of prethrombin 2 and is corrected by factor Va only in the presence of fragment 2. Without factor Va, fragment 2 has no influence on bovine prethrombin 2 activation by GDFXa, but fragment 2 enhances prethrombin 2 activation by the Q192E mutant at least 10-fold. These results indicate that the fragment 2 domain of prothrombin probably alters the conformation of the prethrombin 2 domain, selectively improving its presentation to GDFXa Q192E. With respect to inhibition, tissue factor pathway inhibitor and bovine pancreatic trypsin inhibitor are 30 times poorer inhibitors of GDFXa Q192E than of GDFXa, but these enzymes are inhibited at comparable rates by antithrombin. These results indicate that Gln-192 in factor Xa is a key determinant of substrate/inhibitor specificity.


INTRODUCTION

Factor X is a vitamin K-dependent plasma zymogen(1) . After activation to generate factor Xa, this enzyme can form a Ca-dependent complex with factor Va on membrane surfaces (prothrombinase complex) that rapidly activates prothrombin to thrombin(1, 2) . In comparison to factor Xa alone, formation of the complete complex enhances the rate of prothrombin activation 10-fold(2) . The dramatic increase in catalytic efficiency of prothrombinase is thought to arise from a 100-fold decrease in apparent K and a 3000-fold increase in the k for the reaction(2) . The decrease in K is believed to be the result of negatively charged phospholipid-protein interaction, which raises the local concentration of prothrombin near the enzyme complex, and the increase in k is attributed to the cofactor effect of factor Va(2) .

The mechanism by which factor Va increases the k of prothrombin activation may involve conformational changes in the enzyme, substrate, or both(3, 4, 5, 6) . In support of the first hypothesis, Husten et al.(4) , using fluorescently labeled active site-specific probes, demonstrated that upon association with factor Va, the environment of the probe in factor Xa is altered, suggesting a change in the conformation of the active site of the enzyme. Also consistent with this hypothesis, Krishnaswamy et al.(7) demonstrated that factor Va complex formation with factor Xa enhanced the binding affinity of the tick anticoagulant protease inhibitor. Support for the second hypothesis is provided by the observation that optimal cofactor effect of factor Va requires the presence of the fragment 2 domain of prothrombin in either covalent or noncovalent association with prethrombin 2(8, 9) . The simplest interpretation of the fragment 2 requirement in prothrombin activation is that it provides a binding site for factor Va. Alternatively, fragment 2 may change the conformation of residues near the scissile bonds to resemble the transition state (5) and be complementary to the factor Xa-Va complex.

Prothrombin activation requires two proteolytic cleavages: one to release the activation fragments, which correspond to roughly half of the mass of prothrombin, and the other to generate the two-chain enzyme thrombin(2) . Depending on the order of bond cleavage, two intermediates can be formed, prethrombin 2 or meizothrombin. With factor Xa alone, the reaction intermediate is prethrombin 2 and a large activation fragment 1.2(9) . The order of bond cleavage is altered by the presence of factor Va and membranes(7, 10) . In the presence of factor Va, the majority of the thrombin is derived from the meizothrombin intermediate(7, 10) . Meizothrombin hydrolyzes small peptide chromogenic substrates well, but does not clot fibrinogen effectively(11) . Meizothrombin is also inhibited by antithrombin, but the inhibition is not accelerated by heparin(12, 13) .

Our previous studies (24, 28) have shown that residue 192 in thrombin and activated protein C plays an important role in enzyme specificity. Mutation of Glu-192 to Gln in both thrombin and activated protein C resulted in mutant enzymes that cleaved substrates with the acidic P3 residues better than the wild-type enzymes, indicating that Glu-192 in both thrombin and activated protein C may be responsible for the slow cleavage of substrates with acidic residues at the P3 positions. Residue 192 in factor Xa is Gln. Interestingly, the sequence near the cleavage sites in human and bovine prothrombins contains acidic residues at both the P3 and P3` positions in both cleavage sites (except for a Thr at the P3` position of the first cleavage site in human prothrombin). We hypothesized that if residue 192 in factor Xa interacts with the P3 position of the substrate, then the Q192E mutant would activate prothrombin at a slower rate. Furthermore, if factor Va changes the conformation of the active-site pocket in factor Xa and/or residues near the scissile bonds in prothrombin, then factor Va may overcome the inhibitory interactions in a manner analogous to thrombomodulin with thrombin.

Overall, the results of this study indicate that residue 192 contributes significantly to the substrate and inhibitor specificity of factor Xa and that it may be one of the residues sensitive to conformational changes induced by factor Va binding.


