©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of TAFI, a Thrombin-activable Fibrinolysis Inhibitor (*)

Laszlo Bajzar , Reg Manuel , Michael E. Nesheim (§)

From the (1)Departments of Biochemistry and Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies demonstrated that tissue plasminogen activator-induced fibrinolysis in vitro is retarded in the presence of prothrombin (II) activation and that the anticoagulant-activated protein C appears profibrinolytic by preventing the formation of thrombin (IIa)-like activity during fibrinolysis. To disclose the molecular connection between the generation of IIa and the inhibition of fibrinolysis, a lysis assay that is sensitive to the antifibrinolytic effect of II activation was developed and was used to purify a 60-kDa single-chain protein from human plasma. Because the lysis of a clot, produced from purified components, is retarded when this protein is present and when II activation occurs in situ, the protein was named TAFI (thrombin-activatable fibrinolysis inhibitor). TAFI is cleaved by IIa yielding 35-, 25-, and 14-kDa products. Amino-terminal sequence analyses identified TAFI as a precursor of a plasma carboxypeptidase B (CPB). Formation of the 35-kDa product correlates with both prolongation of lysis time and CPB-like activity. Prolongation of lysis time saturates at about 125 nM TAFI. Activated TAFI inhibits the activation of Glu-plasminogen but does not prolong the lysis of clots formed in the presence of Lys-plasminogen. 2-Guanidinoethylmercaptosuccinic acid, a competitive inhibitor of CPB, completely inhibits prolongation of lysis by activated TAFI in a purified system and the prolongation induced by II activation in barium-adsorbed plasma. This suggests that TAFI accounts for the antifibrinolytic effect that accompanies prothrombin activation and that activated protein C appears profibrinolytic by attenuating TAFI activation.


INTRODUCTION

The activation of II to IIa in vivo is catalyzed by prothrombinase, a tetrameric complex consisting of the serine protease factor Xa (FXa),()a negatively charged phospholipid surface, calcium ion and the cofactor factor Va (FVa)(4) . In response to injury, IIa catalyzes the cleavage of fibrinopeptides A and B from fibrinogen, forming fibrin monomers which spontaneously polymerize to form the insoluble fibrin network of a blood clot and prevent the catastrophic loss of blood(5) . The fibrinolytic cascade works in opposition to the coagulation cascade and is responsible for the removal of a fibrin clot. Plasmin is the terminal protease formed by the fibrinolytic cascade and is formed by the activation of plasminogen by tissue-type plasminogen activator (t-PA). Plasmin catalyzes the cleavage of fibrin to produce soluble fibrin degradation products thereby removing a fibrin clot(6, 7) . Both coagulation and fibrinolysis are in part regulated by the specific inhibitors antithrombin III (AT-III) and -antiplasmin, respectively(8, 9) . Both activation and inhibition of each cascade regulate and maintain a proper balance between fibrin formation and its dissolution(5) .

Activated protein C (APC) is generated by cleavage of the precursor, protein C, a reaction catalyzed by IIa in complex with thrombomodulin (10). APC can down-regulate II activation by catalyzing the proteolytic inactivation of the essential cofactor in prothrombinase, FVa(11) . Protein S, a cofactor for this reaction, is responsible for the species specificity of the anticoagulant activity of APC(12) . APC, however, also appears to up-regulate fibrinolysis(1, 13, 14, 15, 16) . We have shown that this effect is not due to a direct profibrinolytic effect of APC but rather an indirect effect expressed through inhibition of II activation(1) . This explains why others have observed that the profibrinolytic effect of APC appears to be potentiated by protein S in a species-specific manner(17) . Our data also indicated that II activation, when it occurs during fibrinolysis and yields either IIa or meizothrombin, is antifibrinolytic(2) . Those studies also suggested that some preparations of plasminogen contain a contaminant required to reproduce, in a system of defined components, the profibrinolytic effect of APC which is always observed in plasma(1) . Those studies, however, did not provide a molecular explanation for the inhibition of fibrinolysis by II activation during fibrinolysis.

This work was undertaken to characterize further the mechanism by which II activation during fibrinolysis prolongs the lysis time of a clot. This was accomplished by producing a turbidometric lysis assay that is both sensitive to a component in plasma which is thrombin-activated and inhibits fibrinolysis (TAFI) and is buffered with respect to the inhibitors AT-III and -antiplasmin. Using this assay, we purified a 60-kDa zymogen from plasma which, when activated by IIa, yields an enzyme with carboxypeptidase B (CPB)-like activity. The generation of this activity correlates with a prolongation in lysis time and inhibition of GluPgn activation. Inhibition of IIa generation by APC thus attenuates activation of TAFI and thereby links the profibrinolytic effect of APC to its anticoagulant activity.


