An Extensive Interaction Interface between Thrombin and Factor V Is Required for Factor V Activation*

Timothy MylesDagger, Thomas H. Yun, Scott W. Hall, and Lawrence L. K. LeungDagger

From the Division of Hematology, Stanford University School of Medicine, Stanford, California 94305

Received for publication, December 15, 2000, and in revised form, March 5, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu45-Asn57 insertion loop, and the Na+ binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na+ binding loop mutants, E229A and R233A, and the Leu45-Asn57 insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na+ binding loop mutant E229A, and the Leu45-Asn57 insertion loop mutant W50A showed a requirement for both ABEs and the Na+-bound form of thrombin for efficient cleavage at the FV residue Arg709. Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na+-bound form of thrombin is important for its procoagulant activity.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thrombin is a serine protease that interacts with a large number of macromolecular substrate receptors, cofactors, and inhibitors that have both procoagulant and anticoagulant properties. This functional versatility of thrombin is revealed by its structure (1). Thrombin has the characteristic serine protease fold but differs in having a deep narrow active site cleft occluded by the Leu45-Asn57 (60 insertion loop) and Leu144-Gly155 (149 insertion loop) surface loops. Unique to thrombin are two cationic surface domains termed anion-binding exosite I (ABE-I)1 and II (ABE-II) that are important for the binding of ligands to overcome the steric hindrance of the occluded active site cleft. ABE-I is important for the binding of fibrinogen (2-4), fibrin (5), heparin cofactor II (6, 7), PAR1 (8, 9), thrombomodulin (2, 10-12), and hirudin (13-15). ABE-II is important for the binding of platelet glycoprotein Ib (16) and the glycosaminoglycan-bound serpins antithrombin III (6), heparin cofactor II (6), and protease nexin I (17, 18).

Both exosites are involved in the binding of coagulation factors V and VIII, which is important for the amplification of the coagulation cascade (19). Studies with the ABE-I-specific inhibitor hirugen (19, 20) have implicated the thrombin ABE-I in cleavage of factor V, whereas studies using the ABE-II triple mutant thrombin RA (R89A/R93A/R98A) have implicated ABE-II (19). The specific proteolytic cleavage of human factor V (FV) by the serine protease thrombin generates factor Va (FVa), which acts as a cofactor with factor Xa, prothrombin, and Ca2+ ions to form the prothrombinase complex on the surface of anionic phospholipids. This leads to the amplification of the coagulation pathway at the sites of vascular injury. FV, a single chain 330,000-kDa cofactor protein, is activated to FVa with the release of the B domain activation products (E fragment and C1 fragment) by cleavage at the residues Arg709, Arg1018, and Arg1545 (21). FVa is composed of the 105-kDa heavy chain (A1-A2 domain) and the 74-kDa light chain (A3-C1-C2 domains) held together by a calcium ion (22, 23). The crystal structure of the light chain C2 domain implies a Ca2+-independent mode of binding to the phospholipid membrane via immersion of hydrophobic residues into the laminar membrane core, with direct interactions between the basic C2 domain residues and the negatively charged phosphatidylserine head groups and generalized complementary electrostatic interactions (24). The heavy chain interacts with prothrombin, whereas both chains interact with factor Xa. As part of the prothrombinase complex, factor Xa has a 300,000-fold increase in catalytic efficiency in thrombin generation from prothrombin (25). Specific cleavage of FV occurs preferentially first at Arg709, giving rise to the heavy chain, followed by cleavage at Arg1018 and then by the rate-limiting cleavage at Arg1545, which gives rise to the light chain. Cleavage of both Arg709 and Arg1545 is important for the full cofactor activity of factor Va, and the cleavage of Arg1018 enhances the rate of cleavage of Arg1545 (26).

In this study, we used 53 mutant thrombins in which solvent-accessible polar and charged residues were substituted with alanine to fully define thrombin residues important in the recognition and cleavage of FV. This provided insights into the roles of the anion-binding exosites for specificity toward FV and of residues involved in substrate recognition and cleavage within the active site cleft.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Purified human FV and FVa were purchased from Hematologic Technologies Inc. Factor V-deficient plasma and thromboplastin were purchased from Sigma. The expression and purification of wild-type (WT) and alanine-substituted mutant thrombins from Chinese hamster ovary cells and of the mutant R62Q from insect cells have been described in detail previously (27, 28). The concentration of active thrombin molecules was determined by titration with D-Phe-Pro-Arg-chloromethyl ketone using the chromogenic substrate H-D-Val-Leu-Arg-p-nitroanilide (S-2266, Chromogenix, Sweden). The catalytic activity of the purified recombinant WT and mutant thrombins toward H-D-Phe-Pip-Arg-p-nitroanilide (S-2238, Chromogenix, Sweden), fibrinogen, protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI) has been described (27).