EXPERIMENTAL PROCEDURES

Mutagenesis and Expression of Recombinant Proteins

Construction and expression of GDFX()in the RSV-PL4 vector were described previously(14, 15) . Mutagenesis of the GDFX cDNA fragment for preparing the Q192E mutant was performed by the polymerase chain reaction. The mutant codon and the native ApaI restriction enzyme site were included in the 3`-antisense primer. The sense primer started from the native StuI restriction enzyme site and was designed to contain a silent mutation to eliminate the second ApaI restriction enzyme site. The sense mutagenesis primer was 5`-CCTGCATTCCCACAGGGCCTTACCCCTGT-3`, and the antisense primer was 5`-CGTGCGGGCCCCCGCTGTCCCCCTCGCAGGCATCCT-3`. After amplification of factor X cDNA with these two primers, the resulting DNA fragment was ligated into the StuI and ApaI sites of the GDFX DNA fragment in the RSV-PL4 expression vector(14, 15) . After confirmation of the mutation by DNA sequencing(16) , the expression vector was transfected into 293 cells, and the mutant protein was isolated from cell culture supernatants by immunoaffinity chromatography as described previously(14, 15) .

Protein Preparation

Human factor X(17) , human thrombin (9), human meizothrombin(3) , human prothrombin(18) , bovine prothrombin and prethrombin 2(9) , bovine prothrombin fragment 2(9, 19) , bovine factor Va(20) , bovine antithrombin(21) , and the factor X-activating enzyme from Russell's viper venom (22) were isolated as described. Recombinant full-length tissue factor pathway inhibitor was expressed and isolated from Escherichia coli and was a generous gift of Dr. Gerald Gallupi (Monsanto). Bovine pancreatic trypsin inhibitor was purchased from Sigma.

Activation of GDFX and GDFX Q192E and Analysis of Active-site Concentration

The relative concentrations of factor X mutants were determined based on their relative A values assuming a molecular mass of 55,000 Da for the zymogen forms and 45,000 Da for the active forms and an E of 10 for both forms of mutants. Activation was performed by incubating 20 µg of zymogens with 500 ng of the factor X activator from Russell's viper venom in 0.1 M NaCl, 0.02 Tris-HCl, pH 7.5, 0.02% NaN (TBS) containing 0.1% gelatin and 1 mM CaCl at 37 °C for 4 h, which is sufficient to obtain complete activation as demonstrated by SDS-polyacrylamide gel electrophoresis analysis and amidolytic activity assays using the chromogenic substrate Spectrozyme FXa (American Diagnostica Inc., Greenwich, CT). The active-site concentration was determined by an active site-specific immunoassay using BioCap-EGR-ck (Haematologic Technologies Inc.) as described(23) . This procedure was used as an alternative to the conventional active-site titration method using p-nitrophenyl p`-guanidinobenzoate. In contrast to the p-nitrophenyl p`-guanidinobenzoate burst, the BioCap-EGR-ck immunoassay method is sensitive to <1 ng/ml factor Xa. Briefly, factor Xa and the active mutant derivatives (1 µg/ml) were incubated with a 10-fold molar excess of BioCap-EGR-ck for 15 min at 37 °C, after which no factor Xa activity was detected by an amidolytic activity assay using Spectrozyme FXa. A 96-well microtiter plate was coated with 2 µg/ml goat anti-factor X polyclonal antibody in TBS and placed at 4 °C overnight. The wells were washed with TBS containing 0.1% Tween 20 and then blocked with TBS containing 1% bovine serum albumin at room temperature for 1 h. After removing the blocking buffer, BioCap-EGR-ck-treated wild-type factor Xa of known concentrations as a standard or BioCap-EGR-ck-treated GDFXa and GDFXa Q192E as unknowns were added. After a 1-h incubation at room temperature and washing with TBS, streptavidin-alkaline phosphatase conjugate was added for 1 h at room temperature. The substrate (the reduced form of NADPH) was added, and the signal was amplified according to the manufacturer's direction using the enzyme-linked immunosorbent assay amplification system kit (Life Technologies, Inc.). This enzyme-linked immunosorbent assay detected active site-inhibited factor Xa concentrations as low as 300 pg/ml.

Prothrombin Activation and Apparent Affinity for Factor Va

The initial rates of prothrombin (5 µM) activation by GDFXa and GDFXa Q192E (2.5 nM in the presence of 500 nM factor Va and 50 nM in the absence of factor Va) were measured in TBS containing 5 mM Ca and 0.1% gelatin at room temperature. The initial rate was determined by measuring the increase in amidolytic activity as a function of time. The initial rates of prothrombin activation were also measured as a function of enzyme concentration. In this case, prothrombin (5 µM) was incubated with 500 nM factor Va and different concentrations of GDFXa or GDFXa Q192E (2.5, 5, 10, 20, 30, and 50 nM) for 3 min at room temperature in the same buffer. The rate of prothrombin activation was also used to determine the apparent affinity of factor Va for GDFXa and GDFXa Q192E. In this case, prothrombin (1 µM) was incubated with 5 nM enzymes and 5 mM Ca in the same buffer at room temperature in the presence of different concentrations of factor Va. The rate of prothrombin activation was determined from a standard curve in an amidolytic assay using the chromogenic substrate S2238 (Kabi Pharmacia/Chromogenix, Franklin, OH) and plotted as a function of the free factor Va concentration as described previously(14) .