EXPERIMENTAL PROCEDURES

Materials and Methods

The recombinant t-PA, Activase, was generously provided by Dr. Gordon Vehar of Genentech (South San Francisco, CA). The lyophilized powder was dissolved in water to a final concentration of 1.0 mg/ml and working stock solutions were prepared as described previously(1) . Porcine CPB was purchased from Boehringer Mannheim (Laval, PQ) as a lyophilized powder that was dissolved in water to 5.0 mg/ml and stored in 20-µl aliquots at -20 °C. 2-Guanidinoethylmercaptosuccinic acid (GEMSA), a specific CPB inhibitor, was purchased from Calbiochem. Dulbecco's modified Eagle's medium, nutrient mixture F-12 (1:1) (DMEM/F-12), Opti-Mem, newborn calf serum, and G418 (Geneticin) were purchased from Life Technologies Inc., and methotrexate was purchased from David Bull Laboratories (Vaudreuil, PQ). Phosphatidylcholine/phosphatidylserine vesicles (PC/PS, 3:1) were prepared according to the method of Barenholz et al.(18) as described by Bloom et al.(19) . II and factor X (FX) were purified as described previously(13) . IIa was prepared from II and isolated by a modification of the procedure of Lundblad et al.(20) as described previously(21) . FXa was prepared from FX utilizing the FX activator purified from Russell's viper venom and isolated as described by Krishnaswamy et al.(22) . Protein C was purified and activated as described previously(13) . Factor V (FV) was purified by immunoaffinity chromatography according to the method of Nesheim et al.(23) . A concentrate of AT-III was kindly provided by Alpha Therapeutics (Los Angeles, CA) and was subjected to affinity chromatography on immobilized heparin as described by Damus and Rosenberg (8) with the modifications of Nesheim(24) . Fibrinogen, >95% clottable, was isolated by the procedure of Straughn and Wagner (25) and subsequently passed over Lys-Sepharose to remove traces of plasminogen. Glu-plasminogen (GluPgn) was isolated by Lys-Sepharose chromatography, as described previously(26) . GluPgn was coupled to CNBr-activated Sepharose CL-4B (Sigma) by a modification of the procedure described by Cuatrecasas(27) . Briefly, Sepharose CL-4B (Pharmacia) (5 ml) was washed at 22 °C with 50 ml of both HO and 2.0 M NaCO under vacuum. The cake of washed Sepharose CL-4B was then activated with 0.5 g of CNBr (0.5 g of CNBr dissolved in 0.5 ml of acetonitrile) for 1.0 min at 22 °C and then washed with 100 ml each of ice-cold 0.1 M NaHCO, HO, and 0.1 M sodium citrate, pH 6.5, in succession. Five ml of 10 mg/ml GluPgn in 0.1 M sodium citrate, pH 6.5, was then incubated with the activated resin at 4 °C for 24 h and for a further 24 h after the addition of 1.0 ml of 1.0 M Tris, pH 8.0. This resulted in a 98% coupling efficiency producing 5.0 ml of a 9.8 mg/ml Pgn-Sepharose resin. Lys-plasminogen (LysPgn) was prepared as described by Nesheim et al.(26) by incubation of GluPgn (33 µM) with plasmin (0.1 µM) in 6.2 ml of 50 mM Tris, pH 8.0, and 10.0 mM ACA. The reaction was stopped after 2.0 h by passing the mixture over benzamidine-Sepharose, and the flow-through was incubated for 2.0 h at 22 °C with 1.0 µM valyl-phenyl-alanyl-lysyl chloromethyl ketone (Calbiochem). LysPgn was precipitated with 80% (NH)SO. The pellet was dissolved in 50% glycerol/HO and stored at -20 °C. The LysPgn preparation contained no GluPgn as assessed by acid-urea gel electrophoresis(28) . Plasmin was prepared by adding 10.0 ml of Lys-Sepharose to 20 ml of 1.0 mg/ml GluPgn and 50 units/ml human urine urokinase (Calbiochem) in 20 mM Tris-HCl, 150 mM NaCl, pH 8.0 (TBS) and incubating with gentle agitation at 37 °C for 1.0 h. The resin was then poured into a column; after washing with 70 ml of TBS, plasmin and residual plasminogen were eluted with 20 mM ACA in TBS, collecting 1.0-ml fractions at 22 °C. Protein containing fractions, identified by absorbance at 280 nm, were pooled and passed over a 2.0-ml p-aminobenzamidine Sepharose 6B (Sigma) column in order to separate plasminogen from plasmin. The column was washed with TBS, collecting 1.0-ml fractions at 22 °C until the A was less than 0.01. Plasmin was then eluted with 20 mM benzamidine in TBS, collecting 1.0-ml fractions. Protein-containing fractions were identified using the BCA assay (Pierce Chemical Co.). Appropriate fractions were pooled (8.0 ml), and plasmin was precipitated by dialysis against 250 ml of ammonium sulfate at 80% saturation with one change overnight at 4 °C. After centrifugation (13,000 g, 5 min, 22 °C) of the retentate, the pellet was dissolved in 50% glycerol/water and stored at -20 °C until use. From this procedure 400 µl of 17.1 mg/ml plasmin was isolated, amounting to a 34% recovery of 100% active plasmin as determined by active site titration with p-nitrophenylguanidinobenzoate(29) .

