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Originally published In Press as doi:10.1074/jbc.M306916200 on August 25, 2003

J. Biol. Chem., Vol. 278, Issue 45, 44489-44495, November 7, 2003
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Role of Prothrombin Fragment 1 in the Pathway of Regulatory Exosite I Formation during Conversion of Human Prothrombin to Thrombin*

Patricia J. Anderson{ddagger} and Paul E. Bock§

From the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received for publication, June 29, 2003 , and in revised form, August 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin54–65 labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir54–65()) or 5-(carboxy)fluorescein ([5F]Hir54–65()). [NBD]Hir54–65() distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir54–65(). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir54–65() by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin·F1.2 complex bound [5F]Hir54–65() with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the penultimate step of blood coagulation, thrombin is generated by factor Xa cleavage of Pro at Arg271-Thr272 and Arg320-Ile321. Pro activation is regulated by the protein cofactor, factor Va, phospholipids, and calcium, which together elicit a ~300,000-fold increase in the activation rate through formation of the membrane-bound, factor Xa·factor Va, prothrombinase complex (1–4). In the absence of factor Va, factor Xa cleavage of Arg271-Thr272 results in accumulation of prethrombin 2 (Pre 2)1 and activation fragment 1.2 (F1.2) noncovalent complex as an activation intermediate. Factor Va confers a substrate specificity change such that factor Xa cleavage of Arg320-Ile321 is preferred, and the catalytically active intermediate meizothrombin (MzT) is generated predominately (5–8). Cleavage of the alternative sites in the intermediates generates the products thrombin and F1.2.

The physiological substrate specificity of thrombin and the localization of thrombin activity are mediated by one or both of two exosites (I and II) distinct from the catalytic site (9, 10). Exosite I is in a precursor state on Pro called proexosite I (11, 12). Activation of the catalytic site in the formation of thrombin is accompanied by an overall ~100-fold increase in affinity of exosite I for hirudin54–65 (Hir54–65()). The proexosite has been implicated in the mechanism of factor Va rate acceleration of Pro activation and cofactor-mediated Pro substrate recognition (13, 14). The role of proexosite I in substrate recognition is supported further by recent site-directed mutagenesis studies demonstrating that mutation of proexosite I residues in prethrombin 1 (Pre 1) results in loss of factor Va cofactor activity and is correlated with loss of affinity of the proexosite for Hir54–65() (15). A natural mutation of Arg67 to His in proexosite I of Pro isolated from a patient with a severe procoagulant defect and mild bleeding phenotype also showed reduced factor Va acceleration of its activation (16). In the preceding paper, proexosite I on Pre 1, a Pro analog that lacks the fragment 1 (F1) domain, and Pre 2 was shown to be activated by cleavage of Arg320-Ile321, generating the active intermediate, meizothrombin des-fragment 1 (MzT(-F1)), and the product, thrombin, respectively. Removal of F1 from Pro by thrombin cleavage to yield Pre 1 resulted in a 6-fold increase in affinity for hirudin peptides. This result suggested a new role for F1 in modulating activation of exosite I. Previous studies also suggested a role for F1 in expression of exosite I in the observation that macromolecular exosite I ligands bound to MzT(-F1) but not meizothrombin (MzT) (17).

The roles of F1, F2, and the catalytic domain (Pre 2) in factor Va regulation of Pro activation are not fully understood. Early studies demonstrated a rate-enhancing effect of F2 in factor Va-accelerated Pre 2 activation (18), suggesting that F2 mediated Pro binding to factor Va. Subsequent studies demonstrated similarly significant rate-enhancing effects of F2 and F1.2 on Pre 2 activation in solution (19). A kinetic analysis of Pre 2 activation by the membrane-bound factor Xa·factor Va complex, however, does not support a significant role for F2 (19) and indicates that Pre 2 substrate recognition is mediated by exosites expressed on factor Xa in the factor Va-assembled complex (20–22). Productive binding of Pro to factor Va in the prothrombinase complex has also been linked to the Gla domain (23) and kringle domains of both F1 and F2 (24, 25). Our studies of thrombin- and Pro-factor Va interactions support a direct role for proexosite I on the Pro catalytic domain in mediating productive binding to the heavy chain subunit of factor Va within the factor Xa·factor Va complex (13, 26, 27). The proexosite I-factor Va interactions are thought to be influenced by changes in exosite I expression during Pro activation.