Activation of Prethrombin 2 and Meizothrombin Des-fragment 1 by GDFXa and GDFXa Q192E

The initial rate of bovine prethrombin 2 (2 µM) activation by GDFXa or GDFXa Q192E (200 nM) was measured in the presence or absence of bovine prothrombin fragment 2 (2 µM) at 37 °C in TBS containing 5 mM Ca and 0.1% gelatin. Prethrombin 2 + fragment 2 activation was also carried out in the presence of factor Va. In this case, prethrombin 2 + fragment 2 (2 µM each) was incubated with GDFXa or GDFXa Q192E (10 nM) and factor Va (100 nM) at room temperature in TBS containing 5 mM Ca and 0.1% gelatin. The initial rate of activation was measured from the rate of thrombin generation as a function of time by an amidolytic activity assay as described above.

Since the activity of meizothrombin des-fragment 1 toward fibrinogen is considerably less than that of thrombin, the initial rate of meizothrombin des-fragment 1 activation was measured in a fibrinogen clotting assay using an ST4 Bio coagulometer (Diagnostica/Stago, Asnieres, France). In this case, meizothrombin des-fragment 1 (2 µM) was incubated with GDFXa or GDFXa Q192E (15 nM) at 37 °C in TBS containing 5 mM Ca and 0.1% gelatin (in the presence of factor Va (300 nM), the concentration of GDFXa or GDFXa Q192E was 4 nM). At different time points, the activation mixture was diluted into TBS buffer, and 100 µl of this dilution was added to 100 µl of 6 mg/ml human fibrinogen (Kabi Diagnostica, Stockholm, Sweden) in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM Ca at 37 °C. The rate of thrombin generation was measured from the clotting time and a clotting reference curve made with known amounts of thrombin. At each time point, the background clotting time of meizothrombin des-fragment 1 was subtracted from the samples.

Amidolytic Activity

Steady-state kinetic analysis of commercially available chromogenic substrate hydrolyses were performed in the presence of 2.5 nM GDFXa or GDFXa Q192E in TBS containing 5 mM Ca and 0.1% gelatin. The k and Kvalues were determined individually for S2765 and S2222 (Kabi Pharmacia/Chromogenix), Spectrozyme PCa (American Diagnostica Inc.), and Chromozym tPA (Boehringer Mannheim). The rates of hydrolysis were monitored at 405 nm at room temperature in a V kinetic plate reader (Molecular Devices, Menlo Park, CA). The concentration of synthetic substrate ranged from 15 µM to 2 mM.

Inhibition by Kunitz Inhibitors and Antithrombin

Bovine pancreatic trypsin inhibitor (BPTI) and recombinant TFPI were incubated at room temperature with 2.5 nM GDFXa or GDFXa Q192E for 30 min. The inhibitor concentration ranged from 0 to 300 µM BPTI for both GDFXa and GDFXa Q192E, from 0 to 100 nM TFPI for GDFXa, and from 0 to 1 µM TFPI for GDFXa Q192E. Residual amidolytic activity was determined at equilibrium, and the inhibition constant (K) for each inhibitor was estimated as described previously(24) . The second-order rate constant for antithrombin inhibition of GDFXa and GDFXa Q192E (2.5 nM) was estimated from inhibition time courses performed at antithrombin concentrations ranging from 0 to 1.6 µM as described previously(24) .

Electrophoresis

SDS-polyacrylamide gel electrophoresis was performed on a 10% polyacrylamide gel as described by Laemmli (25) and stained with Coomassie Blue R-250.

Data Analysis

The apparent affinity of factor Va for GDFXa and GDFXa Q192E and the k and K values were determined by nonlinear regression analysis using the one-site ligand binding and the Michaelis-Menten equation in the ENZFITTER program (Elsevier-Biosoft, London) with simple weighting.


