definition of a factor va binding site in factor xa*

Definition of a Factor Va Binding Site in Factor Xa*

Amy E. Rudolph1, Rhonda Porche-Sorbet, and Joseph P. Miletich2

From the Departments of Pathology and Medicine, Division of Laboratory Medicine, Washington

University School of Medicine, St. Louis, Missouri 63110

* This work was supported in part by grants from the National Institutes of Health Grant (NHLBI

HL14147) and the Monsanto/Searle Company

1 To whom correspondence and requests for reprints should be addressed. Present address: Pharmacia

Corp., 800 North Lindbergh Boulevard, St. Louis, Missouri 63167. Tel: 314-694-9017; Fax: 314-694-

8153;

e-mail:[email protected]

2Present Address: Merck & Co., Inc., West Point, PA 19486

1The abbreviations used are: TF, human tissue factor; HEPES, (N-[2-Hydroxyethyl]piperazine-N’-[2-

ethanesulfonic acid]); PEG, polyethylene glycol 8000; BSA, bovine serum albumin; Tris,

(Tris[hydroxymethyl] aminomethane); EDTA, (Ethylenedinitrilo)- tetraacetic acid; SDS-PAGE, sodium

dodecyl sulfate polyacrylamide gel electrophoresis; RVV-X, X activating protein from Russell’s viper

venom

Running title: A Factor Va Binding Site in Factor Xa

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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ABSTRACT

We previously reported that residue 347 in activated fX (fXa) contributes to binding of the

cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39,

2861-2867). Four additional residues that participate in fVa binding have now been identified by

mutagenesis. All five resulting fX species, fXR306A, fXE310N, fXR347N, fXK351A, and fXK414A,

are activated and inhibited normally. However, the rate of inhibition by ATIII in the presence of

submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the

enzymes bind and activate prothrombin similarly except fXaE310N, which has a reduced apparent

affinity (~3-fold) for prothrombin compared to wild type fXa(fXaWT). In the absence of phospholipid,

fVa enhances the catalytic activity of fXaWT significantly, but the response of the variant enzymes was

greatly diminished. On addition of 100nm PC:PS(3:1) vesicles, fVa enhanced fXaWT, fXaR306A, and

fXaE310N similarly, whereas fXaR347N, fXaK351A, and fXaK414A demonstrated near normal catalytic

activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to

fXaWT on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity

for fVa as a result of the substitutions in residues Arg347, Lys351, and Lys414 was verified by a

competition binding assay. Thus, Arg347, Lys351, and Lys414 are likely part of a core fVa binding site,

whereas Arg306 and Glu310 serve a less critical role.

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INTRODUCTION

The vitamin K-dependent plasma serine protease, activated factor X (fXa) serves as the

physiological activator of prothrombin. The zymogen precursor, fX, is activated to fXa by complexes in

the intrinsic (factor IXa/factor VIIIa) and extrinsic (factor VIIa/TF1) coagulation pathways and by RVV-

X in the presence of calcium (1-6). Once activated, fXa is inhibited by antithrombin III (ATIII) and

tissue factor pathway inhibitor (TFPI, 7-10). The light chain of fX contains 11 γ-carboxylated glutamic

acid residues that compose the gla domain and two epidermal growth factor-like domains (EGF-1 and

EGF-2). The activation peptide and the serine protease domain form the heavy chain of fX which is

bound to the light chain via a single disulfide bond.

Factor Xa interacts synergistically with cofactor (fVa), substrate (prothrombin), and a

phospholipid surface to form the prothrombinase complex which supports maximally efficient

prothrombin activation (11). Several of these interactions were independently evaluated for a variant

recombinant fX, fXaR347N (residue 165 in chymotrypsin numbering) and it was found that substitution

of Arg347 selectively reduces fVa affinity (12). The current study describes further delineation of the fVa

binding site of fXa through targeted mutagenesis of additional residues in the surface epitope that includes

Arg347.