The cDNA for -antiplasmin in the expression vector pPAB (30) was a kind gift of Sharon Busby of Zymogenetics (Seattle, WA). The expression vector pNUT and baby hamster kidney cells were provided by Dr. Ross MacGillivray of University of British Columbia. Baby hamster kidney cells were cotransfected with pPAB, containing the antiplasmin insert, and pNUT using CaPO coprecipitation technique(27, 31) . The DNA (10 µg of each vector) was precipitated at pH 6.95 and added to the cells and incubated for 4 h. Cells were allowed to recover overnight in DMEM/F-12, supplemented with 5% newborn calf serum. Positive clones were selected in DMEM/F-12 supplemented with 5% newborn calf serum, 440 µM methotrexate, and 400 µg/ml G418. Positive clones were screened for antiplasmin production by enzyme-linked immunosorbent assay using goat polyclonal capture antibody GA2AP-1IG (Affinity Biologicals, Hamilton, ON) and detected with horseradish peroxidase-conjugated monoclonal murine antibody (Dr. A. R. Giles, Queen's University, Kingston, ON). One clone (25 µg of antiplasmin/ml of medium/24 h from confluent cells on 10-cm wells) was used for roller bottle production of antiplasmin. Antiplasmin was routinely isolated from 24-h conditioned medium from two roller bottles, each containing 150 ml of Opti-Mem supplemented with 80 µM ZnCl, 220 µM methotrexate, and 200 µg/ml G418. Conditioned medium was diluted 1/3 with 20 mM HEPES, pH 7.4 (HB) and passed over a 10-ml Q-Sepharose (Pharmacia) column. The column was washed with 5 column volumes of HB, and antiplasmin was eluted with 0.2 M NaCl in HB. Appropriate fractions were pooled and diluted 1/4 and passed over a 1.0-ml DEAE fast flow (Pharmacia) column at 22 °C. The column was washed with 20 ml of HB, and the antiplasmin was eluted with 0.15 M NaCl in HB. Five mg of antiplasmin is typically recovered by this procedure, and the antiplasmin inhibits active site titrated plasmin with a 1:1 stoichiometry.

Lysis Assay and Measurement of the Antifibrinolytic Effect of Purified TAFI

A concentrated stock solution (fibrinogen solution) was produced containing 15.4 µM fibrinogen, 4.2 µM GluPgn, 1.8 µM -antiplasmin, 52.6 µM PC/PS vesicles, 44 nM FV, 3.65 µM II, and 5.0 µM AT-III in 20 mM HEPES, 0.15 M NaCl, pH 7.4 (HBS). The assay was performed by adding 40 µl of the fibrinogen solution to 160 µl of assay sample or purified TAFI. Two clots were produced for each sample by pipetting 95 µl of each assay mixture into wells containing 2.0-µl aliquots of IIa (6.0 nM, final), Ca (10 mM, final) and t-PA (441 pM, final) and either buffer or FXa (100 pM, final). Turbidity at 405 nm was then monitored at 2.5-min intervals in a microtiter plate reader thermostatted at 37 °C. Lysis time, defined as the time to achieve a 50% reduction of the maximum turbidity, for each clot was determined from the plot of Aversus time. Relative prolongation was then defined as the lysis time determined for clots produced in the presence of FXa relative to the lysis time determined for clots produced in the absence of FXa.

Purification of TAFI

Two units of fresh frozen plasma (500 ml), obtained from the Canadian Red Cross (Kingston General Hospital, ON) anticoagulated with acid citrate dextrose, were thawed at 37 °C for 0.5 h. The plasma was brought to 80 mM BaCl by dropwise addition of 1.0 M BaCl with continuous stirring at 4 °C. Barium citrate and adsorbed vitamin K-dependent proteins were pelleted by centrifugation at 10,000 g for 20 min at 4 °C (these conditions were used in all subsequent centrifugation steps). An aliquot (10 ml) of the supernatant was removed and dialyzed against 2.0 liters of HBS with four changes, for not less than 2.0 h/change, at 4 °C to produce barium-adsorbed plasma (BAP). To remove remaining Ba ion, solid (NH)SO was added directly to the remaining portion of the supernatant to a concentration of 10% saturation. The mixture was stirred for 0.5 h at 4 °C, BaSO was pelleted by centrifugation, and the supernatant was recovered. The supernatant was brought to 45% saturation (NH)SO and stirred for 0.5 h at 4 °C. Following precipitation and centrifugation, the pellet was discarded, and the supernatant was brought to 70% (NH)SO, stirred for 0.5 h at 4 °C, and centrifuged. The resulting pellet was dissolved in 150 ml of HB and dialyzed against 4.0 liters of the same buffer at 4 °C with four changes and minimally 2.0 h between changes. Following dialysis the retentate was passed over a 450-ml Q-Sepharose column (5.5 19 cm) at 22 °C which had been washed with 0.5 M NaCl, HB, and then equilibrated in HB. The eluate during the load was collected into a beaker, and the column then was washed with 600 ml of HB, collecting 10.0-ml fractions. TAFI eluted in a 0-0.2 M NaCl, linear gradient in HB (600 ml/side). The fractions containing activity were pooled, and the sample was concentrated 5-fold using an Amicon concentrator with a PY-10 membrane (Amicon, Oakville, ON). The 15.0-ml concentrate (approximately 100 mg/ml) was then subjected to gel filtration on three columns linked in tandem (2.5 102 cm) containing AcA-54 equilibrated in HBS. The column was developed at 4 °C with HBS at a flow rate of 0.42 ml/min, collecting 4.4 ml/fraction. Fractions containing activity were pooled and subjected to affinity chromatography at room temperature on a 5.0-ml Pgn-Sepharose CL-4B (9.8 mg/ml resin) column equilibrated in HBS. After washing with 5 column volumes of HBS and then a similar volume of HB, TAFI was eluted with 50 mM ACA, 0.01% Tween 80, HB. Since ACA is antifibrinolytic, these fractions could not be assayed. Therefore, ACA was separated from TAFI by passing the pooled ACA eluate over a 1.0-ml DEAE fast flow column equilibrated in HB and washing with 40 ml of HB. TAFI was then eluted with 0.2 M NaCl, HB. Peak fractions were pooled and stored at 4 °C, and subsequent experiments were performed within 48 h of isolation.

