©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium Ion Modulation of Meizothrombin Autolysis at Arg-Asp and Catalytic Activity (*)

(Received for publication, December 14, 1995)

Willem K. Stevens (1) Hélène C. F. Côté (2) Ross T. A. MacGillivray (2) Michael E. Nesheim (1)(§)

From the  (1)Departments of Biochemistry and Medicine, Queen's University, Kingston, Ontario K7L 3N6 and the (2)Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

When a recombinant variant of prothrombin with the cleavage site mutations R155A, R271A, and R284A (rMZ) is exposed to either prothrombinase or ecarin, a form of meizothrombin (rMZa) is generated that is stable for weeks in the presence of Ca (Côté, H. C. F., Stevens, W. K., Bajzar, L., Banfield, D. K., Nesheim, M. E., and MacGillivray, R. T. A.(1994) J. Biol. Chem. 269, 11374-11380). In the absence of Ca however, rMZa is rapidly cleaved within a disulfide bonded loop in the F1 domain at Arg in the sequence RTPRDKL, yielding a molecule with 3 chains joined by two disulfide bonds (rMZa*). Cleavage kinetics are first order regardless of the rMZa concentration, indicating an intramolecular cleavage. This cleavage does not occur at Ca concentrations in excess of 1.0 mM. To assess the role of the F1 domain in rMZa activity, another variant lacking the R155A mutation (rMZdesF1) was expressed, which when activated yields meizothrombin lacking the F1 domain (rMZdesF1a). Rates of hydrolysis of the tripeptide substrate S2238 by rMZa or rMZa* increase from 60% to 90% that of recombinant thrombin as Ca, Mg, or Mn concentrations are varied from 0 to 10 mM. K and k values for rMZa in the absence and presence of 5 mM Ca are 1.9 and 2.2 µM and 65 and 105 s. TAME esterase activity of rMZa also increases with 5 mM Ca. No such metal ion-dependent effects are obtained with either thrombin or rMZdesF1a. Fibrinogen clotting activities, relative to that of thrombin, increase in a manner analogous to those obtained with small substrates, for rMZa and rMZa* but not rMZdesF1a. Complexes of the active site probe dansylarginine N-(3-ethyl-1,5-pentanediyl)amide with rMZa and rMZa*, but not thrombin or rMZdesF1a exhibit large cation-dependent decreases in fluorescence intensity, suggesting that metal ion binding in the F1 domain alters the environment of the probe at the active site. These results indicate that in the absence of divalent cations, the activity of rMZa is inhibited, perhaps by obstruction of the active site by the F1 domain, and that Ca binding to the F1 domain modulates the properties of not only the F1 domain but also the protease domain.


INTRODUCTION

Prothrombin is one of a group of plasma proteins involved in blood coagulation that require vitamin K-dependent carboxylation of several amino-terminal glutamic acid residues for full biological function(1) . In prothrombin, 10 of the first 33 residues are -carboxyglutamic acid (Gla) (^1)residues and define the Gla domain. This domain mediates the metal ion-dependent interaction of prothrombin and the other vitamin K-dependent coagulation factors with negatively charged membrane surfaces(2) . Unlike other Gla-containing coagulation factors such as factor IX, factor X, and protein C, prothrombin loses its Gla domain following activation to the serine protease thrombin.

Prothrombin activation is catalyzed by the prothrombinase complex(3) , composed of factor Xa (a serine protease), factor Va (an essential protein cofactor), negatively charged phospholipids, and calcium ion. Prothrombinase-catalyzed activation of prothrombin to thrombin results in peptide bond cleavage at two sites in the molecule (Fig. 1). Cleavage occurs initially at Arg, within a disulfide loop, forming the active intermediate meizothrombin (4) . Further cleavage at Arg produces thrombin and the amino-terminal activation peptide referred to as fragment 1.2. Thrombin itself can cleave prothrombin. Two major sites are recognized, one at Arg, between the kringle domains of prothrombin, and a second at Arg, within the A chain of the protease domain. Thrombin has also been reported to cleave prothrombin at a third site, at Arg within a disulfide loop between the Gla and first kringle domains(5) . Two of the intermediates of prothrombin activation, meizothrombin and meizothrombin des-fragment 1, may also catalyze the cleavage of prothrombin at these sites.