Arg306, Lys351, and Lys414 (125, 169, and 230 in chymotrypsin numbering, respectively) have

been substituted by alanine. Glutamate310 (129 in chymotrypsin numbering) was substituted by

asparagine. The fVa affinity of the resulting enzymes was probed using functional and binding studies. It

was determined that Arg347, Lys351, and Lys414 compose a core cofactor binding epitope and Glu310

and Arg306 form an extended region of the epitope.

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EXPERIMENTAL PROCEDURES

Reagents and ChemicalsCrude snake venoms, L-α-phosphatidylcholine, and L-α-

phosphatidylserine were purchased from Sigma. Spectrozyme FXa and Spectrozyme TH" were

purchased from American Diagnostica Inc. (Greenwich, CT). Full-length heparin was obtained from

Elkins-Sinn Inc. (Cherry Hill, NJ). Heparin pentasaccharide was a generous gift of J.C. Lormeau of

Sanofi Recherché (Gentilly Cedex, France). 100 nm phosphatidylcholine:phosphatidylserine (PC:PS,

3:1) vesicles were prepared as described (12-14).

ProteinsProthrombin (13-14) and fVII (15) were purified from human plasma as described.

Prothrombin was immunodepleted of residual fX (<1 nM) using a anti-fX monoclonal antibody. ATIII

was purchased from Kabi Pharmacia Diagnostics (Piscataway, NJ) and recombinant tissue factor pathway

inhibitor was kindly provided by Monsanto/Searle Company (St. Louis, MO). Human fVa and fIXa were

purchased from Haematologic Technologies (Essex Jct., VT). Thrombin was prepared as described (13-

14). Porcine fVIII was purchased from Porton Products (Agoura Hill, CA) and purified as described (13-

14). Recombinant, lipidated tissue factor, Innovin, was purchased from Baxter Diagnostics Inc.

(Deerfield, IL). RVV-X was purified from Russell’s viper venom (16). SDS-PAGE was performed by

the method of Laemmli (17). All recombinant fX species were purified as described (14).

ImmunoglobulinsAnti-fX murine monoclonal antibodies utilized in the study were developed

by standard methods. 3698.1A8.10 reacts with the gla domain in the presence of calcium, 3514.5H12.10

reacts with the gla domain independent of calcium, and 3448.1D7.20 binds to EGF-2. Activation of fX

was monitored by a two-site immunofluorescent assay utilizing 3448.1D7.20 and 3514.5H12.10 as

described (13). Murine anti-fX monoclonal antibody directed against the heavy chain was purchased

from Enzyme Research Laboratories, Inc. (South Bend, IN). For immunoblotting, an antibody cocktail of

3448.1D7.20, 3514.5H12.10, and the anti-fX heavy chain monoclonal were utilized.

Mutant construction and cell cultureThe fXWT cDNA was cloned and modified as previously

described (13-14). Sequence changes for the variants include: fXR306A,

(CCGAGCGTG→CCGAAGCTG); fXE310N, (GCCGAGTCC→

GCAAATTCC); fXR347N, (GACCGCAAC→GACAACAAC); fXK351A, (TGCAA-

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GCTG→TGTGCACTG); fXK414A, (ACCAAGGTC→ACCGCGGTC); All sequence changes and the entire fXWT

sequence were verified by sequence analysis. Mutant and fXWT sequences were shuttled into the

expression vector (ZMB3) kindly provided by Dr. Don Foster. All constructs were transfected into

human kidney cells (293) using calcium phosphate precipitation (18). 293 cells were cultured as

described (13-14). Isolated colonies were screened for fX production by immunoassay and expanded.

ActivationThe assay buffer used was 10 mM HEPES, pH 7.0, 100 mM NaCl, 5 mM CaCl2, 1

mg/ml BSA, 1 mg/ml PEG 8000. All variant zymogens were activated as described previously for

fXR347N (12). Briefly, variants or fXWT (100nM) were activated with 2nM RVV-X for endpoint activation

or 50 pM RVV-X for initial rate measurements. Each fX was activated by fVIIa (40 pM) in the presence

of 0.5 nM lipidated tissue factor, and by fIXa (2 nM) in the presence of fVIIIa (4 U/ml) on PC:PS vesicles

(20 µM). All concentrations were subsaturating for the activators and initial rates of activation (< 10% of

substrate utilized) were determined. Reactions were monitored over time by quenching aliquots of the

reaction in EDTA buffer (10 mM HEPES pH 7.0, 100 mM NaCl, 5 mM EDTA, 1 mg/ml BSA, 1 mg/ml

PEG8000) and measuring the rate of hydrolysis of Spectrozyme FXa (100 µM).