Amino-terminal Sequence, Amino Acid Composition, and Extinction Coefficient Determinations

Microamino acid sequence analyses were performed by the Core Facility for Protein/DNA Chemistry at Queen's University. Sequences were determined following electroblotting of bands, resolved by SDS-PAGE, to polyvinylidene difluoride (Millipore, Bedford, MA)(32) , using an Applied Biosystems model 475A amino acid analyzer with on-line phenylthiohydantoin analysis and reverse-phase separation on a C18 column. Quantitative amino acid analysis was performed by HSC Biotechnology Service Centre (Toronto, ON). To determine the extinction coefficient of TAFI, duplicate samples of known A, containing norleucine as an internal standard, were subjected to hydrolysis with 6.0 N HCl followed by derivatization with phenylisothiocyanate. Separation and quantitation of the derivatized amino acids were accomplished using the Picotag processing method and using absorbance at 254 nm, respectively. The , using the peptide molecular weight of 48,442(3) , is 26.4 ± 3.6, and this value was used to calculate all concentrations for TAFI. (The molar extinction coefficient is 1.28 10M cm, which is 1.44-fold greater than that calculated from the tyrosine and tryptophan content(33) .)

Radiolabeling Plasminogen and TAFI

GluPgn and TAFI were iodinated with I using IODO-BEADS (Pierce Chemical Co.). IODO-BEADS were first washed with 1.0 ml of TBS; 500 µl of TBS and 1.0 mCi of NaI (ICN, Mississauga, ON) were added and incubated at room temperature for 5 min. The solution was then removed and added directly to another tube containing GluPgn (2.0 mg/ml) in 500 µl of TBS, and incubation was carried out for 10 min at room temperature. The reaction was quenched by the addition of 10 µl of 1.0 M sodium metabisulfite, and unincorporated I was removed by gel filtration on a 10.0-ml Sephadex G-25 column (Pharmacia). The final concentration of I-plasminogen was 0.89 mg/ml, and the specific radioactivity was 4.5 cpm/ng. Iodination of TAFI was performed identically except the buffer was HBS and the final concentration was 0.064 mg/ml with a specific radioactivity of 234 cpm/ng.

Thrombin Cleavage of TAFI

The reaction was carried out in a 1.5-ml Eppendorf tube containing 100 µl of TAFI (0.145 mg/ml, 90 µl of 0.153 mg/ml TAFI plus 10.0 µl of 0.064 mg/ml I-TAFI) in HBS. CaCl (1.0 M) was added to a final concentration of 10 mM. A 10-µl aliquot was removed for the zero time point, and the remaining 100 µl was brought to 545 nM IIa by the addition of 9.0 µl of 6.0 µM IIa. The reaction mixture was then incubated at 37 °C, and at various times a 10.0-µl aliquot was removed and immediately prepared for SDS-PAGE. Preparation for electrophoresis included the addition of 10.0 µl of HBS and 5.0 µl of a solution consisting of 25% glycerol, bromphenol blue, 5% SDS, 75 mM EDTA, pH 7.4, and heating the mixture for 3.0 min at 90 °C. Approximately 1.5 µg of TAFI and 15,000 cpm were loaded per well and run under the conditions of Neville (34) on a 5-15% gradient minigel (Hoefer, model SE250, Technical Marketing, Ottawa, ON). The gel was stained with Coomassie Blue, destained, dried in Biowrap (BioDesign Inc., Carmel, NY), and subjected to autoradiography using Kodak XAR-5 x-ray film overnight at -70 °C. Densitometry was performed using an LKB-2202 densitometer (Pharmacia).

Activation of TAFI by Thrombin

The effect of preincubation of TAFI with IIa on lysis time in the presence and absence of FXa and on the rate of hydrolysis of hippuryl-Arg was determined. Incubation of 3.14 µM TAFI with 545 nM IIa in 10.0 mM Ca, HBS was carried out in a final volume of 33 µl for various periods of time (0-2.0 h) at 37 °C. For each incubation time point a clot was produced by the addition of the fibrinogen solution to wells in which a 1.25-µl aliquot of the TAFI activation medium had been placed, giving a final concentration of 40 nM TAFI and 6.8 nM IIa. Included also in the wells were Ca (10.0 mM, final) and t-PA (441 pM, final) plus or minus FXa. Simultaneously, an aliquot (30 µl) of the TAFI activation medium was removed and added to 1.0 ml of 0.1 mM hippuryl-Arg in 25 mM Tris, 0.1 M NaCl, pH 7.65, and absorbance at 254 nm was monitored over time in a Perkin-Elmer Lambda 4b spectrophotometer(35) .