To resolve the role of F1 in the activation of exosites I and II during Pro activation, binding of ligands specific for either exosite were characterized quantitatively for the Pro activation intermediates. Fluorescent derivatives of Hir54–65() labeled at the amino termini with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir54–65()) or 5-carboxy(fluorescein) ([5F]Hir54–65()) were used as specific probes of exosite I formation. [NBD]Hir54–65() displayed fluorescence spectral changes upon binding to the Pro activation intermediates that reported different proexosite I environments on Pro, Pre 1, and Pre 2. Expression of exosite I on the Pro activation intermediates, as measured by the increase in affinity for the peptides, displayed a similar pattern as for the Pre 1 activation intermediates, where initial cleavage at Arg320-Ile321 caused simultaneous activation of the active site and full activation of exosite I (28). Binding of F1.2 to Pre 2 decreased the affinity of the fluorescein-labeled peptide for exosite I by 3-fold, whereas no effect of F1.2 was observed for thrombin. The attenuating effect of F1 was correlated with binding of F1.2 to Pre 2 with a 16-fold higher affinity compared with thrombin. The results indicate that F1.2 interacts with the zymogen proteinase domain with greater affinity than the active proteinase and decreases the affinity of exosite I for hirudin peptides only in the zymogen forms. The results characterize a new role of F1 in the expression of exosites I and II, which is disengaged on folding of the proteinase "activation domain" (29) into the catalytically active form, simultaneous with activation of exosite I. These observations suggest that changes in (pro)exosite I interactions mediated by the F1 domain and differential expression of exosite I on the Pro activation intermediates may regulate factor Va-Pro interactions that control the activation pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Purification and Characterization—Human Pro, Pre 2, and thrombin were prepared as described in the preceding paper (28). Hir54–65() was labeled with 5-carboxy(fluorescein) ([5F]Hir54–65()) or succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoate ([NBD]Hir54–65()) and characterized as previously described (30). Traces of thrombin were removed from Pro by chromatography on sulfopropyl-Sephadex. Thrombin was active-site labeled with 6-(iodoacetamido)fluorescein and characterized as previously described (31). Active-site blocked MzT was prepared by activation of 20 µM Pro with 5 units/ml of ecarin (Sigma) in the presence of 400 µM FPR-CH2Cl at 25 °C in 50 mM Hepes, 110 mM NaCl, 5 mM CaCl2, pH 7.4. After 1 h, FPR-MzT was concentrated by YM10 ultrafiltration and dialyzed against buffer containing 0.1 µM FPR-CH2Cl.

Preparation of Fragment 1.2—F1.2 was prepared by activation of 20 µM FPR-MzT with 30 nM factor Xa in the presence of 3 µM Thromstop (American Diagnostica) for 2 h at 25 °C. The reaction was chromatographed on sulfopropyl-Sephadex (2.5 x 21 cm) equilibrated with 20 mM Mes, 0.1 M NaCl, 0.1 mM EDTA, 1 mM benzamidine, pH 6.5. F1, F2, and F1.2 co-eluted from the resin in the void volume. FPR-thrombin was eluted from the column with a 1-liter gradient of buffer up to 0.8 M NaCl. The fragments were dialyzed against 20 mM Hepes, 0.25 M NaCl, 1 mM benzamidine, 2 mM EDTA, 0.02% NaN3, pH 7.4, and F1.2 was separated from F1 and F2 by chromatography on fast-flow Q-Sepharose (2.5 x 13 cm) (Amersham Biosciences). The column was washed with 20 mM Hepes, 0.25 M NaCl, 1 mM benzamidine, 0.02% NaN3, pH 7.4, eluting F2. F1 and F1.2 were separated by a 360-ml gradient of buffer up to 20 mM CaCl2. The purified activation fragments were concentrated by YM3 (F2) or YM10 (F1 and F1.2) ultrafiltration and dialyzed against 50 mM Hepes, 110 mM NaCl, 5 mM CaCl2, 1 mg/ml polyethylene glycol 8000, pH 7.4, containing 0.1 µM FPR-CH2Cl. Protein concentrations were determined by absorbance at 280 nm with the same absorption coefficients ((mg/ml–1)/cm–1) and molecular weights as in the previous paper (28) or with the following constants (7, 32, 33): MzT, 1.47, 71,600; F1.2, 1.12, 34,500.

Fluorescence Studies—Fluorescence was measured with an SLM 8100 fluorometer using cuvettes coated with polyethylene glycol 20,000. All experiments were performed in 50 mM Hepes, 110 mM NaCl, 5 mM CaCl2, 1 mg/ml polyethylene glycol 8000, pH 7.4, containing 0.1 µM FPR-CH2Cl at 25 °C. Emission spectra (4-nm band pass) of 0.4 µM [NBD]Hir54–65() with near-saturating concentrations of Pro, Pre 1, and the activation intermediates were recorded with excitation at 480 nm (16-nm band pass) and normalized to the initial fluorescence intensity. Corrections for background (<=1%) were made from parallel measurements on blanks lacking the probe, and corrections for dilution were <= 5%.