InhibitionInhibition of all variants was measured using established methodologies (12-13, 19-

20). All zymogens (100 nM) were activated by RVV-X (2 nM). Each fXa (0.5 nM) was incubated with

Spectrozyme FXa (100 µM) in the presence of inhibitor: ATIII alone (0-4 µM), ATIII (0-50 nM) in

the presence of heparin pentasaccharide (0.5 µM), ATIII (0-16 nM) in the presence of 2.5mU/ml full-

length heparin, or TFPI (0-50 nM). Concentrations of heparin pentasaccharide and full length heparin

were emperically determined to support half-maximal acceleration of inhibition. Residual fXa activity

was quantitated from Spectrozyme FXa hydrolysis. First and second order rate constants were derived

as described (12).

Thrombin formationAssays were performed in assay buffer as described (12). Kinetic values

were calculated from the least squares fit of the data to the equation:

v=Vmax [Z]/(Km+[Z])

where v= the observed initial rate of thrombin formation, Vmax= the maximal initial rate of thrombin

formation, [Z]= the concentration of the component being varied in the experiment, and Km= the

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apparent concentration of the variable component required to reach half maximal thrombin formation

under the conditions specified. Data are expressed as mol thrombin (IIa)/sec/mol Xa. Samples were

removed from each reaction at various times and quenched into EDTA buffer. Thrombin formed was

quantitated by incubating quenched samples with 500 µM Spectrozyme TH, a thrombin chromogenic

substrate. The conditions for the experiments have been described in detail (12). In summary, 10 nM of

each fXa was incubated with prothrombin alone (0-450 µM). In the absence of phospholipid, varying

concentrations of fVa (0-250 nM) were incubated with 10 µM prothrombin and fXa (0.5 nM). In the

presence of PC:PS vesicles (3:1, 20 µM), 20 pM fXa was incubated with fVa (0-1 nM) and the reaction

was initiated with 1 µM prothrombin. Thrombin-activated platelets (108/ml) were incubated with fXa

(0-1 nM) and thrombin formation was initiated by the addition of 1 µM prothrombin.

Competition binding assayReagents for the latex bead-based binding assay were prepared as

described (12). Briefly, fXS379A was labeled with 125I using Bolton-Hunter reagent, Amersham

(Arlington Heights, IL). Radioactivity not incorporated into protein was removed using a Bio-Spin 6

column, Bio-Rad Laboratories (Hercules, CA). The specific activity of the labeled protein was typically

2000 cpm/ng. Labeled FXS379A was activated using RVV-X as described above.

1.0 µm latex beads (Interfacial Dynamics Corp., Portland, OR) were coated with PC:PS (3:1)

according to published methods (12). Beads coated with PC only were also prepared and used in pilot

experiments to demonstrate specificity; i.e., no fVa-dependent binding without PS. Non-specific binding

accounted for <10% of binding, and approximately 50% of 125I-fXaS379A binding was prevented by

the addition of 1 nM unlabeled fXaWT.

The concentrations of all assay components were empirically determined as described (12). In

the assay, 1 nM fVa was incubated with 0.2% (v/v) PC:PS beads and 0.8% (v/v) uncoated beads for 10

min. 125I-fXaS379A (final concentration 1.0 nM) was mixed with various concentrations of either wild

type or mutant fXa, added to the fVa/bead mixture, and incubated with mixing for an additional 10 min.