Inhibition of Activated TAFI and Porcine CBB by GEMSA

Fibrinogen solution diluted 1/5 or a solution of BAP, diluted 1/3 with HBS plus PC/PS vesicles (10.0 µM, final) and II (0.73 µM), were supplemented with GEMSA at various concentrations ranging from zero to 2.0 mM. The correlation between lysis time obtained in the absence and presence of FXa (100 pM) and the time of incubation of TAFI with IIa was determined. Lysis times of clots prepared from 95-µl aliquots (see under ``Lysis Assay'') in the presence and absence of FXa (i.e. plus or minus II activation) were determined for each of the GEMSA concentrations. These experiments thus constituted an evaluation of the effect of GEMSA on TAFI-mediated prolongation of lysis. In a similar series of experiments, fibrinogen solution, diluted 1/5, supplemented with GEMSA at various concentrations, and porcine CPB (7.4 nM, final), was used in place of TAFI, and lysis times were determined in the absence of FXa. These experiments were performed to evaluate the effect of GEMSA on the porcine CPB-mediated prolongation of lysis.

Activation of TAFI during Fibrinolysis

To investigate the activation of TAFI in a fibrin clot during fibrinolysis, two sets of 12 identical clots were formed from fibrinogen solution containing 110 nMI-TAFI, in the presence of IIa (6.0 nM, final), Ca (10.0 mM, final) and t-PA (441 pM, final) in the wells of a microtiter plate. One set was formed in the presence of FXa (100 pM) and one set in its absence. At various times a fibrin clot from each series was solubilized by the addition of 100 µl of 0.2 M acetic acid, thereby quenching all reactions. Once the clot was solubilized, 55 µl of a solution composed of 5% SDS, 20% mercaptoethanol, 50 mM EDTA, 20% glycerol/HO containing bromphenol blue, and 40 mM Tris-HCl, pH 8.3, was added, and the solution was heated for 5 min at 90 °C. One clot from each series was allowed to lyse completely, and the turbidity profile from these clots was used to determine lysis times. After lysis, this clot was similarly prepared for SDS-PAGE. The samples were then subjected to SDS-PAGE; the resulting gel was stained, destained, and dried, and an autoradiogram was produced.

Effect of LysPgn on TAFI-induced Prolongation of Lysis Time

To determine the effect of LysPgn on TAFI-induced prolongation of lysis time, two fibrinogen solutions were produced, each identical, except one contained 0.86 µM GluPgn and the other 0.90 µM LysPgn. From each stock solution, six 200-µl samples were produced to which various concentrations of TAFI were added, such that the final concentrations ranged from 0 to 31 nM for each group of six. Clots (100 µl) then were formed in the presence and absence of FXa (see under ``Lysis Assay''), and lysis times were determined.

Effect of TAFI on Plasminogen Activation

Two series of identical clots were formed from fibrinogen solution containing 140 nM TAFI and 0.47 µMI-plasminogen by adding 95-µl aliquots to microtiter wells containing either FXa (100 pM, final) or buffer, and IIa (6.0 nM, final), Ca (10.0 mM, final) and t-PA (441 pM, final). Each clot was incubated at 37 °C, and turbidity was monitored to determine lysis time. At various times between 0 and 200 min the reactions in one clot from each series (plus and minus FXa) were quenched, and the fibrin was solubilized by adding 25 µl of 10.0 M urea, 2.0 M acetic acid and heating at 90 °C for 3 min. All samples were then frozen at -20 °C prior to acid-urea electrophoresis(28) . Samples were thawed by heating for 3 min at 90 °C, and a 5.0-µl aliquot was counted in a LKB mini-gamma counter (Pharmacia) to account for gel loading differences. Twenty µl from each sample was subjected to acid-urea 7.5% polyacrylamide gel electrophoresis (28) at 120 V for 1.5 h in a mini-slab gel electrophoresis apparatus. The gel was stained with Coomassie Blue, destained, and dried in Biowrap prior to autoradiography. Areas on the gel corresponding to bands on the autoradiogram were excised, and the normalized counts were plotted as a function of time. The species that were excised included GluPgn, LysPgn, and plasmin--antiplasmin complexes.


RESULTS

Assay and Generation of a Standard Curve

To assess the efficiency of each step in the purification of TAFI a turbidometric fibrinolysis assay was developed. The assay utilizes purified components and is based on the prolongation of lysis which accompanies II activation. Therefore, TAFI activity was assessed by the prolongation of lysis time in the presence of FXa relative to the lysis time in the absence of FXa. Neither the fibrinogen solution nor BAP individually exhibited any relative prolongation. Prolongation, however, could be reconstituted in clots formed from BAP supplemented with II and PC/PS vesicles, or from the fibrinogen solution supplemented with BAP. (Not all isolates of GluPgn, however, yielded identical lysis times in fibrinogen solution in the presence and absence of FXa, which suggests that some plasminogen preparations contain traces of contaminating TAFI, as inferred previously(1) ). Fig. 1shows the effect of various volumes (0-25 µl/100-µl clot) of BAP on the lysis time of clots formed from fibrinogen solution in the presence and absence of FXa. The differential in lysis times plus and minus FXa increases linearly over the concentration of BAP studied and provides a measure of the TAFI concentration.