RESULTS

Expression and Activation of Recombinant Proteins

GDFX and GDFX Q192E were expressed in 293 cells and purified from cell culture supernatants as described under ``Experimental Procedures.'' The SDS-polyacrylamide gel electrophoresis analysis indicated that the isolated proteins are essentially homogeneous (data not shown). Under nonreducing conditions, both derivatives migrated as a single chain with a similar apparent molecular mass of 62 kDa. When reduced, a heavy and a light chain of 50 and 25 kDa were observed in both cases, indicating that 293 cells processed both factor X mutants completely to the two-chain form characteristic of plasma factor X. GDFX and GDFX Q192E were activated separately with the factor X-activating enzyme from Russell's viper venom. The concentration of active enzymes was determined by an active site-specific immunoassay as described under ``Experimental Procedures.'' As expected from homogeneity on SDS-polyacrylamide gel electrophoresis, the concentration of both enzymes as determined by this assay was in good agreement with that calculated by their absorbance values at 280 nm (0.89 and 0.87 mol of active site/mol for GDFXa and GDFXa Q192E, respectively).

Factor Va can accelerate GDFXa activation of prothrombin at least 1000-fold(14) . To determine the affinity of factor Va for the Q192E mutant of GDFXa, the rates of human prothrombin activation by GDFXa and GDFXa Q192E were compared as a function of factor Va concentration (Fig. 1). Analysis of the data in Fig. 1indicated that factor Va interacted with both enzymes with a similar affinity (K = 3.6 ± 0.4 µM for GDFXa and 3.7 ± 0.8 µM for GDFXa Q192E). We next compared the time course of activation of prothrombin in the absence and presence of factor Va. As shown in Fig. 2A, the rate of human prothrombin activation by GDFXa Q192E in the absence of factor Va was 6-10-fold slower than that by GDFXa, but in the presence of factor Va, both derivatives of factor Xa activated prothrombin at a similar rate (Fig. 2B). These results indicate that factor Va essentially overcomes the catalytic deficiency in GDFXa Q192E. Similar results were obtained with bovine prothrombin. However, GDFXa and GDFXa Q192E activated human prothrombin 3- and 9-fold faster than bovine prothrombin in the presence of factor Va and 3- and 8-fold faster than bovine prothrombin in the absence of factor Va, respectively (data not shown). This may be due to sequence differences near the cleavage sites. The P3-P3` sequences of the two cleavage sites of human prothrombin are 1) Glu-Gly-Arg-Thr-Ala-Thr and 2) Asp-Gly-Arg-Ile-Val-Glu. In bovine prothrombin, the corresponding sequences are 1) Glu-Gly-Arg-Thr-Ser-Glu and 2) Glu-Gly-Arg-Ile-Val-Glu. Similar results were obtained when the concentration of prothrombin was kept constant, and the initial rates were compared as a function of different concentrations of factor Xa derivatives (data not shown).


Figure 1: Comparison of the apparent affinity of GDFXa and GDFXa Q192E for factor Va. 2.5 nM GDFXa () or GDFXa Q192E () was incubated at room temperature with 1 µM prothrombin in TBS containing 0.1% gelatin, 5 mM Ca, and the concentrations of factor Va indicated on the xaxis. Apparent dissociation constants for factor Va were measured from the rate of thrombin formation as described under ``Experimental Procedures.'' Three independent estimates of K were obtained with GDFXa. With prothrombin as the substrate, the K values were 3.0, 4.0, and 3.3 µM (average of 3.6 µM). With GDFXa Q192E and prothrombin as the substrate, two independent estimates of K were performed, and both gave K = 3.7 µM. With prethrombin 1 as the substrate, the K for GDFXa was 1.3 µM and for GDFXa Q192E was 1.5 µM.




Figure 2: Initial rates of prothrombin activation by GDFXa and GDFXa Q192E with and without factor Va. A, 50 nM GDFXa () or GDFXa Q192E () was incubated at room temperature with 5 µM human prothrombin in TBS containing 5 mM Ca and 0.1% gelatin. At the indicated time points, samples were removed into 20 mM EDTA on ice, and the amidolytic activities were determined with S2238 as described under ``Experimental Procedures.'' B, in the presence of 500 nM factor Va, 2.5 nM GDFXa () or GDFXa Q192E () was incubated at room temperature with 5 µM human prothrombin in TBS containing 5 mM Ca and 0.1% gelatin. Similar to the conditions described for Fig. 4A, at the indicated time points, samples were removed, and the amidolytic activities were determined with S2238.



Thrombin generation is the result of two peptide bond cleavages, Arg-274-Thr-275 and Arg-323-Ile-324 (bovine prothrombin numbering). The order in which these bonds are cleaved varies with activation conditions(7, 10) . One pathway generates prethrombin 2 (a single-chain thrombin precursor) as the reaction intermediate; the other generates meizothrombin, a two-chain amidolytically active protein of the same mass as prothrombin(9) . Since, in the absence of factor Va, prothrombin activation by the mutant was 6-10-fold slower than the wild type and, in the presence of factor Va, the activation rates were comparable, we decided to determine whether cleavage of the Arg-274 bond or the Arg-324 bond is impaired by the Q192E mutation. To address this issue, the activation of both intermediates by GDFXa and the Q192E mutant was studied separately in the presence and absence of factor Va.