125I-fXaS379A was utilized as the labeled fXa species for greater stability over time; i.e. to minimize

fXa-dependent proteolysis. The beads were collected by centrifugation and the pellets and supernatants

were counted separately. Non-specific binding was determined from reactions not containing fVa and

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subtracted from counts bound in the pellets. The percentage of bound fXa was quantitated relative to the

amount bound in reactions with no unlabeled fXa. The apparent affinity (Kdapp) represents the

concentration of unlabeled fXa required to displace 50% of 125I-fXaS379A from the fVa-bound beads

and is calculated from the following equation:

y=100%/1+(x/Kdapp)s

In the equation, y=% bound 125I-fXaS379A, x=concentration of added, unlabeled fXa, and s=a slope

factor.

fXa model of the fVa binding siteThe model of fXa was generated from the published

coordinates (21) using RIBBONS v3.0, developed by M. Carson at the University of Alabama

(Birmingham, AL). In the structure, the Gla domain is missing and EGF1 is disordered.

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RESULTS

Mutagenesis We previously demonstrated that the employed expression system is efficacious for

the production of fully functional recombinant wild-type and variant fX (12-14). Construction of the

variant molecules in the current study was based on two mutational strategies. Residues Arg306, Lys351,

and Lys414 were substituted with alanine whereas Glu310 and Arg347 were substituted with asparagine,

resulting in the creation of a potential N-linked glycosylation site, N X (C/S/T), (Table I). The apparent

molecular weight of fXE310N was elevated, indicating the addition of a carbohydrate group at this

residue (Fig. 1). However, as previously described, asparagine substitution of Arg347 did not result in an

added carbohydrate, as the electrophoretic mobility of fXR347N was not altered as compared to fXWT

(Fig 1, ref 12).

Activation All variants hydrolyzed a peptide substrate, Spectrozyme FXa, normally (DATA

NOT SHOWN). Initial rates of activation by RVV-X and the extrinsic (fVIIa/TF) and intrinsic

(fIXa/fVIIIa) activation complexes were quantitated by monitoring Spectrozyme FXa hydrolysis.

Activation of all variant zymogens by all activators was very similar to the activation of fXWT.

Inhibition The impact of the substitutions on the active site of fXa was probed by studying the

interaction between the enzymes and the physiologic inhibitors, ATIII and TFPI. All variant enzymes

were inhibited normally by these inhibitors as compared to fXaWT and second order rate constants for

inhibition of fXaWT were consistent with reported values (Table II, 22). Therefore, the active site of fXa

was not compromised by these mutations. Inhibition of each enzyme by ATIII was also examined in the

presence of heparin pentasaccharide and full-length heparin. The second order rate constants for ATIII

inhibition in the presence of full-length heparin were reduced for all variants as compared fXaWT (Table

II). Rate constants for inhibition of fXaR306 and fXaE310N were modestly reduced (66% and 64% of

fXaWT, respectively), whereas those for fXaR347N (12.6% of fXaWT, 12), fXaK351A (20.1%), and

fXaK414A (21.1%) were markedly reduced. In contrast to these effects with full-length heparin, inhibition of

all variants in the presence of the pentasaccharide was equivalent to that of fXaWT (Table II). Thus, the

basic residues at position 347, 351, and 414 likely contribute to the heparin binding site of fXa.

Prothrombin activation in the absence of fVa and phospholipid The catalytic function of the

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enzymes was evaluated using the physiological substrate, prothrombin (Fig. 2). Multiple concentrations

of prothrombin were incubated with the mutant enzymes and the apparent affinity for prothrombin and

catalytic turnover were compared to that of fXaWT. In the absence of cofactor and phospholipid, all

variant enzymes, with the exception of fXaE310N, interact similarly with prothrombin as indicated by

similar Kmapp values (range Kmapp=88.4 to 96.4 µM). fXaE310N demonstrated a 3-fold reduction in

the apparent affinity for prothrombin (fXaWT Kmapp=91.9 µM versus fXaE310N Kmapp=299 µM).

The variation in the range of maximum prothrombin turnover rates for all enzymes is less than 2-fold

(0.026 to 0.047 sec-1) indicating that all variant enzymes have similar catalytic activity compared to

fXaWT.