Figure 1: Standard curve: effect of various concentrations of BAP on lysis time of clots produced from fibrinogen solution in the presence and absence of FXa. Lysis times were determined for clots (100 µl) that were formed from fibrinogen solution with various concentrations of BAP (0-25% v/v) in the presence of IIa, Ca, and t-PA and incubated at 37 °C in the absence () or presence () of FXa. The absorbance at 405 nm was monitored at 2.5-min intervals in an enzyme-linked immunosorbent assay reader, and the time corresponding to the transition midpoint in the reduction in turbidity is the value reported as lysis time.



Purification of TAFI

BAP was subjected to differential ammonium sulfate precipitation, followed by anion exchange chromatography on Q-Sepharose. TAFI eluted at 50 mM NaCl and was located in fractions that were typically orange in color. Gel filtration chromatography was then performed. TAFI eluted on the trailing edge of the second of two protein peaks. Affinity chromatography was then performed on Pgn-Sepharose (Fig. 2). The flow-through contained less than 2.0% of the TAFI activity. TAFI was eluted as a small trailing protein peak. The eluate was passed over a 1.0-ml DEAE fast flow Sepharose column, and TAFI was eluted with 0.2 M NaCl, 20 mM HEPES, pH 7.4. The protein and TAFI activity loaded on the column eluted with quantitative recovery in a single peak of 3.0-ml total volume (Fig. 2, inset). This procedure produced a 14,300-fold purification with a 12.1% recovery of TAFI activity (Table) I. The isolated material migrated as a single band on SDS-PAGE with a mass of 60 kDa (Fig. 3).


Figure 2: Affinity chromatography of TAFI on Pgn-Sepharose. The pooled material recovered from gel filtration was passed over 5 ml of Pgn-Sepharose. The absorbance profile is shown. The column was washed with HBS (fractions 15-26), then HB (fractions 27-37), and eluted with 50 mM ACA, 0.01% Tween 80 in HB. Fractions 40-60 were pooled and subjected to anion exchange chromatography on a 1.0-ml DEAE fast flow column at 22 °C, collecting 1.0-ml fractions. Both the absorbance profile () and the prolongation activity () are shown (inset). A peak of activity, coincident with an absorbance peak, eluted with 0.2 M NaCl in HB.




Figure 3: Coomassie Blue-stained gel of fractions from Pgn-Sepharose and DEAE chromatography. Samples were subjected to SDS-PAGE on a 5-15% gradient gel under nonreducing conditions. Shown are 20 µg of protein from Pgn-Sepharose load (lane 1); Pgn-Sepharose flow-through (lane 2); and 2.0, 1.5, and 0.5 µg of protein from the 0.2 M NaCl/HB eluate of DEAE-Sepharose fractions 41, 42, and 43 (Fig. 2, inset), respectively (lanes 3, 4, and 5).



Antifibrinolytic Effect of TAFI

Fig. 4shows the lysis time, in both the presence and absence of FXa, and relative lysis time for each concentration of TAFI. A dose-dependent increase in lysis time in the absence and presence of FXa was observed. In the absence of FXa lysis time increased, modestly and linearly, from 30 to 50 min; however, in the presence of FXa lysis time increased substantially more, in a saturable manner, from 30 to 130 min. A similar phenomenon was also observed in clots produced from a fibrinogen solution not containing the inhibitors antiplasmin and AT-III in the presence of a reduced concentration of t-PA (14.7 pM). The control times without FXa, however, were more sensitive to the concentration of TAFI. In the absence of the inhibitors the lysis time with FXa (Lt+FXa) increased, in a saturable manner, from 55 to 100 min by 125 nM TAFI. The lysis time in the absence of FXa (Lt-FXa) increased linearly from 55 to 133 min over the concentration range of TAFI. The TAFI-dependent increase in Lt-FXa in both the presence and absence of the inhibitors AT-III and antiplasmin indicates that the IIa (6.0 nM), present to initiate clot formation, is capable of activating TAFI, at least partially. This effect is more pronounced in the absence of AT-III and antiplasmin than in their presence. The observation that, in the absence of inhibitors, at high concentrations of TAFI the Lt-FXa (where the IIa concentration is low, 6.0 nM) exceeds Lt+FXa (where the IIa concentration is high, maximally 730 nM) suggests that TAFI is both activated and inactivated by IIa.


Figure 4: Antifibrinolytic effect of TAFI. Lysis times were determined for 100-µl clots produced from fibrinogen solution containing various concentrations of TAFI (0-313 nM), produced in the absence () and presence of FXa (), from which relative lysis times were calculated (). All clots were formed in the presence of IIa (6.0 nM), Ca (10.0 mM), and t-PA (441 pM) and maintained at 37 °C while absorbance at 405 nm was monitored.