Activation of Factor V by WT and Mutant Thrombins-- Cleavage assays were performed in 50-µl volumes containing 300 nM human FV and 100 pM thrombin in assay buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM CaCl2, and 0.1% polyethylene glycol 6000) at 37 °C for 30 min, and the reactions were then placed on ice. The concentration of FVa generated in the cleavage assay was determined by a one-stage clotting assay. Cleavage reactions were diluted 1000-fold in assay buffer on ice, and 50 µl was then added to 50 µl of FV-deficient plasma and left at room temperature for 10 min. The clotting assay was started with 100 µl of thromboplastin (containing 5 mM CaCl2 and equilibrated at room temperature), and the clotting reaction was monitored over time at 600 nm in a SoftmaxTM plate reader (Molecular Dynamics) at room temperature until maximum clotting was achieved (no further change in absorbance at 600 nm). The t1/2 value was recorded as the time at which 50% clot formation occurred. The effective concentration of diluted human factor Va from the cleavage reaction in the clotting assay was determined from a calibration curve using various concentrations of purified human FVa versus the t1/2 value for clotting. For mutants showing decreased clotting ability, dose-response curves were constructed over a range of thrombin concentrations using the above protocol.

SDS-PAGE Analysis of Cleavage Reactions-- Cleavage reactions containing 300 nM human FV and 0.5 nM thrombin in assay buffer were incubated from 1 to 120 min before being terminated by the addition of SDS loading buffer and boiling for 5 min. Cleavage products were resolved by electrophoresis on 5-18% gradient SDS-polyacrylamide gels (Bio-Rad) and then stained with biosafe Coomassie Blue (Bio-Rad). The intensity of Coomassie Blue-stained bands was determined by direct scanning of stained SDS-PAGE gels using the UMAX Astra 4000U scanner and Umax VistaScan 3.5.2 software. Scanned images were saved as TIFF files at a resolution of 600 dots per inch. Pixel densities were calculated for each band from TIFF files imported into Scion Image 1.62c.

Molecular Modeling of Thrombin-- The x-ray crystal structure of thrombin (EC 3.4.21.5) bound with the active site inhibitor D-Phe-Pro-Arg-chloromethyl ketone was obtained from the Protein Data Bank (entry 1PPB). Thrombin is depicted as a space-filling (CPK) model with solvent removed using the RasMol V2.5 software package.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Screening for Thrombin Mutants Defective in Human FV Activation-- A site-directed mutagenesis strategy was used to determine residues on thrombin important for the interaction between thrombin and human FV necessary for activation of FV to FVa. Fifty-three thrombin mutants (Table I), where solvent-exposed polar and charged residues were mutated to alanine (27), were used in a two-stage clotting assay using purified human FV to assay FV activation. Seventeen thrombin mutants with ~50% or less FV activation compared with WT thrombin were identified and mapped to ABE-I, ABE-II, the Leu45-Asn57 insertion loop, and the Na+ binding loop (Fig. 1, Table II). ABE-I had six residues (K21A, K65A, H66A, R70A, R73A, K77A) with less than 50% of WT activity. Two mutant thrombins showed less than 15% of WT activity (R68A, Y71A) (Fig. 1, Table II). The double mutant K106A/K107A showed only 24% of the WT type activity, indicating that the effects of the single mutations (K106A, 62%; K107A, 52%) were additive for the double mutant. Interestingly, substitution of Arg62 with Gln gave a greater decrease in clotting activity (23% of WT activity) compared with the alanine-substituted form of Arg62 (65% of WT activity). ABE-II showed one mutant (R98A) with greatly reduced FV activation (27% of WT activity), whereas two triple mutants (with a total of six residues substituted) showed less than 50% of WT activation (R89A/R93A/E94A and R245A/K248A/Q251A). Both the ABE-I and ABE-II mutants have normal kcat/Km values toward the chromogenic substrate S-2238, suggesting that the reduced activity is due to impaired binding to the exosites rather than to an effect on catalysis (Table I). The residues Glu229, Arg233, and Asp234 form part of a surface-exposed Na+ binding loop that modulates the activity of thrombin (29). Mutation of each residue resulted in 5, 15, and 32% of WT activity, respectively. Interestingly, the E229A mutant shows a 13-fold reduction in the kcat/Km for S-2238 (Km = 36.3 µM; kcat = 33.2 s-1) compared with WT thrombin (Km = 4.7 µM; kcat = 55.9 s-1), whereas the remaining two mutants have normal catalysis toward S-2238, suggesting that substitution of Glu229 affects substrate binding within the active site cleft. Two residues, situated on the Leu45-Asn57 loop, also have reduced FV activation. Trp50 forms part of a hydrophobic "YPPW lid" that restricts the specificity of thrombin by occluding the active site, and Lys52 protrudes and contributes to the specificity of the S' subsite (30, 31). Alanine substitution at these residues showed FV activation of 29%. The mutant thrombin W50A has a 5-fold reduction in kcat/Km for S-2238, reflected by a 6-fold increase in Km, whereas K52A has normal catalysis toward S-2238. Mutation of Trp50, like Glu229, appears to alter the active site cleft, affecting substrate binding.