Figure 1: Schematic representation of the activation of recombinant prothrombin and the mutants rMZ and rMZdesF1. Activation of recombinant prothrombin (rII) to thrombin (rIIa) proceeds via the intermediate meizothrombin (rmIIa), formed by cleavage within the protease domain at Arg. Further cleavage can occur at Arg, Arg, and Arg, to yield thrombin (rIIa) and the activation peptides, fragments 1 and 2. The variant rMZdesF1 has the cleavage site mutations R271A and R284A and, upon activation, proceeds through meizothrombin to meizothrombin des-fragment 1 (rMZdesF1a). A stable recombinant form of meizothrombin (rMZ) was also produced with the additional mutation R155A. This species, when activated and stored in the presence of calcium ion, remains as meizothrombin (rMZa), but rapidly autocatalyzes cleavage at Arg in the absence of calcium ion to form a three-chain form of meizothrombin (rMZa*).



In contrast to thrombin, the prothrombin activation intermediate meizothrombin retains an intact Gla domain. Although naturally unstable, this intermediate was recently obtained as a stable product by introducing three mutations (R155A, R271A, R284A) which remove susceptibility to cleavage by prothrombinase at Arg and by thrombin at the two feedback cleavage sites(6) . If Arg is retained, cleavage by either thrombin or meizothrombin produces a species without the Gla domain and the first kringle, known as meizothrombin des-fragment 1 (Fig. 1).

Recent studies of the epidermal growth factor-like and Gla domains of coagulation factors IXa and X suggest that calcium ion binding in the Gla domain may affect properties of the other domains of these proteases. For example, calcium ion binding in the Gla domain of factor IXa alters the conformation of the serine protease domain(7) , and a study of amino-terminal fragments of factor X (8) revealed that Ca binding in the Gla domain of factor X influences Ca binding in the amino-terminal of the epidermal growth factor-like domain. Previous studies of meizothrombin activity (6, 9, 10) have not examined activities at varying levels of Ca.

To understand the consequences of retaining the amino-terminal portion of the prothrombin molecule on meizothrombin activity, recombinant prothrombin and two site-directed mutants were prepared. One mutant was prothrombin (R155A,R271A,R284A) designated rMZ, which upon activation with either the prothrombinase complex or ecarin yields full-length meizothrombin(6) . A second mutant prothrombin (R271A,R284A) designated rMZdesF1 yields meizothrombin des-fragment 1 upon activation. These and recombinant prothrombin (rII) were expressed, isolated, and activated, and their activities against small substrates and fibrinogen were compared in the presence of varying amounts of Ca.


EXPERIMENTAL PROCEDURES

Expression Vectors

For expression in mammalian cells, the cDNAs for rII and rMZ were ligated into the pNUT expression vector (11) downstream of the zinc-inducible mouse metallothionein promoter and upstream of the human growth hormone polyadenylation signal, as described previously for rMZ(6) . The expression vector for rMZdesF1 was prepared by digesting the pNUT rMZ vector with the restriction enzyme BstEII, liberating a 1-kilobase fragment corresponding to a small portion of the vector backbone and the first 690 base pairs of the rMZ cDNA. This fragment, containing the R155A mutation, was then replaced with the analogous fragment obtained from pNUT containing the wild-type prothrombin cDNA, yielding a construct coding for a mutant prothrombin (R271A,R284A), with the thrombin cleavage site at Arg restored.