Direct binding of the labeled peptides to Pro, the Pro activation intermediates, and thrombin was measured by titrating the labeled peptide with each protein. Changes in fluorescence ({Delta}F/F0 = Fobs – F0/F0) were monitored as described for [5F]Hir54–65() (28). For [NBD]Hir54–65(), fluorescence was measured with excitation at 480 nm (16-nm band pass) and emission at 540 nm (8-nm band pass). The data were fit by the quadratic binding equation to obtain the maximal fluorescence change ({Delta}Fmax/F0) and the dissociation constant (KD) for peptide binding. Competitive binding of labeled and unlabeled Hir54–65() was measured by titrations of fixed concentrations of labeled peptide and protein as a function of competing ligand concentration. The direct and competitive binding data were analyzed simultaneously with the cubic binding equation (28, 34, 35).

Binding of F1.2 to Thrombin and Pre 2—Binding of F1.2 to thrombin was measured from the change in fluorescence of 190 nM [6F]FPR-thrombin as a function of F1.2 concentration. Competitive binding of Pre 2 was measured by titration of mixtures of 190 nM [6F]FPR-thrombin and 20 µM Pre 2 with F1.2. The fluorescence changes as a function of total F1.2 concentration were fit by the competitive binding model.

Effect of F1.2 on Binding of Hirudin Peptides to Pre 2 and Thrombin—The effect of F1.2 on binding of [5F]Hir54–65() to Pre 2 was determined by titration of 50 nM [5F]Hir54–65() with Pre 2 in the absence and presence of 20 µM F1.2. The change in fluorescence as a function of total Pre 2 concentration was fit by the random addition ternary complex binding model to determine the dissociation constants for [5F]Hir54–65() (H) binding to free Pre 2 (P2) and the Pre 2·F1.2 (P2F1.2) complex (KP2(H) and KP2F1.2(H)), and the maximum fluorescence changes for each of the fluorescent species ({Delta}Fmax P2(H)/F0 and {Delta}Fmax P2F1.2(H)/F0) as described previously (28, 36). The dissociation constant for F1.2 binding to Pre 2 (KP2(F1.2)) was fixed at the value determined as described above. The effect of F1.2 on binding of [5F]Hir54–65() to thrombin was determined similarly and analyzed to determine the binding constants for [5F]Hir54–65() binding to free thrombin (T) and thrombin·F1.2 complex (KT(H) and KTF1.2(H)), and the maximum fluorescence changes for each of the fluorescent species ({Delta}Fmax T(H)/F0 and {Delta}Fmax TF1.2(H)/F0). The dissociation constant for F1.2 binding to thrombin (KT(F1.2)) was fixed at the determined value.

Free Energy Calculations for [5F]Hir54–65() Binding to Pro and Pre 1 Activation Intermediates—The change in free energy of association upon [5F]Hir54–65() binding to Pro and the activation intermediates was calculated from {Delta}G = RT ln KD. The change in free energy of peptide binding to each of the activation intermediates relative to Pro was calculated from: {Delta}{Delta}G = RT ln(KInt/KPro), where KInt is the dissociation constant for [5F]Hir54–65() binding to one of the intermediates and KPro is the dissociation constant for [5F]Hir54–65() binding to Pro (37, 38). Nonlinear least squares analysis was performed with SCIENTIST (MicroMath). Reported errors represent ± 2 S.D.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Fluorescence Spectral Properties of [NBD]Hir54–65() Binding to Pro Activation Intermediates— Fluorescence emission spectral studies of [NBD]Hir54–65() peptide binding were carried out to investigate further the small changes in the environment of proexosite I on Pro and the Pre 1 activation intermediates observed with the fluorescein-labeled peptide (28). With the more environmentally sensitive NBD probe, the fluorescence emission spectra for [NBD]Hir54–65() binding to Pro, Pre 1, and the Pro activation intermediates were distinctly different for each of the proteins (Fig. 1). Pro enhanced the fluorescence of the NBD-labeled peptide by 20 ± 1%, blue-shifting the maximum from 544 to 540 nm, while binding of the probe to Pre 1 produced a similar blue shift of ~4 nm, but a very small change in fluorescence at 540 nm (0.6%) compared with Pro. This indicated that removal of F1 from Pro had a significant effect on the environment of the bound fluorescent peptide. Peptide binding to Pre 2 quenched the NBD fluorescence by 19 ± 1%, whereas MzT, MzT(-F1), and thrombin displayed indistinguishable spectral properties, with maxima at 539 nm and larger quenching of the fluorescence of 44–50%. The results indicated that NBD-labeled hirudin54–65 reported differences in the environments of proexosite I on the Pro, Pre 1, and Pre 2 zymogen forms and the activated enzymes. The probe-peptide environment of exosite I on the enzymes MzT and MzT(-F1) was indistinguishable from that of thrombin.