                              
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Table I
Thrombin nomenclature and S-2238 amidolytic activities of purified thrombin mutants
Table 1 has been reported previously (27).


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Fig. 1.   Human FV activation by thrombin mutants. The ability of purified WT and mutant thrombins to activate human FV was determined using a two-stage clotting assay as described under "Experimental Procedures." The effect of alanine substitutions on FV activation is reported relative to 100% WT activity. The error bars represent the standard deviation of at least two separate independent experiments (carried out in duplicate).

                              
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Table II
Mutant thrombins with less than 50% of human factor V activation

Dose-response Curves for Mutants with Diminished Human FV Activation-- For mutants with a marked decreased in FV activation (less than 20% of WT activity), dose-response curves were constructed, because these responses in the screening assay may not be in the linear range for cleavage (Fig. 2). The mutants R68A and R233A had 12.5 and 15.4% of WT activity in the initial screen, each requiring 6-fold more thrombin to generate the same amount of human FVa generated by cleavage with 100 pM WT thrombin. The 6-fold more thrombin required for cleavage by the mutants corresponded well with the initial screen of the clotting assays. Consistent with this, the 5.2% of WT activity of E229A in the initial screen also corresponded well in requiring 12-fold more thrombin to generate the same amount of FVa obtained by 50 pM WT thrombin. The mutant W50A required 4-fold more thrombin to generate the same amount of FVa compared with 100 pM WT thrombin. Of all the mutants tested, Y71A appears to have had the greatest effect on cleavage of FV. Even at a concentration of 600 pM, only 25 nmol of FVa were produced in 30 min by Y71A, which is equivalent to a 30-fold decrease in FV activation by Y71A as compared with WT thrombin.


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Fig. 2.   Dose dependence of thrombin cleavage of human FV. FV (390 nM) was incubated with several concentrations of WT and mutant thrombins ranging from 50 to 600 pM at 37 °C for 30 min. The concentration of human FVa generated after the 30-min cleavage reaction was determined using a two-stage clotting assay as outlined under "Experimental Procedures." A shows the dose dependence curves for WT thrombin (squares) and the ABE-I mutants R68A (circles) and Y71A (triangles). B shows the dose dependence curves for WT thrombin (squares) and the Na+ binding loop mutants E229A (circles) and R233A (triangles) and the Leu45-Asn57 loop mutant W50A (diamonds).