Proteins

Human plasma factor V(12) , factor X(13) , and fibrinogen (13) were isolated, and factors V and X were activated as described previously. The prothrombin activator of Echis carinatus venom, ecarin, was purified from crude venom (Sigma) by anion exchange chromatography and polyacrylamide preparative gel electrophoresis as described previously(14) . The recombinant proteins rMZ and rMZdesF1 were expressed and isolated using the methods described for rMZ by Côtéet al.(6) . Conditioned medium (Opti-Mem (Life Technologies Inc.), supplemented with 50 µM ZnCl(2)) was collected from stably transfected lines of BHK cells cultured at 37 °C in roller bottles. Sodium citrate was added to a final concentration of 0.025 M, and the prothrombin was adsorbed with the addition of 1 M BaCl(2) (80 ml/liter) and subsequent formation of a barium citrate precipitate. The barium citrate pellet was eluted with 0.2 M EDTA, and the eluate was concentrated and dialyzed against 0.02 M Tris, 0.15 M NaCl, pH 7.4. Subsequent ion exchange using a salt gradient and pseudo-affinity chromatography with a calcium ion gradient on a Pharmacia Mono Q column yielded homogenous species(6) . Samples of the recombinant proteins rMZ and rMZdesF1 (1 mg at 0.1 mg/ml) were activated by incubation with ecarin (1.5 µg/ml) at 22 °C in the presence of 5 mM CaCl(2) for approximately 30 min. The activation mixtures were passed at 22 °C over a 2-ml column of benzamidine Sepharose, previously equilibrated in 0.02 M HEPES, 0.15 M NaCl, 5 mM CaCl(2), pH 7.4. The column was washed with 20 ml of the equilibration buffer, and the enzymes were eluted with the same buffer supplemented with 10 mM benzamidine. Fractions containing rMZa or rMZdesF1a were identified using a Bio-Rad protein assay, mixing 90 µl of each fraction with 20 µl of the dye reagent, and monitoring color development at 595 nm. Fractions containing protein were pooled and dialyzed against 0.02 M HEPES, 0.15 M NaCl, 5 mM CaCl(2), pH 7.4 and subsequently stored at 4 °C. Recombinant thrombin was isolated by activating rII directly in the conditioned media. Conditioned medium (40 ml of Opti-Mem) was treated with 5 nM factor Va, 5 nM factor Xa in the presence of 10 µM phosphatidylcholine/phosphatidylserine (3:1) vesicles and 5 mM CaCl(2). The activation mixture was passed over a 2 ml column of SP-C50 cation exchange resin, previously equilibrated with 0.02 M HEPES, 0.15 M NaCl pH 7.4, and the column was subsequently washed with 20 ml of the equilibration buffer. The rIIa was eluted from the column with 0.02 M HEPES, 0.5 M NaCl, pH 7.4. Fractions containing recombinant thrombin were identified by amidolytic activity against S2238. Aliquots of each fraction (10 µl) were incubated with 90 µl of 0.5 mM S2238, and color development at 405 nm was monitored. Fractions containing S2238 activity were pooled and stored at 4 °C until used (within 5 days). The isolated rIIa migrated as a single band (nonreduced) on NaDodSO(4)-polyacrylamide minigels, which comigrated with thrombin prepared from human plasma.

Active Site Titration

Concentrations of rIIa, rMZa, and rMZdesF1a were determined by titrating an aliquot of the protein (1.6 ml, 100 nM) with 5 µM PPACk in the presence of 200 nM DAPA, while measuring the fluorescence energy transfer from the proteases to DAPA ( 280 nm, 545 nm). Reaction of the protease with PPACk displaced DAPA from the active site resulting in a decrease in fluorescence intensity. The decrease was linear with respect to the concentration of PPACk, and extrapolation to the baseline fluorescence value yielded the active site concentration. The PPACk stock was dissolved shortly prior to use, and the concentration provided by the manufacturer was assumed to be correct. Titration of wild-type thrombin using this method yielded equivalent results to a p-nitrophenyl-p-guanidinobenzoate determination of active site titer(15) .

Time Course of Proteolysis at Arg

Stock solutions of rMZa at 25,50, and 100 µg/ml were prepared in 0.02 M HEPES, 0.15 M NaCl, 2 mM CaCl(2). Immediately prior to, and 4, 8, 12, 16, 20, and 36 min after addition of a small aliquot of 0.5 M EDTA, pH 7.4, to chelate the Ca present in the solution, aliquots equivalent to 2.5 µg of protein were withdrawn and quenched with the addition of 2 volumes of 0.2 M acetic acid. The samples were reduced to dryness using a Savant Speed Vac, dissolved in gel loading buffer, and analyzed on NaDodSO(4)-polyacrylamide gradient (5-15%) minigels under reducing conditions(16) . After staining with Coomassie Blue, the gels were destained and scanned with an LKB 2202 laser densitometer, and the traces were subjected to gravimetric analysis. For this experiment and subsequent experiments where EDTA was added to buffered solutions containing Ca, the pH of an equivalent mixture was monitored to ensure that the pH of the solution did not significantly change upon chelation of Ca.

Calcium Ion Dependence of Cleavage of Activated Meizothrombin

A series of microcentrifuge tubes were prepared containing 0.02 M HEPES, 0.15 M NaCl, pH 7.4, with calcium ion concentrations ranging from 0 (1 mM EDTA, pH 7.4) to 5 mM CaCl(2). A fixed amount (2.5 µg) of rMZa in 0.02 M HEPES, 0.15 M NaCl, 5 mM CaCl(2), pH 7.4, was added to all the tubes, resulting in solutions with varying Ca concentrations with a minimum of 0.37 mM. The samples were then incubated for 1 h at 22 °C, quenched with acetic acid, reduced to dryness, run on gels, and analyzed as described above.