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  		FIG. 1.
Fluorescence emission spectra of [NBD]Hir54–65() binding to Pro activation species. Fluorescence emission spectra ({Delta}F) are shown for 0.44 µM [NBD]Hir54–65() alone (black) and in the presence of 15 µM Pro (magenta, 70% saturation), 15 µM Pre 1 (light green, 97% saturation), 14 µM Pre 2 (dark blue, 94% saturation), 1 µM MzT (light blue, 97% saturation), 1 µM MzT(-F1) (dark green, 98% saturation), and 1 µM thrombin (red, 97% saturation). Spectra were obtained as described under "Experimental Procedures."

 

Binding of [NBD]Hir54–65() to Pro, Pre 1, and Thrombin—Titrations of NBD-labeled peptide with Pro yielded a dissociation constant of 3.7 ± 0.8 µM (Fig. 2A), which was in excellent agreement with the previously determined value of 3.2 ± 0.3 µM for [5F]Hir54–65() (11). Binding of [NBD]Hir54–65() to Pre 1 resulted in a 12 ± 1% quench of the fluorescence (measured at 580 nm) instead of the enhancement seen with Pro, indicating a significant effect of the removal of F1 on the change in probe environment. [NBD]Hir54–65() bound to Pre 1 with a 7-fold increased affinity in comparison to Pro (data not shown, Table I), confirming the 6-fold increase in affinity observed for [5F]Hir54–65() (28). Pre 2 bound the NBD-labeled peptide with a slightly lower (<2-fold) affinity compared with Pre 1, which was not considered significant. In contrast to the enhancement seen with Pro, thrombin quenched the fluorescence of [NBD]Hir54–65() by 50 ± 1% and bound with a ~250-fold tighter dissociation constant of 15 ± 3 nM, indistinguishable from the affinity determined for the fluorescein-labeled peptide (Fig. 2B and Table I). Comparison of the results for binding of the two peptides to Pro and thrombin showed a 250-fold increase in affinity for the NBD-labeled peptide binding to thrombin, similar to the 130-fold increased affinity for [5F]Hir54–65() seen previously (11). Comparison of Pre 2 and Pre 1 showed that, although the spectral changes were distinct, there was no correlation with a change in exosite affinity. The results suggested that removal of F1 from Pro resulted in either a conformational change in proexosite I on formation of Pre 1 or that F1 interfered directly with the binding of peptides to proexosite I on Pro.



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  		FIG. 2.
Fluorescence titrations of [NBD]Hir54–65() with Pro and thrombin. A, the fractional change in fluorescence ({Delta}F/F0) of 0.11 µM [NBD]Hir54–65() as a function of total Pro concentration ([Pro]o). B, fluorescence changes for 0.11 µM [NBD]Hir54–65() as a function of total thrombin concentration ([T]o). The lines represent the nonlinear least-squares fit of the binding equation with the parameters listed in Table I. Titrations were performed and analyzed as described under "Experimental Procedures."

 

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  		TABLE I
Parameters determined for binding of hirudin54–65 peptides to Pro, the activation intermediates, and products

The dissociation constants (KD), stoichiometric factors (sites), and maximum fluorescence changes ({Delta}Fmax/F0) determined from direct and competitive titrations, and ternary complex analysis for labeled and unlabeled hirudin54–65 peptides are listed for Pro and Pro activation species, alone and in the presence of near-saturating levels of F1.2, as indicated. Titrations were performed and analyzed as described under "Experimental Procedures."

 

Binding of Fragment 1.2 to [6F]FPR-thrombin and Pre 2— Interactions of F1.2 with exosite II on thrombin and Pre 2 were characterized in competitive binding experiments with fluorescein-labeled thrombin ([6F]FPR-thrombin). The fluorescence of [6F]FPR-thrombin was quenched by 28 ± 3% upon binding of F1.2 with a dissociation constant of 8 ± 2 µM (Fig. 3). Competitive titrations of [6F]FPR-thrombin and unlabeled Pre 2 with F1.2 gave a dissociation constant of 0.5 ± 0.3 µM for F1.2 binding to Pre 2, displaying a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin. By contrast, the affinities of F2 binding to exosite II on Pre 2 and thrombin are the same (28). The results indicated that F1 increased the affinity of F1.2 for Pre 2 by 16-fold, whereas no effect of F1 on F1.2 binding to thrombin was seen.