Prothrombin activation in the presence of fVa ± phospholipid To examine the interaction between

the variant enzymes and cofactor, thrombin formation was first quantitated in the presence of fVa, but in

the absence of phospholipid. All enzymes demonstrated markedly reduced function as compared to

fXaWT (Fig. 3). fVa enhanced the catalytic function of fXaWT e.g., at 10µM prothrombin, fXaWT was

catalytically more than 1000-fold faster in the presence of 250 nM fVa. The catalytic activity of the

variants was also enhanced by the addition of cofactor, although not nearly to the same extent. In an

effort to increase the concentration of cofactor in the presence of the enzymes, prothrombin turnover was

evaluated on 100nm PC:PS (3:1) vesicles which bind fXa and fVa and co-localize the enzyme with

cofactor. Under these conditions, all variant enzymes displayed measurable function (Fig. 4). Here, the

apparent fVa affinity for fXaR306A and fXaE310N were only slightly reduced relative to fXaWT

(fXaWT Kdapp=106 pM, fXaR306A=203 pM, and fXaE310N=157 pM), whereas fXaK351A and fXaK414A

demonstrated a greater reduction in cofactor affinity (fXaK351A Kdapp=366 pM, and fXaK414A=556

pM). As previously reported, the relative fVa affinity of fXaR347N was markedly reduced under these

conditions (fXaR347N Kdapp=2299 pM). Maximal turnover of prothrombin by all variant enzymes was

found to be within 2-fold that of fXaWT (Range 18.9 to 28.9 sec-1) in the presence of cofactor and

phospholipid vesicles. These data are most easily explained by the hypothesis that the major effect of

substitution of Arg347, Lys351, or Lys414 is the reduction in fVa affinity in the presence of PC:PS

vesicles with little or no impact on catalytic activity of the fVa-fXa complex.

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If the mutations only impact cofactor affinity, the full catalytic potential should be realized under

ideal conditions. This hypothesis was evaluated using activated platelets which provide an ideal

phospholipid surface and saturating fVa. Indeed, under these conditions, all fXa species are very similar

to fXaWT (Fig. 5).

Competition binding assay In order to confirm the results of the functional studies and more

directly probe the role of these residues in fVa binding, we employed a previously described binding

assay (12). In the assay, active-site modified, radiolabeled fXa (125I-fXaS379A) was mixed with

varying concentrations of unlabeled, mutant enzyme. Both enzymes were then allowed to compete for

fVa binding on a PC:PS-coated latex bead. Factor Va binding is expressed for the variant enzymes in

figure 6 as the percentage of 125I-fXaS379A bound to the bead in the presence of increasing

concentrations of added, unlabeled fXa. fXaR306A and fXaE310N demonstrated modest reductions in

fVa affinity in the assay (fXaWT Kdapp=1.83 nM, fXaR306A=2.96 nM, fXaE310N=4.03nM), whereas

the relative fVa affinity of fXaK351A, fXaK414A, and fXaR347N was markedly reduced (Kdapp=8.81,

12.17, and 19.45 nM, respectively). Consistent with the functional studies on phospholipid vesicles,

substitution of residues Lys351, Lys414, and Arg347 resulted in a selective loss of fVa affinity in the

absence of prothrombin, whereas mutation of Arg306 and Glu310 reduced fVa binding to a much lower

extent.

fVa binding site of fXa The juxtaposition of the targeted residues is apparent from the fXa

structure (Fig. 7, 21). Residues found to contribute to the fVa binding site are clustered in a defined

region on the fXa surface in the serine protease domain. The impact of surrounding residues (Arg273,

Ile357, Arg406, Lys406, and Lys420) on fVa affinity was evaluated using the fVa-sensitive functional assays

described above i.e. in the presence and absence of PC:PS vesicles. Substitution of these residues had no

impact on fVa affinity in these assays (DATA NOT SHOWN), thereby delineating the fVa binding

epitope.

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DISCUSSION

The current study describes substitutions in the surface epitope of fX that includes Arg306,

Glu310, Arg347, Lys351, and Lys414. All mutants were synthesized and secreted in the expression system in

a manner equivalent to that of fXWT. Activation of these zymogens by fVIIa/TF, fIXa/fVIIIa, and

RVV-X, hydrolysis of a small, peptide substrate, and inhibition by TFPI and ATIII all occurred at a

near-normal rates. Based on these studies, substitution of these residues does not compromise the active

site structure of fXa.