Thrombin Cleavage of TAFI

A reaction solution composed of TAFI and I-TAFI in HBS and 10 mM Ca was incubated at 37 °C with IIa for up to 2.0 h. At various times an aliquot was removed, prepared, and subsequently subjected to SDS-PAGE followed by autoradiography. Densitometry of both the Coomassie Blue-stained gel and the autoradiogram shows that the cleavage patterns of TAFI and I-TAFI by IIa were identical (data not shown). The autoradiogram (Fig. 5) shows that TAFI is cleaved to produce three major species that migrate with a molecular mass of 35, 25, and 14 kDa. Densitometry of the autoradiogram indicated that initially the 35- and 14-kDa bands appear, followed by the 25-kDa band. The intensity of the 35-kDa band increased to a maximum of 24% of the total radiolabel by 30 min. This level is maintained until 60 min, after which it begins to decrease and approaches 14% by 120 min. This is in contrast to both the 25- and 14-kDa bands, which increase over the duration of the experiment to 56 and 18%, respectively. The protein migrating at 60 kDa decreases continuously over the same time period to 13%. The cleavage pattern suggests that the 60-kDa protein is cleaved to form the 35- and 14-kDa fragments. The 35-kDa fragment can be proteolyzed further to yield products that migrate at 25 and 14 kDa. The 60-, 35-, and 25-kDa bands were sequenced, and the first five residues of each were (FQSGA; 60 kDa), (ASASY; 25 kDa) and (XQAG(A or Y); 35 kDa). The sequences corresponding to the 60- and 25-kDa proteins were unambiguously determined, whereas the sequence data of the 35-kDa protein did not permit the identification of the first amino acid, and there appeared to be two possibilities for the identity of the fifth amino acid. The sequences unambiguously assigned correspond to that of plasma CPB(3) . However, our inability to fit the sequence XQAG(A or Y) to the published sequence of plasma CPB suggested that the band migrating at 35 kDa is composed of two unresolved proteins, with respective sequences corresponding to those of the 60- and 25-kDa proteins. Since serine is not well quantitated by the sequencing method utilized, the single sequence obtained may be accounted for by combining the sequences of both the 60- and 25-kDa NH termini and disregarding the serines. The cleavage patterns also are consistent with the identification of TAFI as the plasma CPB precursor reported by Eaton et al. (3).


Figure 5: Thrombin-catalyzed cleavage of TAFI. The time course of product formation by IIa-catalyzed cleavage of TAFI was determined by SDS-PAGE and both Coomassie Blue staining and autoradiography. I-TAFI was incubated with IIa (545 nM) in the presence of Ca (10.0 mM). At various times an aliquot was removed, quenched, and subjected to SDS-PAGE on a 5-15% gradient gel. The gel was stained and dried and then subjected to autoradiography. The autoradiogram shows TAFI (lane 1), TAFI with IIa quenched immediately after the addition of IIa (lane 2), and the cleavage products after 10, 20, 30, 45, 60, 75, 90, and 120 min of incubation (lanes 3-10), respectively.



Activation of TAFI by Thrombin

Activation of TAFI by IIa was investigated by incubating TAFI with IIa for various lengths of time and then assaying the activated TAFI for prolongation activity in the lysis assay and for CPB activity using hippuryl-Arg. Lysis times are plotted as a function of incubation time of TAFI with IIa in Fig. 6a. Without incubation of TAFI with IIa, Lt+FXa was 73 min compared with Lt-FXa of 37 min and corresponds to a relative prolongation of 1.97. Lt+FXa was invariant at approximately 70 min for all incubation times less than 45 min. Longer incubation times, however, were accompanied by decreased values of Lt+FXa. In contrast, Lt-FXa increased to 47 min at the earliest time point measurable (0.5 min) and attained a value of 70 min by 10 min of incubation, which is equal to the Lt+FXa at that point. Lt-FXa then paralleled Lt+FXa. These data correspond to a reduction in the relative lysis time from 2 to 1 by a 10-min incubation, which is maintained for the remaining periods of incubation. The relative lysis time, however, does not reflect the initial activation followed by inactivation observed with the absolute lysis times. Profiles of both the CPB activity, as reported by rates of hippuryl-Arg hydrolysis, and Lt-FXa as a function of incubation time of TAFI with IIa were similar (Fig. 6b), indicating that prolongation activity correlates with CPB activity. By comparing both the prolongation activity and the CPB activity with the banding patterns on gel in Fig. 5, it is apparent that the only band that has a time course similar to these activities is the band that migrates at 35 kDa, suggesting that formation of this species correlates with activation of TAFI.


Figure 6: Activation of TAFI by thrombin. Activation of TAFI by IIa was investigated by both prolongation of lysis time (panel a) and cleavage of hippuryl-Arg (panel b). TAFI (3.14 µM) was incubated with IIa (545 nM) in the presence of Ca (10.0 mM) at 37 °C for various periods of time (0-120 min). Shown in panel a are both the absolute lysis times (,) and relative lysis times (squlo]) of clots produced from fibrinogen solution containing 1.25 µl of the TAFI activation solution, IIa (6.8 nM), and both Ca (10.0) and t-PA (441 pM). The lysis times were measured in the absence () and presence () of FXa (100 pM, final). For each incubation time with IIa, a third aliquot (30 µl) was removed and added to 1.0 ml of a 1.0 mM hippuryl-Arg solution. Absorbance at 254 nm was monitored at 37 °C, and rates of hippuryl-Arg hydrolysis were determined. Shown in panel b are rates of hippuryl-Arg hydrolysis () and lysis times in the absence of FXa (), each as a function of incubation time of TAFI with IIa.