Amidolytic Assays

Aliquots of rIIa, rMZa, and rMZdesF1a (50 µl) to yield final concentrations of 0.25 nM were incubated with 150 µl of 0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80 with varying amounts (0-10 mM) of either CaCl(2), MgCl(2), or MnCl(2) for 5 min at 37 °C in microtiter wells in the sample compartment of a TiterTek Twinreader. The substrate S2238 (0.5 mM in 0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80, pH 7.4) was added, and initial rates of hydrolysis were determined by measuring the absorbance at 405 nm at 1-min intervals.

Esterase Activity

Samples (30 µl) of rIIa or rMZa were diluted with 0.05 M Tris, pH 8.1, to a final concentration of 40 nM, and placed in quartz cuvettes in the sample compartment of a Perkin-Elmer Lambda 4B spectrophotometer. The reaction was initiated by the addition of 100 µl of 0.01 M TAME in water, and the absorbance at 247 nm was monitored continuously. Initial slopes of substrate consumption were used to determine reaction rates, either in the absence or presence of 5 mM CaCl(2).

Fibrinogen Clotting

Concentrations of rIIa (1 nM), rMZa/rMZa* (15 nM), or rMZdesF1a (3.6 nM) that yielded approximately equal clotting times in the presence of 5 mM CaCl(2) were established. Samples of concentrated stocks of these proteins (50 µl in 0.02 M HEPES, 0.15 M NaCl, 5 mM CaCl(2), 0.01% Tween 80, pH 7.4) were added to wells of a microtiter plate, containing 50 µl of buffer supplemented with varying levels of EDTA to yield Ca concentrations between 0 and 10 mM. The mixture was allowed to incubate for 1 min, and then 100 µl of 2 mg/ml fibrinogen in 0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80, pH 7.4, was added, and the time to onset of clotting was determined by measuring turbidity at 340 nm, at 10-s intervals. Results were analyzed by comparing times of onset of clotting for rMZa, rMZa*, or rMZdesF1a to the time obtained with rIIa at the equivalent calcium ion concentration.

Energy Transfer to DAPA

Samples of rIIa, rMZa, rMZdesF1a, and cleaved rMZa (rMZa*) (100 nM) in 200 nM DAPA, 0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80, pH 7.4, were placed in quartz cuvettes in the sample compartment of a Perkin-Elmer LS-50B fluorescence spectrophotometer. The samples were excited at 280 nm, and the DAPA fluorescence was monitored continuously at 545 nm, as aliquots of CaCl(2) were added.


RESULTS

Isolation and Characterization of Recombinant Proteins

Recombinant prothrombin and the variants rMZ and rMZdesF1 were expressed in stably transfected lines of BHK cells. The prothrombin variants rMZ and rMZdesF1 were isolated and were homogeneous as judged by analysis of Coomassie Blue-stained NaDodSO(4)-polyacrylamide gradient minigels. These proteins were activated by the snake venom protease ecarin, and the active species was captured on benzamidine Sepharose. Migration on 5-15% NaDodSO(4)-polyacrylamide gradient gels of these proteins (reduced and nonreduced) was consistent with a single cleavage at Arg for rMZa and additional loss of the F1 domain for rMZdesF1a (data not shown).

Stability of rMZa

Although rMZa was shown previously to be stable for up to 28 days in the presence of the components of the prothrombinase complex at 4 °C (6) and is stable when purified for 6 months at -20 °C in 50% glycerol, storage of the purified protein in the absence of Ca yielded rapid conversion to a species with a cleavage in the F1.2A chain visible on reduced gels (Fig. 2), but co-migrating with active meizothrombin under nonreducing conditions. Following NaDodSO(4)-polyacrylamide gel electrophoresis and blotting onto a polyvinylidene difluoride membrane, amino-terminal sequence analysis of the cleaved F1.2A chain revealed a new amino terminus with the sequence DKLAA, consistent with cleavage within a disulfide bonded loop, between the Gla domain and the kringle of Fragment 1. Cleavage of rMZa at Arg, within the sequence RTPRDKLA yields a species consisting of three peptide chains held together by two disulfide bonds; this species is referred to as rMZa*.


Figure 2: Calcium ion protection of rMZa from autocatalysis to rMZa*. A sample of rMZ (100 µg/ml) in 0.02 M HEPES, 0.15 M NaCl, 5 mM Ca, pH 7.4, was incubated with ecarin (3 µg/ml), and samples were withdrawn and quenched with acetic acid (0.2 M, 2 volumes) at 1, 5, 20, and 60 min as indicated on the gel. After 60 min, the sample was split in two, and to one aliquot EDTA was added (10 mM final concentration). Subsequent samples from the incubations with (+) and without(-) EDTA were quenched 1, 5, 20, and 60 min later. The samples were lyophilized, dissolved in gel loading buffer, and analyzed by NaDodSO(4)-polyacrylamide gel electrophoresis under reducing conditions. Bands corresponding to intact rMZ, the two chains of rMZa (F1.2A and B chains), and the cleaved F1.2A chain from rMZa* (F1.2A*) are indicated.