Heparin can accelerate the rate of fXa inhibition by ATIII by mediating conformational changes

in the inhibitor and by acting as a template that binds both inhibitor and enzyme (23). The individual

contributions of structural alterations in ATIII and binding to fXa were evaluated using heparin

pentasaccharide which binds ATIII, but does not act as a template. All variant enzymes were inhibited

normally by ATIII in the presence of heparin pentasaccharide. However, the rate of inhibition by ATIII

in the presence of full-length heparin was reduced for all variants, indicating that these residues

contribute to the heparin binding capacity of fXa. Substitution of the glutamic acid at position 310 results

in the addition of a carbohydrate group which could sterically hinder the binding of heparin to the fXa

surface. Substitution of the basic residues at positions 347, 351, 414, and 306 likely attenuates ionic

interactions between heparin and fXa. These data are corroborated by structural analysis of thrombin and

fXa. Lys351 and Lys414 are adjacent to the analogous epitope defined in thrombin as the heparin binding

exosite (24). Moreover, Padmanabhan and coworkers have described the heparin binding domain of fX

as a large basic region which either includes or is adjacent to these residues (25). Delineation of the entire

heparin binding domain of fXa will require additional mutagenesis.

All variant enzymes bound and activated prothrombin in a manner similar to fXaWT with one

exception, fXaE310N. The apparent affinity of fXaE310N for prothrombin was 3-fold lower than

fXaWT. It is conceivable that the added carbohydrate sterically hinders substrate binding. Alternatively, the

added sugar may restrict conformational mobility of fXa and prevent the enzyme from adapting a

favorable conformation for substrate binding in the absence of a lipid surface.

This study examined the independent interactions between fXa and other prothrombinase

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complex constituents. It was found that the sensitivity to aberrant fVa binding is heightened under sub-

optimal conditions for prothrombin turnover i.e. in the presence of fVa ± PC:PS vesicles allowing clear

delineation of the residues that compose the fVa binding site. As expected, substitutions of residues that

comprise the core binding site result in a more severe phenotype as compared with mutations in the

residues that form the extended regions of the site. The power of this methodology is exemplified by

fXaR306A and fXaE310N which displayed a reduced function in the absence of phospholipid. On addition of

PC:PS vesicles, which increase the local concentration of prothrombinase complex components and

maximize productive interactions, these enzymes bind fVa with a similar apparent affinity to fXaWT. In

contrast, the phenotype of fXaR347N, fXaK351A, and fXaK414A was overcome under the ideal

conditions of activated platelets suggesting a more critical role of these residues in fVa binding.

Therefore, Arg306 and Glu310 may form part of the binding site that extends beyond the core epitope

whereas Arg347, Lys351, and Lys414 likely contribute to the core of the binding epitope.

It has been proposed that fVa mediates conformational changes in fXa that contribute to the

cofactor-driven enhancement of prothrombin activation (26-29). In the absence of a phospholipid

surface, cofactor enhanced the activity of fXaWT, but had little effect on the variant enzymes. There are

at least two possible explanations for these data. Either the mutations compromised the functional

capacity of fXa by attenuating these proposed cofactor-mediated structural rearrangements, or fVa

binding is impaired by the mutations. If this structural rearrangements are attenuated by the substitutions,

the aberrant phenotype would not likely be overcome even under optimal conditions for substrate

turnover. It was demonstrated that mutant enzymes with less severe phenotypes (fXaE310N and

fXaR306) can function nearly normally on a PC:PS vesicle surface in the presence of fVa and the function of all

mutant enzymes was similar to the wild-type enzyme under the conditions provided by activated

platelets. Moreover, the competition binding assay, which directly probes cofactor binding, demonstrated

a more marked loss of fVa affinity for variant enzymes with more severe phenotypes in the functional

assays. The data do not support the postulate that substitution of these residues prevented fVa-mediated

changes in the fXa structure, but rather that the fVa affinity is selectively attenuated by the mutations.