In meizothrombin des-fragment 1, the Arg-323 bond is already cleaved, and cleavage of Arg-274 bond leads to thrombin generation. Meizothrombin des-fragment 1 activation by GDFXa and GDFXa Q192E in the presence and absence of factor Va is shown in Fig. 3. In the absence of factor Va, GDFXa activated meizothrombin des-fragment 1 10-fold faster than GDFXa Q192E (Fig. 3A). In the presence of factor Va, however, the activation by GDFXa was also 5-fold faster than that by GDFXa Q192E (Fig. 3B), indicating that factor Va has little effect in correcting the defect of the Q192E mutation through cleavage of the Arg-274 bond.


Figure 3: Initial rate of meizothrombin des-fragment 1 activation by GDFXa and GDFXa Q192E. A, meizothrombin des-fragment 1 (2 µM) was incubated with GDFXa () or GDFXa Q192E () (15 nM) at 37 °C in TBS containing 5 mM Ca and 0.1% gelatin. At different time points, aliquots of the meizothrombin des-fragment 1 activation mixture were removed, and the rate of thrombin generation was measured from the rate of fibrinogen cleavage as described under ``Experimental Procedures.'' B, meizothrombin des-fragment 1 (2 µM) was incubated with GDFXa () or GDFXa Q192E () (4 nM) in the presence of factor Va (300 nM) in the same buffer, and the rate of thrombin generation was measured as described for A.



In prethrombin 2, the Arg-274 bond is cleaved, and only the cleavage of the Arg-323 bond is required for thrombin formation. In the absence of factor Va, the Q192E mutation decreases the prethrombin 2 activation rate at least 10-fold (Fig. 4A). It has been demonstrated in the past that the optimal cofactor function of factor Va in prothrombin activation requires the fragment 2 domain(8) . In the presence of prothrombin fragment 2 and factor Va, the activation rate by the Q192E mutant was nearly similar to that by GDFXa (Fig. 4B). These results indicate that in the presence of fragment 2, factor Va largely overcomes the inhibitory effect of the Q192E mutation.


Figure 4: Initial rate of prethrombin 2 activation by GDFXa or GDFXa Q192E. A, bovine prethrombin 2 (2 µM) was incubated with GDFXa () or GDFXa Q192E () (200 nM) at 37 °C in TBS containing 0.1% gelatin. At the indicated time points, samples were removed into 20 mM EDTA on ice, and the amidolytic activities were determined with S2238 as described under ``Experimental Procedures.'' B, prethrombin 2 (2 µM) and an equimolar concentration of fragment 2 were incubated with GDFXa () or GDFXa Q192E () (10 nM) in the presence of factor Va (100 nM) at room temperature in the same buffer. The rate of thrombin generation was measured as described for A.



To determine whether fragment 2 by itself would change the properties of prethrombin 2, we examined prethrombin 2 activation by GDFXa and GDFXa Q192E in the presence and absence of the fragment 2 domain. As shown in Fig. 5A, the presence or absence of fragment 2 has no influence on prethrombin 2 activation by GDFXa, consistent with previous results reported for full-length factor Xa (8). Interestingly, with GDFXa Q192E, the presence of the fragment 2 domain enhanced prethrombin 2 activation at least 10-fold (Fig. 5B).


Figure 5: Initial rates of prethrombin 2 activation by GDFXa and GDFXa Q192E in the presence or absence of prothrombin fragment 2. A, 200 nM GDFXa was incubated at 37 °C with 2 µM bovine prethrombin 2 in the absence () or presence () of 2 µM bovine fragment 2 in TBS containing 5 mM Ca and 0.1% gelatin. At the indicated time points, samples were removed into 20 mM EDTA, and the rate of thrombin generation was measured with the chromogenic substrate S2238. B, all the experimental conditions were the same as described for A, except that GDFXa Q192E was used to activate prethrombin 2 in the absence () or presence () of fragment 2.



Comparison of the Enzyme Specificities toward Peptide Chromogenic Substrates

To examine if the Q192E substitution in factor Xa influences the p-nitroanilide substrate specificity, kinetic analysis was performed with S2765, S2222, Spectrozyme PCa, and Chromozym tPA. Comparison of the ratio of the second-order rate constants of GDFXa Q192E to GDFXa for each substrate indicates that GDFXa Q192E cleaves the chromogenic substrates with basic residues at the P3 positions (Arg in S2765 and Lys in Spectrozyme PCa) better than GDFXa (). In contrast, GDFXa Q192E cleaves the substrates with an acidic (Glu in S2222) or a hydrophobic (Phe in tPA substrate) residue less effectively that GDFXa. These results are consistent with the concept that residue 192 in factor Xa influences the P3 substrate specificity.