SDS-PAGE Analysis of Cleavage Reactions-- SDS-PAGE analysis of cleavage reactions on FV by representative thrombin mutants of the major interaction interfaces (ABE-I, ABE-II, the Na+ binding loop, and the Leu45-Asn57 loop) was employed to assess their role in the differential cleavage of the three thrombin cleavage sites Arg709, Arg1018, and Arg1545 (Fig. 3, A and B). Cleavage of FV at Arg709 by WT thrombin occurred within 1 min, with the appearance of the heavy chain (VaH). The 75-kDa band appearing within 2-5 min represents both the light chain (VaL) and the E fragment (generated by cleavage at Arg709 and Arg1018), which are difficult to resolve under these conditions. Within 5 min the majority of the 330-kDa FV was converted into VaH and other intermediate protein species. Within 30 min there was almost complete conversion to the VaL and VaH species. For the mutant thrombins W50A, R68A, and R98A, the VaH appeared to be delayed by 1-2 min compared with WT thrombin, with the intact protein persisting for 10 min, suggesting that cleavage at Arg709 is delayed. Generation of the VaH protein species in cleavage reactions for the mutants Y71A and E229A was further delayed, requiring 5-10 min before detection of the heavy chain. The appearance of the VaH chain by specific cleavage at Arg709 between WT and mutant thrombins was assessed by running cleavage products from 1-min and 5-min reactions on the same gel. This was performed to avoid making comparisons between several gels showing differing degrees of staining. The amount of VaH generated was quantified by scanning stained gels directly, the mean pixel density for each band was determined, and the results are presented as a percentage of the VaH band intensity generated by cleavage with WT thrombin (Fig. 4). The effect of the Y71A and E229A substitutions on cleavage at Arg709 was most prominent, with almost undetectable levels of VaH at 1 min compared with WT thrombin (0.2 ± 0.5 and 2.4 ± 2.4% band intensity, respectively). After 5 min, the Y71A and E229A mutants generated only 5.1 ± 1.1 and 24.9 ± 3.0% of the VaH compared with WT thrombin. The effects of the R68A, W50A, and R98A substitutions were less severe, showing 26.0 ± 2.8, 34.8 ± 2.1, and 43.8 ± 2.7%, respectively, of the VaH band intensity respective to WT thrombin within 1 min. Cleavage reactions with the mutant thrombin W50A showed persistence of the E-C1-VaL protein species over the entire time course, suggesting impairment of cleavage at Arg1018. To confirm this, cleavage reactions of WT and W50A were run on the same gel (Fig. 3C), which clearly shows the persistence of the E-C1-VaL fragment over 120 min.


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Fig. 3.   SDS-PAGE analysis of cleavage reactions for ABE-I, ABE-II, and Na+ binding and Leu45-Asn57 loop mutants. Cleavage reactions containing 390 nM human FV and 0.5 nM thrombin were performed at 37 °C at several time points ranging from 1 to 120 min. Cleavage products were resolved by SDS-PAGE on a 5-18% gradient gel. A shows the expected sizes of cleavage products. B shows gels for WT thrombin, W50A, R68A, Y71A, R98A, and E229A. C shows time course experiments for the cleavage of WT and W50A thrombins over 0, 1, 5, 10, and 60 min.


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Fig. 4.   Comparison of human FV heavy chain release by WT and mutant thrombins. The intensity of Coomassie Blue-stained FV heavy chain bands (VaH) generated from cleavage reactions at 1 and 5 min for WT and thrombin mutants (W50A, R68A, Y71A, R98A, and E229A) was determined by direct scanning of stained SDS-PAGE gels as described under "Experimental Procedures." The band intensity for each mutant is presented on the bar graph as the percent VaH band intensity with respect to WT thrombin.

The appearance of the VaL species for the cleavage reactions by all the mutant thrombins was difficult to interpret, because cleavages at Arg709 and Arg1018 generate the E fragment, which comigrates with the VaL chain. In addition, cleavage at Arg1018 enhances cleavage at Arg1545 (32). Hence, it is difficult to assess the effect of the substitutions on cleavage at Arg1545.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using a total of 53 mutant thrombins, which have a total of 78 alanine substitutions, we were able to map the interaction interface between thrombin and FV using a two-stage clotting assay. This revealed an extensive binding surface that utilizes ABE-I, ABE-II, the Na+ binding loop, and the Leu45-Asn57 insertion loop (Fig. 5).


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Fig. 5.   Space-filling model of thrombin residues with decreased human FV activation. Thrombin is depicted as a space-filling model using the RasMol V2.5 software package showing residues, when substituted with alanine, with less than 50% of WT human FV activation. Mutants with <= 15% of WT activity are depicted in a darker color. Residues of interest are labeled using the single amino acid codes. The top panel shows the classic front view of thrombin (31) with the active site cleft running horizontally left to right, with the Leu45-Asn57 insertion loop on the top of the cleft occluding the active site Ser205 (red). The active site is shown with the bound active site inhibitor D-Phe-Pro-Arg-chloromethyl ketone as a brown stick model. The Leu45-Asn57 insertion loop residues are colored magenta, and the Na+ binding loop residues are green. ABE-I runs to the right of the active site cleft, and ABE-II is on the opposite side of the molecule. The middle and bottom panels show the same molecule but rotated 90° to the left and right, respectively, to show ABE-I and ABE-II. ABE-I residues are blue, and ABE-II residues are depicted in yellow.