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Analysis of the tertiary structure of fXa provides compelling structural support for the existence

of a fVa binding epitope in the serine protease domain of fXa. Using peptide mapping studies,

Chattopadhyay et al. identified three regions of fXa that were found to contribute to fVa binding: 211-

222, 254-269, and 263-274 (30). Each of these regions was evaluated by mutagenesis using functional

assays and found not to contribute to fVa binding (DATA NOT SHOWN). The discrepancy between the

studies may reflect differences in methodologies. The region identified by the current study has also been

described as a cofactor binding site for other coagulation enzymes. For example, the analogous surface

loop of fIXa, which corresponds to residues 344-352 in fXa, was recently identified to form a binding

epitope for fVIIIa (31-32. Moreover, Banner et al. identified multiple regions of the fVIIa surface that

contribute to TF binding including one region analogous to fX residues 346-349 (33).

Targeted mutagenesis of the residues that surround the proposed cofactor binding epitope was

also conducted as part of this study. It was found that residues Arg273, Ile357, Arg406, Lys406, and

Lys420 do not contribute to fVa binding, but rather outline the fVa binding epitope. Based on this survey of

adjacent epitopes, structural examination, and comparisons to other cofactor binding epitopes, it is likely

that Arg347, Lys351, and Lys414 form the core of a fVa binding epitope of fXa which is extended to

include Arg306 and Glu310.

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Acknowledgments- We would like to thank Drs. Tom Girard and Kevin Conricode for their critical

review of the manuscript and Dr. Ravi Kurumbail for his assistance with the RIBBONS program.

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FIGURE LEGENDS

Fig.1. Gel electrophoresis of fX species. Each lane represents 2.5 ng of fX. Proteins were analyzed by

SDS/12.5% polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted using

monoclonal antibodies directed against the light chain and heavy chain. Molecular weight standards are

as indicated. Lanes 1-6: (1) fXWT, (2) fXK414A, (3) fXR347N, (4) fXE310N, (5) fXR306A, and (6)

fXK351A.

Fig. 2. Prothrombin activation in the absence of fVa and phospholipid. 10 nM of fXaWT (m),

fXaR306A (o), fXaE310N (t), fXaR347N (∇), fXaK351A (n), or fXaK414A (l) was incubated with varying

concentrations of prothrombin (0-450 µM). Aliquots were removed from the reactions for initial rate

measurements, diluted 10-fold in EDTA buffer, and incubated with 500 µM Spectrozyme TH. The

initial rate of hydrolysis was monitored at 405 nm. The concentration of thrombin was determined from a

standard curve prepared from dilutions of maximally activated prothrombin. The rate of thrombin

formation was calculated for the indicated prothrombin concentrations and expressed mol thrombin

(IIa)/mol Xa/sec. The maximal rate of thrombin formation is expressed as kcat (sec-1).

Fig. 3. Prothrombin activation in the presence of fVa without phospholipid. 10 µM prothrombin was

pre-incubated with varying concentrations of fVa (0-250 nM). Reactions were initiated by the addition

of 0.5 nM of fXaWT (m), fXaR306A (o), fXaE310N (t), fXaR347N (∇), fXaK351A (n), or fXaK414A

(l). Samples were removed from the reactions for initial rate measurements, quenched in EDTA buffer, and

incubated with 500 µM Spectrozyme TH. Initial rates of Spectrozyme TH hydrolysis were

determined at 405 nm and thrombin formation was quantitated as described in the legend to figure 2 for

the fVa concentrations indicated.

Fig. 4. Prothrombin activation in the presence of fVa and PC:PS vesicles. Reactions contained 20 µM

PC:PS vesicles (3:1), fVa (0-1 nM) and 20 pM fXaWT (m), fXaR306A (o), fXaE310N (t), fXaR347N

(∇), fXaK351A (n), or fXaK414A (l). Thrombin formation was initiated by the addition of 1 µM

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prothrombin and quantitated by incubating quenched reaction aliquots with 500 µM Spectrozyme TH.