Inhibition by the Kunitz-type Inhibitors and Antithrombin

TFPI is a member of the Kunitz family of inhibitors and is a competitive inhibitor of factor Xa(26) . TFPI inhibited GDFXa (K = 6 10M) 2 orders of magnitude better than GDFXa Q192E (K = 5 10M) in TBS containing either 2.5 mM Ca or EDTA (Fig. 6). BPTI inhibited GDFXa with a K of 30.0 µM (data not shown). With up to 300 µM BPTI, no inhibition of GDFXa Q192E was detected (data not shown). In contrast to these Kunitz inhibitors, the serine protease inhibitor antithrombin inhibited both GDFXa and GDFXa Q192E at comparable rates with second-order association rate constants of 1.0 10 and 7.6 10M s, respectively (data not shown). The presence of 5 units/ml heparin accelerated the antithrombin inhibition of GDFXa and GDFXa Q192E 720- and 728-fold, respectively (data not shown).


Figure 6: Inhibition of GDFXa and GDFXa Q192E by TFPI. GDFXa () or GDFXa Q192E () (2.5 nM) was incubated with TFPI at room temperature for 30 min at the concentrations indicated. At equilibrium, the residual activity was measured from the rate of hydrolysis of Spectrozyme FXa, and the K was determined as described under ``Experimental Procedures.''




DISCUSSION

This study indicates that residue 192 in factor Xa plays a key role in determining the macromolecular substrate and inhibitor specificity of factor Xa. Particularly interesting is that in comparison with GDFXa, GDFXa Q192E by itself activates prothrombin poorly. In the presence of factor Va, however, the activation rates with both derivatives of factor Xa are similar. Inspection of residues surrounding the scissile bonds may reveal an explanation for the slower rate of activation of prothrombin by GDFXa Q192E alone. All but one of the P3 and P3` residues in human prothrombin and all of these residues in bovine prothrombin are acidic. It is our hypothesis that charge repulsion between Glu-192 in the factor Xa mutant and any one of these P3 and/or P3` acidic residues present on prothrombin activation peptides prevents optimal activation by GDFXa Q192E. This is consistent with the relative specificities of the two enzymes toward chromogenic substrates. It appears that factor Va alleviates this inhibitory interaction.

The mechanism by which factor Va influences the k of prothrombin activation has been hypothesized to be the result of either alteration in the active-site pocket of factor Xa or stabilization of the structure near the scissile bonds in prothrombin to better approximate the transition state(4, 5) . The first mechanism of cofactor function is reminiscent of thrombomodulin changing the active-site conformation of thrombin(27) . Thrombomodulin-induced conformational change appears to move Glu-192 in the catalytic pocket of thrombin to a location that it is no longer inhibitory for activation of substrates (such as protein C) with acidic residues at the P3 and/or P3` sites(28) . Interaction of residue 192 in thrombin with P3 and P3` residues is consistent with kinetic studies (28) of thrombin and thrombin mutants with protein C and protein C mutants (15) and with the thrombin crystal structure as analyzed by Stubbs et al.(29) . Similar to thrombomodulin, therefore, a function for factor Va in prothrombin activation could very well be the alteration of the active-site conformation of factor Xa for efficient catalysis. This hypothesis is consistent with studies employing fluorescently labeled active-site probes that demonstrated that upon association with factor Va, the environment of the probe in factor Xa was altered, suggesting a change in the conformation of the active site (or substrate-binding site) of the enzyme(4, 5) . Also consistent with this hypothesis is the observation that factor Va complex formation with factor Xa enhances the binding affinity of the tick anticoagulant protease inhibitor(7) . Factor Xa assembly into the prothrombinase complex is also shown to enhance the reactivity with TFPI(30) .

It should be noted, however, that factor Va binding to the substrate also occurs and could change the conformation near the scissile bond and overcome inhibitory interactions in a similar way(5, 31) . Previous studies demonstrated that prothrombin fragment 2 was necessary for factor Va to accelerate prethrombin 2 activation effectively(8) . In principle, at least two mechanisms could account for this requirement. Fragment 2 could alter the conformation near the scissile bond so that it was recognized better by the factor Xa-Va complex, or it could only provide a binding site for factor Va. The results of the present study suggest that fragment 2 alters the conformation near the scissile bond since it selectively enhances prethrombin 2 activation by GDFXa Q192E. It is therefore likely that fragment 2 binding to prethrombin 2 (or these interactions within prothrombin) contributes to an altered conformation near Arg-323 that is complementary to the active site of factor Xa within the factor Xa-Va complex. These results indicate that a function for fragment 2 in prethrombin 2 activation may involve substrate presentation.