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  		FIG. 3.
Competitive binding of F1.2 to [6F]FPR-thrombin and Pre 2. The fractional change in fluorescence ({Delta}F/F0) of 190 nM [6F]FPR-thrombin as a function of total F1.2 concentration ([F1.2]o) is shown in the absence (•) and presence ({circ}) of 20 µM Pre 2. The lines represent the nonlinear least-squares fit of the competitive binding model with the parameters described in the text. Titrations were performed and analyzed as described under "Experimental Procedures."

 

Binding of Hirudin Peptides to Pre 2 in the Absence and Presence of F1.2—The effect of F1.2 binding on proexosite I of Pre 2 was characterized by direct titration of [5F]Hir54–65() (H) with Pre 2 (P2) in the absence and presence of 20 µM F1.2. The results were analyzed by the random addition ternary complex model (28, 36), which yielded a fitted dissociation constant for the P2·H binary complex (KP2(H)) of 0.43 ± 0.03 µM (Fig. 4 and Table I), the same as the dissociation constant determined by direct titration of Pre 2 alone (0.44 ± 0.04 µM) (28). The dissociation constant for [5F]Hir54–65() binding to P2·F1.2 complex to form the P2·F1.2·H ternary complex corresponded to a 3-fold weaker affinity (KP2F1.2(H) 1.3 ± 0.2 µM) and slightly smaller fluorescence change compared with free Pre 2 (Fig. 4 and Table I). In view of the previous results demonstrating no effect of F2 on binding of peptides to Pre 2, the decrease in affinity of the peptide for the Pre 2·F1.2 complex indicated an effect of the presence of F1 in F1.2 on peptide binding to (pro)exosite I, which approached that seen in comparison of Pro and Pre 1.



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  		FIG. 4.
[5F]Hir54–65() binding to Pre 2 in the presence and absence of F1.2. The changes in fluorescence ({Delta}F/F0) of 50 nM [5F]Hir54–65() as a function of total Pre 2 concentration ([Pre2]o) are shown in the absence (•) and presence of 20 µM F1.2 ({circ}). The lines represent the nonlinear least-squares fit of the ternary complex model with the parameters described in Table I. Titrations were performed and analyzed as described under "Experimental Procedures."

 

Binding of Hirudin Peptides to Thrombin in the Absence and Presence of F1.2—The effect of F1.2 on binding of [5F]Hir54–65() to exosite I on thrombin was investigated in titrations of thrombin (T) in the absence and presence of near-saturating F1.2 (20 µM) (Fig. 5 and Table I). The data were analyzed with the ternary complex model to obtain the dissociation constants for T·H (KT(H) 31 ± 4 nM) and T·F1.2·H (KTF1.2(H) 38 ± 9 nM), which were indistinguishable from the previous results for [5F]Hir54–65() binding (28) (Table I). The results demonstrated that binding of F1.2 to exosite II on thrombin did not affect binding of labeled hirudin peptides to exosite I, similar to results seen with F2 alone (28, 36). The presence of F1 in F1.2 lowered exosite I affinity in the zymogen forms, Pro and the Pre 2·F1.2 complex, but not in the active species, MzT and T·F1.2 complex.



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  		FIG. 5.
Binding of [5F]Hir54–65() to thrombin in the absence and presence of fragment 1.2. The fractional change in fluorescence ({Delta}F/F0) of 50 nM [5F]Hir54–65() plotted as a function of total thrombin concentration ([T]o) in the absence (•) and presence ({circ}) of 18.5 µM F1.2. The lines represent the nonlinear least-squares fit of the ternary complex model with the parameters described in the text. Titrations were performed and analyzed as described under "Experimental Procedures."

 

Binding of Hirudin Peptides to MzT(-F1) and MzT—Exosite I on MzT(-F1) had a similar affinity for [NBD]Hir54–65() in comparison to [5F]Hir54–65() (data not shown, Table I), and the dissociation constant for the NBD-labeled peptide was indistinguishable from that for thrombin. Similarly, MzT quenched the fluorescence of [5F]Hir54–65() by 32.2 ± 0.3% and bound with a dissociation constant of 22 ± 2 nM (Fig. 6A and Table I). Competitive titrations of [5F]Hir54–65() and unlabeled Hir54–65() with MzT displayed dissociation constants for the unlabeled and labeled peptides that were indistinguishable (Fig. 6B and Table I). [NBD]Hir54–65() bound to MzT with an indistinguishable dissociation constant (data not shown, Table I). These results showed that cleavage of Pro at Arg320-Ile321 altered the conformation of exosite I such that binding of the hirudin peptides was increased by 50- to 250-fold. The results confirmed that peptide bond cleavage in the proteinase domain of Pro resulted in simultaneous activation of exosite I and the catalytic site.