The ABE-I of thrombin is a surface patch with a high density of surface-exposed basic amino acids. This region extends away from the active site cleft and is essential for the binding of fibrinogen (2-4), heparin cofactor II (5, 6), PAR1 (8, 9), thrombomodulin (2, 10-12), and the inhibitor hirudin (13-15). Binding to ABE-I is important to overcome steric hindrance to the occluded active site. Basic residues in ABE-I have a dual role in binding its many ligands. Studies with PAR1 (33) and hirudin (34) show that the ABE-I residues contribute to a positive electrostatic field that extends into the solvent. This is important for enhancing the rate of complex formation with complementary electrostatic fields generated by its ligands (electrostatic steering). Secondly, basic residues are used for direct interactions in complex formation (ionic tethering), although which residue is involved differs between the various ligands (33, 34). The ABE-I also has a number of hydrophobic residues important for hydrophobic interactions with PAR1 (9), thrombomodulin (35), and hirudin (14, 15). Alanine substitution of the ABE-I residues Arg68, His66, Arg70, and Tyr71 significantly reduced FV activation, which was due to impaired binding within ABE-I rather than to a direct effect on catalysis. The mutations either affect direct interactions with FV or indirectly affect local protein structure. Both Arg68 and Arg70 have been shown to form direct interactions with ligands in the crystal structure of thrombin bound to either a peptide based on the N-terminal domain of the PAR1 receptor (Arg68) (9) or the C-terminal tail of hirudin (Arg68 and Arg70) (14, 15). Hence, it is possible that these residues may also be in direct interactions with FV. Likewise, the residue Tyr71 is important for hydrophobic interactions with the C-terminal tail of hirudin (14, 15) and the thrombomodulin 4-5-6 epidermal growth factor-like domain (35). The substitution of residue His66 has a large effect on FV activation; however, this residue does not make any direct interactions as seen in the published crystal structures of thrombin bound with various ligands. Furthermore, the H66A mutation has a large effect on the thrombomodulin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) and protein C (27); however, the crystal structure of thrombin bound with the 4-5-6 epidermal growth factor-like domains of thrombomodulin showed that this residue makes no direct major interactions (35). Together with the observation of normal catalysis toward S-2238, would suggest that the H66A substitution has a local effect on the binding in ABE-I. Interestingly, the effect of substitution of Arg62 with Gln is more severe than that seen with Ala. The effect of the R62Q mutation is similar to that of the dysthrombin Quick I (R62C), where the cysteine substitution appears to distort the 60-70 autolysis loop, affecting the binding of hirudin (36), fibrinogen (37), and heparin cofactor II (38) within ABE-I. Hence, it appears that alanine substitution does not affect loop structure to the same degree as glutamine or cysteine substitutions. The role of ABE-I in the recognition and cleavage at the thrombin cleavage sites within FV was difficult to determine because of the complexity of cleavage patterns on SDS-PAGE. What is clear from cleavage studies using the ABE-I mutants of Arg68 and Tyr71 is the importance of ABE-I in the recognition and cleavage of FV at Arg709. These studies suggest a major interaction with a hirudin-like domain N-terminal to the Arg709 cleavage site (21) and are consistent with equilibrium binding studies by Bock and co-workers (39).

The ABE-II, which is located on the opposite side of the molecule, is involved in the binding of glycosaminoglycan-bound serpins antithrombin III (6), protease nexin I (17, 18), heparin cofactor II (6, 7, 28), bovine FV, and recombinant human FVIII (19), as well as in the binding of prothrombin fragment F2 (40) and platelet membrane glycoprotein Ib (16). The mutant thrombins R98A and R89/R93/E94A have an effect on the activation of FV (27-40% of WT activity), suggesting an important role for the interaction of ABE-II with human FV. The importance of these three residues has been shown by Esmon and Lollar (19) using the triple thrombin mutant R89A/R93A/R98A, which had a greatly reduced ability to activate bovine FV. The residues Arg89 and Arg98 lie in close proximity (4 Å), whereas Arg93 is over 12 Å away from these residues (Fig. 5). Mutation of these residues impairs heparin binding and heparin-ATIII-accelerated inhibition of thrombin (41) and chondroitin sulfate-enhanced affinity of thrombomodulin to ABE-II (41). The R98A substitution has a major effect on cleavage of Arg709 on FV, and this effect compares well with cleavage studies of bovine FV using the thrombin triple mutant RA (R89A/R93A/R98A), suggesting a role of ABE-II for cleavage of human FV at Arg709 (19).