It is known that clotting proteases such as activated protein C and thrombin, which have Glu at position 192, are resistant to inhibition by the Kunitz inhibitors TFPI and BPTI, but factor Xa with Gln at this position is inhibited effectively by TFPI. Our previous studies with activated protein C (24) and thrombin (32) indicated that substitution of Glu-192 with Gln increased the reactivity of these enzymes toward Kunitz inhibitors. Consistent with those observations, substitution of Gln-192 with Glu in factor Xa reduces TFPI reactivity by 2 orders of magnitude and completely abolishes BPTI reactivity up to 300 µM inhibitor concentration.

In contrast to Kunitz inhibitors, GDFXa and the Q192E mutant had nearly identical second-order association rate constants with antithrombin in the presence or absence of heparin. This may reflect the strong preference of factor Xa for Gly at the P2 position since antithrombin contains Gly at this site. In support of this hypothesis, S2765 and S2222, two of the best factor Xa synthetic substrates, both contain Gly at the P2 positions, but contain Arg and Glu, respectively, at the P3 positions. The tolerance of two oppositely charged residues at the P3 site may indicate that factor Xa specificity is largely dependent on the S2 specificity pocket.

The studies presented here were performed with GDFXa and the Q192E mutant. Truncation of the Gla domain does alter some of the properties of factor Xa, but the deletion mutants share many properties in common with the wild-type enzyme. The Gla domain of factor X is critical for membrane interaction, partially modulates substrate specificity when associated with factor Va on the membrane surface(2) , and enhances the affinity for factor Va even in the absence of membranes(33) . Factor Va still enhances prothrombin activation by GDFXa and factor Xa to nearly the same extent, however; and as seen in this study, like intact factor Xa, GDFXa requires the presence of prothrombin fragment 2 for maximum acceleration of prethrombin 2 activation. Thus, these forms of factor Xa are useful models for examining enzyme specificity and cofactor function in solution. The deletion mutants avoid the potential for Ca-mediated factor Xa dimerization through the Gla domain, which could complicate interpretations.

Based on the analysis of thrombin, activated protein C, and factor Xa, it is now apparent that residue 192 plays a central role in determining the substrate specificity of the coagulation serine proteases. It also appears that the conformation of this region in the coagulation proteases is altered by binding to the respective cofactors. The combination of enzyme and substrate mutagenesis with x-ray crystallographic studies promises to provide future insights into the mechanisms by which the cofactors accelerate coagulation reactions.

  
Table: Steady-state kinetics of p-nitroanilide hydrolysis by GDFXa and GDFXa Q192E

The K and k for each chromogenic substrate were determined as described under ``Experimental Procedures.'' The numbers in the last column represent the k/k values for GDFXa Q192E divided by GDFXa. Cbz, benzyloxycarbonyl; pNA, p-nitroanilide; Bz, benzoyl.



FOOTNOTES

*
This work was supported by NHLBI Grants R01 HL 29807 and R37 HL30340 (to C. T. E.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Oklahoma Medical Research Found., Cardiovascular Biology Research, 825 N. E. 13th St., Oklahoma City, OK 73104. Tel.: 405-271-7264; Fax: 405-271-3137.

Investigator of the Howard Hughes Medical Institute.

The abbreviations used are: GDFX, Gla-domainless factor X (a deletion mutant of factor X in which the Gla domain corresponding to residues 1-45 has been deleted by recombinant DNA methods); TBS, Tris-buffered saline; BioCap-EGR-ck, biotinyl--aminocaproyl-D-glutamic acid glycylarginine chloromethyl ketone; BPTI, bovine pancreatic trypsin inhibitor; TFPI, tissue factor pathway inhibitor; tPA, tissue plasminogen activator.


ACKNOWLEDGEMENTS

We thank Gary Ferrell and Bronson Sievers for help with cell cultures; Barbara Carpenter and Clendon Brown for isolation of recombinant and plasma proteins used in this study; Naomi L. Esmon for useful discussions; and Jeff Box, Karen Deatherage, and Julie Wiseman for assistance with preparation of the manuscript.