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  		FIG. 6.
Direct and competitive titrations of [5F]Hir54–65() and Hir54–65() binding to MzT. A, the fractional change in fluorescence ({Delta}F/F0) of 50 nM (•) and 250 nM ({circ}) [5F]Hir54–65() plotted as a function of total MzT ([MzT]o) concentration. B, fluorescence titrations of mixtures of 50 nM [5F]Hir54–65() and 50 nM ({circ}) or 250 nM (•) MzT as a function of total, unlabeled, hirudin peptide concentration ([Hir54–65()]o). The lines represent the nonlinear least-squares fit of the quadratic (A) or competitive (B) binding models with the parameters listed in Table I. Titrations were performed and analyzed as described under "Experimental Procedures."

 

Pathway of Exosite I Expression—Changes in free energy of [5F]Hir54–65() association with Pro and the Pro activation intermediates were calculated for each of the species (Table II). Fig. 7 maps the changes in free energy of exosite I binding for the Pre 1 and Pro activation species determined in this study and the companion manuscript (28). The free energy change for [5F]Hir54–65() binding to Pro was –7.5 kcal/mol (Table II). Removal of F1 from Pro to form Pre 1 increased the free energy change of binding by ~1.1 kcal/mol. Peptide binding to Pre 2 in the presence and absence of F2 displayed a free energy change of –8.7 kcal/mol independent of F2, indicating similar effects of F1 and F1 plus F2 removal on the free energy of peptide binding due to F1. A decrease in the free energy change of association was observed for peptide binding to Pre 2 in the presence of F1.2 of ~0.7 kcal/mol, reflecting the decrease in affinity due to the presence of F1. Proteolytic cleavage of Arg320-Ile321, activating the proteinase domain and yielding the active species, MzT, MzT(-F1), and thrombin, increased the binding free energy change by ~3 kcal/mol. These results indicated that F1 had a similar modulating effect on proexosite I of Pro and the Pre 2·F1.2 complex. This effect was specific for the zymogen forms and was alleviated upon the conformational change that activates the catalytic site and exosite I.


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  		TABLE II
Free energy changes for [5F]Hir54–65 () binding to the Pro and prethrombin 1 activation intermediates and products

The calculated free energy changes of association ({Delta}G) for [5F]Hir54–65() binding to the Pro and prethrombin 1 activation species are listed. The differences in free energies of association ({Delta}{Delta}G) for [5F]Hir54–65() binding to the activation intermediates and products relative to Pro are also listed. Results for Pro and thrombin presented for comparison were taken from Ref. 11. Results for [5F]Hir54–65() binding to thrombin in the presence F2 were taken from Ref. 36. Calculations were performed as described under "Experimental Procedures."

 


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  		FIG. 7.
Pathway of exosite I expression during Pro and prethrombin 1 activation. The pathways of Pre 1 activation through Pre 2·F2 or MzT(-F1), to T·F2 (inner oval) and Pro activation through Pre 2·F1.2, or MzT to T·F1.2 (outer oval) are shown. Purple proteins indicate inactive species, whereas green proteins indicate active forms. [5F]Hir54–65()(Hir) binding equilibria are represented vertically, with the changes in free energy of association taken from Table II indicated for each species in kcal/mol.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The pathway of exosite I expression on human Pro activation intermediates and products and the role of F1 in exosite expression were characterized in quantitative equilibrium binding studies for the first time. A new environmentally sensitive NBD derivative of hirudin54–65 was used in fluorescence spectral studies to probe the environment of proexosite I on the Pro species. The fluorescence emission spectra of [NBD]Hir54–65() revealed differences between the environments of the probe in proexosite I on the zymogen forms, Pro, Pre 1, and Pre 2, and on the active enzymes, MzT, MzT(-F1), and thrombin. NBD-peptide binding to Pre 1 showed very little change in fluorescence compared with the enhancement seen for Pro, demonstrating a perturbation of exosite I in Pro by the F1 domain. By contrast to Pro and Pre 1, quenching of the NBD-peptide was observed for Pre 2, and larger and spectroscopically indistinguishable quenching was observed for all of the active enzyme forms. The results indicated that the microenvironment around the N terminus of the hirudin54–65 peptide was altered in distinctly different ways when each of the activation fragments was successively removed from the proteinase zymogen domain and, independently of the activation fragments on zymogen activation.