Further analysis revealed that the cleavage giving rise to rMZa* occurred only in the absence of Ca and was prevented by storage of rMZa in 5 mM CaCl(2). Analysis of the time course of cleavage indicated that rMZa has a half-life in excess of 28 days in the presence of Ca, but only 8 min in the absence of Ca. It is highly unlikely that this cleavage is a result of a trace contaminant in the ecarin preparation, as identical results were obtained when the prothrombinase complex was used as the activator. The cleavage follows first order kinetics, with the percent conversion following Ca removal identical at three concentrations of the enzyme between 25 and 100 µg/ml (Fig. 3). The rate of cleavage is not affected by equimolar concentrations of bovine F1 or a recombinant human prothrombin variant lacking factor Xa and thrombin cleavage sites (data not shown) nor are these species cleaved during the time course of the experiment. Although the concentration range studied was limited for technical reasons, modelling the meizothrombin proteolysis as an intermolecular reaction occurring with either high (geq10 µM), intermediate (1 µM), or low (leq0.1 µM) affinity using Kinsim(17) , failed to produce a reasonable fit to the data, whereas modelling the reaction as an intramolecular event (first order) produced an excellent global fit to the data (Fig. 3). These results indicate that the cleavage at Arg is an intramolecular rather than intermolecular event.


Figure 3: Time course of conversion of rMZa to rMZa* at varying concentrations in the absence of Ca. Samples of rMZa were diluted to 100, 50, and 25 µg/ml (circle, bullet, and down triangle respectively) in buffer containing 2 mM CaCl(2). At time zero, EDTA (5 mM final concentration) was added to chelate the Ca, and, at the indicated times, samples were quenched with 0.2 M acetic acid. Samples were reduced to dryness, redissolved in gel loading buffer, and run on 5-15% polyacrylamide gradient gels under reducing conditions in the presence of NaDodSO(4). The extent of cleavage at each time point was ascertained by laser densitometry of the Coomassie Blue-stained gels. The line represents a global fit determined by regression to a first order rate equation.



Calcium Ion Requirement for Maintenance of Intact rMZa

Samples of rMZa were incubated in buffer containing varying levels of Ca for 60 min, and the extent of cleavage at Arg was analyzed by NaDodSO(4)-polyacrylamide gel electrophoresis. Since the data in Fig. 3clearly indicated that cleavage followed first order kinetics, the single time point measurements obtained in this experiment were used to calculate rate constants for cleavage according to the equation ln([M(0)]/[M])/t = k where [M(0)] and [M] are the levels of intact rMZa at time = 0 and time = t respectively. Analysis of the rate constants for cleavage (Fig. 4) revealed that the minimal Ca concentration required for stability was approximately 1 mM, with cleavage occurring rapidly below this concentration. To account for the possibility that rMZa could be proteolyzed in subsequent experiments to rMZa* during incubation at low calcium ion concentrations, rMZa* was isolated and included as a control.


Figure 4: Calcium ion requirement for maintenance of intact rMZa. Aliquots of rMZa (1 µM) in 0.02 M HEPES, 0.15 M NaCl, 5 mM CaCl(2) were added to 0.02 M HEPES, 0.15 M NaCl, pH 7.4, containing various concentrations of calcium ion. After a 60-min incubation at 22 °C, the samples were quenched and analyzed as described in the legend for Fig. 3. From the determinations of the amount of rMZa remaining intact, the rate constants for cleavage at the varying Ca concentrations were determined. Calculated half-lives are inset.



Metal Ion Dependence of rIIa, rMZa, rMZa*, and rMZdesF1a Activity against the Amidolytic Substrate S2238