Thrombin formation was quantitated as described in the legend to figure 2 and expressed as mol thrombin

(IIa)/mol Xa/sec and the maximal turnover rate in the presence of saturating fVa is represented as kcatVa

(sec-1).

Fig. 5. Prothrombin activation on an activated platelet surface. Washed platelets (108/ml) were

incubated with 0.5 U/ml of thrombin in the presence of varying concentrations (0-1 nM) of fXaWT (m),

fXaR306A (o), fXaE310N (t), fXaR347N (∇), fXaK351A (n), or fXaK414A (l). Thrombin generation

was initiated by the addition of 1 µM prothrombin. Initial rates of thrombin formation were determined

by incubating quenched aliquots of the reactions with 500 µM Spectrozyme TH. Thrombin formed was

quantitated as described in the legend to figure 2 and expressed as thrombin (nM)/sec and the maximal

turnover rate is expressed as Vmax (nM/sec).

Fig. 6. Factor Va binding of fXa variants. Factor Va (1 nM) was incubated with PC:PS-coated latex

beads (0.2%, v/v) for 10 min. 125I-fXaS379A (1 nM) was added in the presence of increasing

concentrations of each fXa species: fXaWT (m), fXaR306A (o), fXaE310N (t), fXaR347N (∇),

fXaK351A (n), or fXaK414A (l) and the reaction was incubated for an additional 10 min. The beads were

collected by centrifugation and the supernatant and pellet were counted separately. The fVa-specific

binding (calcium, phosphatidylserine, and fVa dependent) was quantitated as the percentage of bound

125I-fXaS379A. The concentration of unlabeled fXa required to displace 50% of bound, 125I-fXaS379A is

represented as the apparent affinity (Kdapp). Non-specific binding has been subtracted from all values

shown.

Fig. 7. A fVa binding site of fXa. The model was generated using RIBBONS v3.0. Arg347, Lys351, and

Lys414 are shown in green and were found to contribute to fVa binding. Arg306 and Glu310 are on the

periphery of the binding site and are highlighted in purple. Residues shown in red (Arg273, Ile357,

Arg406, Lys406, and Lys420) were found not to contribute to the fVa binding site of fXa. The active site

residues (Ser379, Asp282, and His236) are shown in gold.

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Table I. fX Mutations

Variant Sequence

fXR306A CLPERDWAE→CLPEADWAE

fXE310N RDWAESTLM→RDWANSTLM

fXR347N PYVDRNSCK→PYVDNNSCK

fXK351A RNSCKLSSS→RNSCALSSS

fXK414A GIYTK VTAF→GIYTA VTAF

Table II. Inhibition of Variant and WTfXa

Second Order Rate Constants

M-1.sec-1X104

(mean±S.E.)

fXa ATIII a ATIII/PS b ATIII/Heparin c TFPIa

WT 0.17±0.03 18.5±1.5 185.1±0.44 14.2±0.64

K414A 0.20±0.01 18.3±0.79 39.7±0.53 9.8±0.18

R347N 0.14±0.03 17.1±2.5 23.4±0.66 10.7±0.59

E310N 0.20±0.06 22.9±0.11 118.3±0.87 18.6±0.51

R306A 0.17±0.03 29.0±0.1.3 122.2±0.40 16.5±0.32

K351A 0.14±0.05 16.5±1.3 37.3±0.58 10.7±0.59

a 0.5 nM fXa was incubated with varying concentrations of rTFPI (0-50 nM) or ATIII (0-4 µM).

b 0.5 nM fXa was incubated with varying concentrations of ATIII (0-50 nM) in the presence of 0.5 µM heparin pentasaccharide.

c 0.5 nM fXa was incubated with varying concentrations of ATIII (0-16 nM) in the presence offull-length heparin (2.5 mU/ml). Second order rate constants were determined as described in Experimental Procedures.

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Amy E. Rudolph, Rhonda Porche-Sorbet and Joseph P. MiletichDefinition of a Factor Va Binding Site in Factor Xa

published online November 21, 2000J. Biol. Chem.

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