[NBD]Hir54–65() bound to Pro and Pre 1 with dissociation constants similar to those seen for the fluorescein-labeled peptide, confirming a 7-fold increase in affinity for hirudin peptides upon removal of F1. On the pathway of exosite I activation, the Pre 2 domain demonstrated no change in affinity for [NBD]Hir54–65() in comparison to Pre 1, confirming the results with [5F]Hir54–65(). Although the complexes with Pre 1 and Pre 2 were spectroscopically different, there was no significant difference in affinity for the peptides. MzT, formed by cleavage of Pro at Arg320-Ile321, displayed a 50- to 150-fold increase in affinity for the fluorescein-labeled and unlabeled peptides in comparison to Pro, similar to the 250-fold increase in affinity seen for the NBD-labeled peptide. These results are consistent with the previous finding that cleavage at Arg320-Ile321 in Pre 1 to activate the catalytic site is accompanied by full activation of proexosite I (28). The results contrast the observations of Liu and colleagues (39) that cleavage of either activation peptide bond results in formation of exosite I. Characterization of exosite I expression in those studies was performed with bovine Pro species and with a different peptide, hirudin53–64. A ~5-fold lower affinity of hirudin54–65 peptides for bovine Pro and thrombin (11) may also translate to differences in the activation intermediates.

Overall, activation of proexosite I affinity for hirudin peptides as model ligands follows a discrete pathway where exosite I and the catalytic site are activated simultaneously. As summarized in Fig. 7, the free energy of peptide binding to each active proteinase species, MzT, MzT(-F1), and thrombin in the presence and absence of either F1.2 or F2 was the same, consistent with the indistinguishable spectroscopic properties of the [NBD]Hir54–65() bound species and a similar conformation of exosite I. The results demonstrate that the presence of the F1 domain decreases the affinity of hirudin54–65 peptides for Pro and Pre 2 in a complex with F1.2. The affinity of the hirudin peptides for Pre 2 was decreased 3-fold by F1.2 binding, in comparison to Pre 2 alone, toward a value that was within ~2.5-fold of the affinity for Pro. This represented similar 0.7- to 1.1-kcal/mol decreases in binding free energy attributable to the presence of F1. The effect of the F1 domain was alleviated upon proteolytic cleavage of Pro at Arg320-Ile321. The results indicated that full expression of exosite I accompanied the conformational change of the proteinase activation domain (29) that activates the catalytic site and eliminated the modulating effect of the F1 domain on (pro)exosite I binding of hirudin54–65. The finding of Wu and colleagues (17) that removal of F1 from MzT to form MzT(-F1) resulted in activation of exosite I toward macromolecular ligands was not observed here with hirudin peptides, which had the same affinity for MzT and MzT(-F1).

An effect of F1 on exosite II binding of F1.2 was demonstrated here for the first time. F1.2 bound to thrombin with the same affinity as F2, whereas Pre 2 displayed a 16-fold increased affinity, demonstrating that F1 aids in the binding of F1.2 to Pre 2. The increased affinity of F1.2 for Pre 2 by contrast to thrombin indicates that the cleavage of Arg320-Ile321 that causes the activating conformational change is also linked to reduced affinity of F1.2 for exosite II. This decrease in affinity on formation of thrombin would be expected to facilitate release of thrombin from the prothrombinase product complex. The more favorable interaction of F1.2 with the catalytic domain only in the zymogen forms is correlated with the effect of F1 to reduce proexosite I affinity. The combined results indicate that F1 either interacts directly with the Pre 2 domain in Pro or induces a conformational change affecting the affinity of F1.2 for exosite II and exosite I affinity for hirudin54–65. Other studies support the possibility that F1 is in proximity to the Pre 2 domain and may interact directly to enhance affinity and reduce access to exosite I. In the absence of calcium, recombinant MzT cleaves the Gla domain at Arg54-Asp55 and in the presence of calcium native MzT cleaves Arg155-Ser156 to release F1 in intramolecular reactions (40, 41). Analysis of the x-ray crystal structure of bovine MzT(-F1) suggests that the Arg54-Asp55 bond in F1 may be within 15 Å of the catalytic site, in a compact structure for Pro in which the F1 and Pre 2 domains are in close proximity (42).