Initial rates of S2238 cleavage for rIIa, rMZa, rMZa*, and rMZdesF1a in 0.5 mM EDTA and varying concentrations of CaCl(2) are displayed in Fig. 5. Relative to rIIa, both rMZa and rMZa* had initial activities of approximately 60%, while rMZdesF1a had a slightly higher activity (70%). While the activity of rMZdesF1a paralleled that of rIIa as the concentration of Ca increased, the activity of both rMZa and rMZa* increased from 60% to 90% that of rIIa over the range of Ca concentrations from 0 to 10 mM. Similar results were obtained when Mn and Mg were used in place of Ca (data not shown). The EC for this change varied with the metal ion, with the pattern Mg (2 mM) > Ca (1 mM) > Mn (0.1 mM). For all species, a decrease in rate was observed at low concentrations (0-0.35 mM) of divalent ion. This may be due to a direct metal ion effect on the protease domain, since the effects are not limited to rMZa or rMzdesF1a. The increases in activity observed for rMZa and its cleaved variant (rMZa*), however, suggest that either the Gla domain or the F1 kringle interacts with divalent metal ions and thus affects the catalytic efficiency of the protease domain. The change in rates of S2238 hydrolysis by rMZa and rMZa* in the presence of divalent metal ions can be attributed to alteration of the k toward S2238. K(m) values for rMZa toward S2238 remain unchanged (1.9 ± 0.2 versus 2.2 ± 0.2 µM) in the presence or absence of 5 mM CaCl(2), whereas the k increases from 65 ± 3 s in the absence of Ca to 105 ± 5 s in the presence of Ca.


Figure 5: Activity of rIIa, rMZa, rMZa*, and rMZdesF1a against the amidolytic substrate S2238 in the presence of varying concentrations of Ca. Aliquots of rIIa, rMZa, rMZa*, and rMZdesF1 (50 µl) (, circle, bullet, and down triangle, respectively) to yield final concentrations of 0.25 nM were incubated for 5 min at 37 °C in microtiter wells with 150 µl of 0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80 with varying concentrations (0-10 mM) of CaCl(2). The substrate S2238 (0.5 mM final concentration in the incubation buffer) was added, and initial rates of hydrolysis were determined by following the absorbance at 405 nm at 1-min intervals.



Hydrolysis of the Esterolytic Substrate TAME by rMZa in the Presence and Absence of Ca

Effects similar to those observed with S2238 hydrolysis are observed with TAME hydrolysis. When monitored by absorbance of the product at 247 nm, cleavage of this ester substrate is enhanced in the presence of Ca with the turnover number increasing from 29 ± 0.9 s to 37 ± 1.2 s in the presence of 5 mM CaCl(2).

Effect of Ca on Clotting of Fibrinogen by rMZa and rMZdesF1a Compared to rIIa

To determine whether Ca has similar effects on the activity of rMZa toward macromolecular substrates, the effect of varying Ca levels on clotting times of purified fibrinogen was examined. Since Ca has a direct effect on the rate of fibrin polymerization(18) , the effects of Ca on clotting times obtained with rMZa and rMZdesF1a were examined relative to those obtained with rIIa. Concentrations of the three proteases required to achieve similar clotting times in 5 mM CaCl(2) were established, and the times to clot at these concentrations were determined at varying Ca levels. The results are presented in Fig. 6. The activity of rMZdesF1a toward fibrinogen was parallel to that of rIIa at all Ca concentrations, with a rate to onset of clot formation that was 27% that of rIIa. In contrast, both rMZa and rMZa* demonstrated a Ca-dependent doubling in rate, from 3.5% that of rIIa in the absence of Ca to 7% that of rIIa in the presence of Ca at levels greater than 2 mM, although the transition appears to require higher levels of Ca for rMZa*. The Ca concentration dependence of the increase in activity of rMZa toward fibrinogen closely parallels that observed in rates of S2238 hydrolysis.


Figure 6: The effect of calcium ion concentration on fibrinogen clotting by rMZa, rMZa*, and rMZdesF1a relative to rIIa. Fixed concentrations of rIIa (1 nM), rMZa (15 nM), or rMZdesF1a (3.6 nM) to give approximately equal clotting times in the presence of 5 mM CaCl(2) were added to the wells of a microtiter plate containing varying concentrations of EDTA to give a range of Ca concentrations (0-5 mM). Fibrinogen cleavage was initiated with the addition of 2 mg/ml fibrinogen in 0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80, pH 7.4, and the time to onset of clotting was determined by monitoring turbidity at 320 nm. Rates of fibrin formation relative to rIIa are plotted for rMZA (circle) and rMZa* (bullet). The clot times for all species (rIIa, rMZa, rMZa*, and rMZdesF1 are , circle, bullet, and down triangle, respectively) are inset.