It is less clear whether both ABE-I and ABE-II are important for cleavage at Arg1018 and Arg1545. Studies by Thorelli et al. (26, 32) show that cleavage at Arg1018 facilitates cleavage at Arg1545. Therefore, the late appearance of the VaL could be a direct consequence of poor cleavage of Arg1018 delaying the appearance of the C1-VaL species and subsequent cleavage at Arg1545, or the mutations may directly affect recognition and cleavage at the individual cleavage sites. The effect of the ABE-I and ABE-II mutations on cleavage of Arg1018 and Arg1545 was difficult to determine because of comigration of the E-fragment and VaL on SDS-PAGE.

Several residues in both ABE-I and ABE-II have reduced FV activation ranging from 52% (K107A) to 65% (R62A). However, the magnitude in reduction suggests that these residues are unlikely to participate in major interactions. Mutagenesis studies support the hypothesis that ABE-I basic residues, not involved in direct interactions with hirudin (33) or PAR1 (34), make small but collectively important contributions to the localized positive electrostatic field generated by ABE-I. Hence, it is likely that both ABE-I and ABE-II positive electrostatic fields are important in enhancing complex formation through the interaction of complementary electrostatic fields with FV (42, 43). Both Arg709 and Arg1545 have sequences N-terminal to the cleavage site with a high density of acidic residues, which, if exposed to the solvent, could contribute to a negative electrostatic potential, allowing rate enhancement by electrostatic steering with complementary electrostatic fields of thrombin ABE-I and -II.

The residues Glu229 and Arg233 make ion pair interactions with residues Lys236 and Asp146, respectively, which are important for maintaining the structural integrity of the Na+ binding loop (44, 45). Mutation of either residue leads to a conformational change of the Na+ binding loop, favoring the anticoagulant form of thrombin (46). Consistent with this, E229A and R233A showed markedly reduced FV activation, suggesting that efficient recognition and cleavage of FV requires the Na+ form of thrombin. The thrombin substitution W50A appears to have an effect on recognition and cleavage at Arg709, but the persistence of the E-C1-Val species shows that recognition and cleavage at Arg1018 is also impaired. Trp50 is spatially well separated (17 Å) from the Na+-binding site; however, mutation of Trp50 may have an indirect effect on the Na+ binding loop. Substitution of Trp50 with serine appears to affect Na+ binding and favors the anticoagulant form of thrombin with enhanced protein C specificity (45). Likewise, it is possible that alanine substitution of Trp50 could favor the anticoagulant form of thrombin with reduced ability to activate FV.

Specificity of the thrombin S1' substrate binding site is restricted to small polar P1' residues, due in part to occlusion by the Lys52 side chain (1). Alanine substitution of this residue resulted in a decrease in FV activation, suggesting a role for this residue and the S1' subsite in defining the specificity of thrombin toward FV. Alanine substitution of this residue has also been shown to be important in defining the specificity of thrombin toward antithrombin III (31) and fibrinogen (12, 47).

In summary, thrombin activation of human FV requires an extensive interaction interface involving ABE-I, ABE-II, the Leu45-Asn57 loop, and the Na+ binding form of thrombin. It will be interesting to analyze and compare the structural requirements for the activation of human factor VIII, which is homologous to human FV and important for the assembly of the intrinsic tenase enzyme complex.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01 HL57530 and the Cheong Har Family Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence may be addressed: Division of Hematology, Stanford University School of Medicine, CCSR (Rm. 1155), Stanford, CA 94305-5156. Tel.: 650-725-4043; Fax: 650-736-0974; E-mail: lawrence.leung@stanford.edu or tmyles{at}stanford.edu.

Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M011324200

    ABBREVIATIONS

The abbreviations used are: ABE, anion-binding exosite; PAR1, protease-activated receptor 1; FV, factor V; WT, wild-type; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; VaH, factor Va heavy chain; VaL, factor Va light chain; serpin, serine protease inhibitor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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