Conformationally distinct exosite I environments on zymogen and active forms of Pro may have importance in Pro substrate recognition by the factor Xa·factor Va complex. Previous studies of Pro domain-deletion mutants and effects of activation fragments on Pro activation have provided evidence that the Gla domain and kringle 1 of F1, and F2 may all participate in Pro-factor Va interactions (23–25). In the absence of membranes, F2 and F1.2 accelerate Pre 2 activation substantially in a factor Va-dependent manner (18, 19), supporting the early hypothesis that F2 mediates Pro-factor Va binding (18). A detailed quantitative analysis of Pre 2 activation by the lipid-assembled factor Xa·factor Va complex, however, demonstrated that acceleration of Pre 2 activation by factor Va is largely independent of F2 (19). These studies concluded that factor Va-dependent substrate recognition exosites that mediate productive binding of Pre 2 are expressed on factor Xa within the factor Xa·factor Va complex. In this model, exosite binding of substrate results in formation of an initial encounter complex with the factor Xa·factor Va complex in which the factor Xa active site is accessible, followed by isomerization of the complex to engage the catalytic site and permit bond cleavage (20–22, 43). The role of factor Va-substrate interactions in the binding and conformational changes of this model is not clearly understood. Our studies support an important role for sites on the Pre 2 domain in factor Va-mediated substrate recognition. Pro and thrombin both bind to the heavy chain subunit of factor Va, and the thrombin interaction is mediated by exosite I (26, 27, 44–46). The demonstrated dependence of factor Va-rate acceleration of Pro activation on the low affinity, proexosite I on Pro is postulated to reflect its mediation of productive binding of Pro substrate species to factor Va within the factor Xa·factor Va complex (13, 14). This interpretation is supported by recent mutagenesis studies demonstrating critical roles of residues in proexosite I on factor Va-dependent Pre 1 substrate recognition (15). On the basis of the available information, substrate recognition exosites expressed on factor Xa upon binding to factor Va and proexosite I-mediated factor Va-Pro interactions both likely play important roles in the recognition mechanism.

The involvement of proexosite I in factor Va interactions suggests that modulation of proexosite I affinity by F1 and full activation of exosite I affect interactions of the Pro activation species with factor Va. Proexosite I interactions may contribute to factor Va regulation of the activation pathway through the differential expression of exosite I on the alternative reaction intermediates. Because the effect of the F1 domain to decrease exosite affinity on the zymogen forms, Pro and Pre 2·F1.2, is also lost on cleavage at Arg320-Ile321, this domain may also alter factor Va-substrate interactions. The interconnectedness of Pro domains and proexosite I expression shown here suggests that domain deletion mutants of Pro are unlikely to have simple additive functional properties. Thus, whether the effects of deletions of the kringles in the F1 and F2 domains truly reflect direct involvement of these domains in factor Va binding or indirect effects due to disruption of domain-domain interactions and changes in exosite affinity remains unclear (24).

Kinetic studies of the four partial reactions of Pro activation have shown that factor Va redirects the activation pathway from one in which Pre 2·F1.2 predominates to one in which MzT is the major intermediate (5–7). This can be explained by differential effects of factor Va on cleavage of Arg320-Ile321 and Arg271-Thr272, where initial cleavage at Arg320 is greatly accelerated by factor Va compared with Arg271 (6, 7, 47). The result is preferential acceleration by factor Va of MzT formation from Pro and thrombin·F1.2 formation from Pre 2·F1.2. As shown here, the cleavage reaction most stimulated by factor Va is also that in which the catalytic site and exosite I are activated, and the modulating effect of F1 is lost. Factor Va has much less effect on cleavage of Arg271 in either Pro conversion to Pre 2·F1.2 or MzT to thrombin·F1.2, for which there is little or no associated change in exosite affinity. The results suggest that expression of proexosite I may regulate the preferred order of bond cleavage by altering factor Va-substrate interactions. On this basis, we speculate that proexosite I-mediated binding of Pro or Pre 2·F1.2 to factor Va may direct cleavage at Arg320, whereas the expression of increased affinity of exosite I on MzT may reorient the factor Va-bound substrate to favor cleavage at Arg271.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL38779 (to P. E. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by a postdoctoral fellowship from the American Heart Association, Southeastern Consortium (SE-9820133V) and subsequently by an Institutional National Research Service Award (DK07186). Present address: Howard Hughes Medical Institute, Washington University School of Medicine, 660 S. Euclid, Box 8022, St. Louis, MO 63110. Back

§ To whom correspondence should be addressed: Dept. of Pathology, Vanderbilt University School of Medicine, C3321A Medical Center North, Nashville, TN 37232-2561. Tel.: 615-343-9863; Fax: 615-343-7023; E-mail: paul.bock{at}vanderbilt.edu.

1 The abbreviations used are: Pre 1, prethrombin 1; Pre 2, prethrombin 2; Pro, prothrombin; F1, fragment 1; F2, fragment 2; F1.2, fragment 1.2; Gla, {gamma}-carboxyglutamic acid; MzT(-F1), meizothrombin des-fragment 1; MzT, meizothrombin; T, thrombin; FPR-CH2Cl, D-Phe-Pro-Arg-CH2Cl; Hir54–65(), Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr()-Leu-Gln; [5F]Hir54–65(), Hir54–65() labeled at the amino terminus with 5-carboxy(fluorescein); [NBD]Hir54–65(), Hir54–65() labeled at the amino terminus with succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate; Mes, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Jennifer L. Ray, Lori Ray-Cox, and Sarah Tomlinson for excellent technical assistance and Margaret Krakowiak for graphic design of Fig. 7.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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