Effect of Ca Concentration on Energy Transfer to DAPA by rIIa, rMZa, rMZa*, and rMZdesF1a

Changes in the active site environment upon exposure to Ca were inferred by tryptophan energy transfer to the fluorescent active site inhibitor DAPA. Tryptophan residues of rIIa, rMZa, rMZa*, or rMZdesF1a were continuously excited at 280 nm, and the fluorescence from DAPA was monitored at 545 nm. The traces presented in Fig. 7represent changes in fluorescence with incremental additions of CaCl(2). Whereas Ca has no effect on the DAPA fluorescence of either rIIa or rMZdesF1a, both rMZa and rMZa* demonstrate a considerable decrease (30%) in DAPA fluorescence intensity in response to Ca addition. Similar to the results obtained for fibrinogen clotting, the transition for the cleaved form of meizothrombin (rMZa*) appears to require higher levels of calcium. Because the DAPA concentration used in these studies was sufficient to nearly saturate the active sites of these enzymes, the change of fluorescence reflects a change in efficiency of energy transfer to the dansyl moiety of DAPA rather than binding affinity and thus is indicative of a change in active site environment. The initial fluorescence signals, obtained in the absence of Ca, from the DAPA-rMZa complex and the DAPA-rMZa* complex, are much higher (2.5-3-fold) than those of either the DAPA-rIIa or DAPA-rMZdesF1a complexes.


Figure 7: Titration of rIIa, rMZa, rMZa*, and rMZdesF1a with calcium ion in the presence of DAPA. Aliquots of rIIa, rMZa, rMZa*, and rMZdesF1a (100 nM) (, circle, bullet, and down triangle, respectively), in the presence of 200 nM DAPA in 0.02 M HEPES, 0.15 M NaCl, 0.01% Tween 80 were titrated with a solution of CaCl(2), while DAPA fluorescence was continuously monitored ( 280 nm, 545 nm). The results are expressed as a percentage of the original fluorescence signal.




DISCUSSION

Previous studies of the binding of prothrombin and its activation intermediates to hirudin tail peptides(19) , thrombomodulin, and exosite antibodies(20) , have suggested that the fragment 1 domain may in some way block access to the primary anion binding exosite. Through measurements of the Ca dependence of meizothrombin autoproteolysis at Arg, activity toward a variety of substrates, and energy transfer to the active site probe DAPA, this study provides further evidence for interactions between the fragment 1 and protease domains of meizothrombin.

Cleavage of prothrombin at Arg has previously been reported following lengthy incubation of prothrombin with thrombin in the absence of Ca(5) . In the present studies, however, the cleavage of rMZa at Arg follows first order kinetics, with the time course of the reaction being identical at three different concentrations of rMZA, indicative of an intramolecular cleavage event. These results imply a direct interaction between the amino-terminal portion of the fragment 1 domain with the active site of meizothrombin. Cleavage of rMZa at Arg occurs rapidly in the absence of Ca with a half-life under 10 min, but is profoundly attenuated in the presence of Ca.

Structural changes in the fragment 1 domain of prothrombin have been shown to accompany divalent metal ion binding by a number of methods including sedimentation velocity(21) , intrinsic fluorescence(21) , circular dichroism(22) , and immunologic techniques(23) . Due to the sensitivity of cleavage at Arg to levels of Ca, and the location of the cleavage site within the fragment 1 domain, this event is likely modulated by metal ion binding in the Gla domain. Indeed, cleavage of prothrombin by thrombin at Arg is also inhibited in the presence of Ca, and the protective effect has been attributed to conformational changes mediated by Ca binding to the Gla domain(24) .

Although the three-dimensional structures for the apo- and Ca-bound forms of human fragment 1 are not available, those of bovine fragment 1 in the absence and presence of Ca have been determined(25, 26) . The structure of the Gla domain is disordered in the absence of Ca, but the disulfide loop containing the cleavage site at Arg is defined in both structures. Interestingly the region around Arg changes significantly upon Ca binding, with a trans to cis conformational change at Pro and a 90° rotation of Arg relative to its neighboring residues, enabling the formation of new salt links between Arg and Gla, Leu, and Gla. These changes at the Arg cleavage site likely explain the insensitivity of Ca-bound meizothrombin to autoproteolysis. Another contributing factor to the stability of rMZa in the presence of Ca may be the disruption of interactions between the negatively charged residues of the Gla domain with positively charged portions of the protease domain (possibly the primary anion binding exosite) upon Ca binding. In any case, autocatalytic cleavage of rMZa at Arg necessitates direct interaction between the fragment 1 and protease domains.

In addition to preventing autoproteolysis of rMZa, Ca increases the activity of rMZa toward small ester (TAME) and amide (S2238) substrates as well as increasing its fibrinogen clotting activity. Equivalent increases in activity are obtained with rMZa*, a species cleaved at Arg, so the increase in activity cannot be simply attributed to a Ca-mediated protection from autoproteolysis. Neither rMZdesF1a, lacking the fragment 1 domain, nor thrombin demonstrate this Ca-dependent increase in activity, suggesting that interactions of Ca with the fragment 1 domain are responsible for the altered activity of rMZa.