REFERENCES
  1. Furie, B., and Furie, B. C. (1988) Cell53, 505-518 [Medline] [Order article via Infotrieve]
  2. Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P., and Krishnaswamy, S. (1990) Blood76, 1-16 [Abstract]
  3. Armstrong, S. A., Husten, E. J., Esmon, C. T., and Johnson, A. E. (1990) J. Biol. Chem.265, 6210-6218 [Abstract/Free Full Text]
  4. Husten, E. J., Esmon, C. T., and Johnson, A. E. (1987) J. Biol. Chem.262, 12953-12962 [Abstract/Free Full Text]
  5. Walker, R. K., and Krishnaswamy, S. (1993) J. Biol. Chem.268, 13920-13929 [Abstract/Free Full Text]
  6. Krishnaswamy, S., Jones, K. C., and Mann, K. G. (1988) J. Biol. Chem.263, 3823-3834 [Abstract/Free Full Text]
  7. Walker, R. K., and Krishnaswamy, S. (1994) J. Biol. Chem.269, 27441-27450 [Abstract/Free Full Text]
  8. Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem.249, 7791-7797 [Abstract/Free Full Text]
  9. Owen, W. G., Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem.249, 594-605 [Abstract/Free Full Text]
  10. Krishnaswamy, S., Church, W. R., Nesheim, M. E., and Mann, K. G. (1987) J. Biol. Chem.262, 3291-3299 [Abstract/Free Full Text]
  11. Franza, B. R., Jr., Aronson, D. L., and Finlayson, J. S. (1975) J. Biol. Chem.250, 7057-7067 [Abstract]
  12. Schoen, P., and Lindhout, T. (1987) J. Biol. Chem.262, 11268-11274 [Abstract/Free Full Text]
  13. Rosing, J., Zwaal, R. F. A., and Tans, G. (1986) J. Biol. Chem.261, 4224-4228 [Abstract/Free Full Text]
  14. Rezaie, A. R., Neuenschwander, P. F., Morrissey, J. H., and Esmon, C. T. (1993) J. Biol. Chem.268, 8176-8180 [Abstract/Free Full Text]
  15. Rezaie, A. R., and Esmon, C. T. (1992) J. Biol. Chem.267, 26104-26109 [Abstract/Free Full Text]
  16. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A.74, 5463-5467 [Abstract]
  17. Le Bonniec, B. F., Guinto, E. R., and Esmon, C. T. (1992) J. Biol. Chem.267, 6970-6976 [Abstract/Free Full Text]
  18. Le Bonniec, B. F., MacGillivray, R. T. A., and Esmon, C. T. (1991) J. Biol. Chem.266, 13796-13803 [Abstract/Free Full Text]
  19. Liu, L.-W., Rezaie, A. R., Carson, C. W., Esmon, N. L., and Esmon, C. T. (1994) J. Biol. Chem.269, 11807-11812 [Abstract/Free Full Text]
  20. Esmon, C. T. (1979) J. Biol. Chem.254, 964-973 [Abstract]
  21. Walker, F. J., and Esmon, C. T. (1979) J. Biol. Chem.254, 5618-5622 [Abstract]
  22. Esmon, C. T. (1973) Prothrombin Activation, Ph.D. dissertation, Washington University, St. Louis, MO
  23. Mann, K. G., Williams, E. B., Krishnaswamy, S., Church, W., Giles, A., and Tracy, R. P. (1990) Blood76, 755-766 [Abstract]
  24. Rezaie, A. R., and Esmon, C. T. (1993) J. Biol. Chem.268, 19943-19948 [Abstract/Free Full Text]
  25. Laemmli, U. K. (1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  26. Broze, G. J., Jr., Girard, T. J., and Novotny, W. F. (1990) Biochemistry29, 7539-7546 [Medline] [Order article via Infotrieve]
  27. Ye, J., Esmon, N. L., Esmon, C. T., and Johnson, A. E. (1991) J. Biol. Chem.266, 23016-23021 [Abstract/Free Full Text]
  28. Le Bonniec, B. F., and Esmon, C. T. (1991) Proc. Natl. Acad. Sci. U. S. A.88, 7371-7375 [Abstract]
  29. Stubbs, M. T., Oschkinat, H., Mayr, I., Huber, R., Angliker, H., Stone, S. R., and Bode, W. (1992) Eur. J. Biochem.206, 187-195 [Abstract]
  30. Huang, Z.-F., Wun, T.-C., and Broze, G. J., Jr. (1993) J. Biol. Chem.268, 26950-26955 [Abstract/Free Full Text]
  31. Guinto, E. R., and Esmon, C. T. (1984) J. Biol. Chem.259, 13986-13992 [Abstract/Free Full Text]
  32. Guinto, E. R., Ye, J., Le Bonniec, B. F., and Esmon, C. T. (1994) J. Biol. Chem.269, 18395-18400 [Abstract/Free Full Text]
  33. Skogen, W. G., Esmon, C. T., and Cox, A. C. (1984) J. Biol. Chem.259, 2306-2310 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.