Similar effects on the activity of rMZa are observed with both small substrates (amide and ester) and fibrinogen, although fibrinogen binding involves considerably more complex interactions with the enzyme than do the small substrates. Thus, obstruction of the active site or reduced turnover for some other reason, rather than a change in substrate binding, is responsible for the reduced activity of rMZa in the absence of Ca. This interpretation is supported by the observation that values obtained for the K(m) for S2238 hydrolysis remain constant (1.9 versus 2.2 µM) in the absence or presence of 5 mM Ca, while k values change (65 versus 105).

Titration of rMZa with Ca in the presence of the reversible fluorescent inhibitor DAPA results in a 30% decrease in fluorescence energy transfer, with a half-maximal effect occurring at 0.5 mM Ca. This result is similar to that obtained with meizothrombin prepared by activation of prothrombin with ecarin in the presence of dansyl-glutamylglycylarginyl chloromethyl ketone, yielding an active site blocked species with a covalently attached dansyl reporter group(27) . In both cases, a change in the active site environment is revealed by a substantial decrease in probe fluorescence upon the addition of Ca. It is unlikely that Ca has a direct effect on the fluorescence of DAPA, as direct excitation of DAPA in the absence of protein yields less than 2% of the signal obtained in the presence of rMZa, and the fluorescence decrement observed with rMZa or rMZa* does not occur with either rMZdesF1a or rIIa. In addition, both rMZa and rMZa* display much greater fluorescence than either rIIa or rMZdesF1a in the absence of Ca (2.5-3-fold). The decreases in DAPA fluorescence of rMZa and rMZa*, but not rMZdesF1a, upon the addition of Ca are consistent with a Ca binding event in the fragment 1 domain affecting the probe environment at the active site.

The Ca concentrations required for half-maximal increase in rMZa activity, protection from autoproteolysis, and decreases in DAPA fluorescence (0.5-1 mM) are significantly higher than those reported necessary for conformational change in the isolated fragment 1 domain (0.2-0.4 mM)(21, 22) . These levels of Ca are, however, consistent with those reported necessary to prevent the proteolytic cleavage of prothrombin at Arg by thrombin (0.6 mM)(24, 28) .

Divalent metal ions have been widely reported to cause self-association of prothrombin fragment 1 (21, 29) and, under some conditions, prothrombin(29, 30) . Metal ion-dependent rMZa dimer formation is unlikely to be responsible for any of the effects observed in these studies, due to the high protein and metal ion concentrations necessary for dimerization to occur. In addition, Mg at the concentrations used in this study does not seem to be capable of inducing fragment 1 or prothrombin dimerization(21, 29, 30) , but will support the increase in S2238 hydrolysis observed with rMZa.

Cleavage of rMZa at Arg has little effect on the rate of S2238 hydrolysis at varying Ca concentrations. There is, however, a marked effect on the concentration necessary to produce half-maximal fibrinogen clotting activity, and decrease in DAPA fluorescence, with higher (1-1.5 mMversus 0.5 mM) levels necessary to produce these changes in the cleaved species. These differences may be indicative of a reduced ability of the cleaved form to undergo the necessary conformational changes to relieve the enzyme from inhibition at a given Ca concentration, with both fibrinogen clotting and DAPA fluorescence being more sensitive to interdomain interactions distant from the active site than S2238 hydrolysis.

These results together provide evidence for interaction between the fragment 1 and protease domains of rMZa at Ca levels below 1 mM. The consequences of this interaction include meizothrombin autoproteolysis at Arg, as well as decreases in catalytic activity toward small ester (TAME) and small and large amide substrates (S2238, fibrinogen). Interestingly, the transition for these effects borders on the range of free Ca in the plasma (1-1.3 mM)(31) . Finally, these results are indicative of flexibility in the structure of prothrombin, allowing for association between the amino-terminal fragment 1 and carboxyl-terminal serine protease domains.


FOOTNOTES

*
This work was supported in part by Grants MA-9781 (to M. E. N.) and MT-7716 (to R. T. A. M.), by an Ontario Graduate Scholarship (to W. K. S.), and a Medical Research Council Studentship (to H. C. F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 613-545-2957; Fax: 613-545-2497.

(^1)
The abbreviations used are: Gla, -carboxyglutamic acid; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; BHK, baby hamster kidney cells; S2238, D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroaniline-dihydrochloride; TAME, p-tosyl-L-arginine methyl ester.


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