Effect of GEMSA on Prolongation of Lysis Time in Reconstituted BAP or a System of Purified Components in Which Either Porcine CPB or TAFI Is Included

GEMSA is a competitive inhibitor of CPB (3, 36) and therefore was used to determine whether the prolongation observed by TAFI in a purified system accounts for a significant proportion of the prolongation of lysis time observed in clots produced from BAP (diluted with HBS, A = 16) reconstituted with II and PC/PS vesicles in the presence of FXa. Porcine CPB prolonged the lysis times of clots produced from the fibrinogen solution without the addition of FXa. Therefore, prolongation is expressed as the ratio of the lysis time with porcine CPB to that in its absence. Lysis time in the absence of porcine CPB was 41 min and increased to 110 min in its presence, giving a relative prolongation of 2.68. TAFI, in the absence of GEMSA, produced a 2-fold increase in lysis time from 36 to 72 min when FXa was included. The Lt+FXa of the clot produced from reconstituted BAP was 2.17-fold longer than Lt-FXa, 60 min. Shown in Fig. 7is relative lysis time, normalized to the maximum value, for each set of conditions. GEMSA completely inhibited the relative prolongation observed with porcine CPB with an EC of 0.34 µM. GEMSA also completely inhibited the prolongation of lysis time induced by FXa, in both the fibrinogen solution with TAFI, and in reconstituted BAP. The EC values were 100 and 115 µM, respectively.


Figure 7: Effect of GEMSA on prolongation of lysis time. Relative prolongation of lysis at various concentrations of GEMSA was determined for clots produced from fibrinogen solution in the presence of 7.4 nM porcine CPB (), fibrinogen solution in the presence of 40 nM TAFI ± FXa(100 pM) (), and BAP supplemented with 0.73 µM II, 10.0 µM PC/PS vesicles ± FXa (100 pM) (). Relative prolongation was normalized to the maximum prolongation observed, i.e. that in the absence of GEMSA, for each set of conditions. Maximum relative prolongations observed for porcine CPB, fibrinogen solution plus TAFI ± FXa, and reconstituted BAP ± FXa were 2.68, 2.0, and 2.17, respectively.



These data indicate that although activated TAFI and porcine CPB are not equally sensitive to GEMSA, activated TAFI can be inhibited by a competitive inhibitor of porcine CPB. These data further support the conclusion that activated TAFI is a carboxypeptidase. As a result of the near identity of the EC values for complete inhibition of prolongation of lysis by GEMSA, in both BAP and a system of defined components including TAFI, it is likely that TAFI accounts for a significant proportion, if not all, of the prolongation of lysis time observed in clots produced from plasma in the presence of FXa.

Effect of APC on FXa-dependent Prolongation of Lysis Time in the Presence and Absence of GEMSA

In a separate experiment the effect of APC (100 nM) on lysis times of BAP (A = 16) supplemented with 0.73 µM II and 10.0 µM PC/PS vesicles in the presence and absence of 500 µM GEMSA was determined. Clots were formed in the presence of IIa (6.0 nM, final) and Ca (10.0 mM) plus and minus FXa (100 pM). Lysis time (20 min) was unaffected by either APC or GEMSA, individually or in combination, in the absence of FXa, and the 2.5-fold prolongation observed in the presence of FXa was not observed when either APC or GEMSA or both were present. In the presence of FXa, the reduction in lysis time by APC in the absence of GEMSA was the same as that observed when GEMSA was included also. Therefore, both APC and GEMSA can inhibit prolongation of lysis time in reconstituted BAP, and they both appear to inhibit prolongation through the same mechanism since the profibrinolytic effect of APC is not observed in the presence of GEMSA.

Activation of TAFI during Fibrinolysis

The autoradiograms shown in Fig. 8, a and b, indicate the time courses of I-TAFI activation during fibrinolysis of clots produced in the absence and presence of FXa, respectively. The lysis times were determined to be 60 min in the absence of FXa and 170 min in the presence of FXa. In the absence of FXa, neither the 35-kDa nor the 25-kDa species is readily apparent until 60 min, whereas in the presence of FXa the 35-kDa band is apparent by 10 min, and the 25-kDa band is apparent by approximately 30 min. Since the 35-kDa band correlates with both prolongation and CPB activity, it is likely that not only is TAFI activated during fibrinolysis in the presence of FXa, but also that TAFI accounts for the prolongation observed when II is activated during fibrinolysis.


Figure 8: Activation of TAFI in a clot. Two series of identical clots were produced from fibrinogen solution containing I-TAFI in the presence of IIa, Ca, and t-PA, except one series was produced in the absence (panel a) and one series in the presence (panel b) of FXa. The turbidity at 405 nm was monitored for each clot at 37 °C. At various times samples were quenched and subjected to SDS-PAGE on a 5-15% gradient gel. Shown are the autoradiograms where lanes 1-12 represent the time points 0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, and 200 min, respectively. Lysis times, indicated by arrows, were 60 min in the absence of FXa and 170 min in the presence of FXa.



Effect of LysPgn on TAFI-induced Prolongation of Lysis Time

The effect of TAFI on fibrinolysis has thus far been described using GluPgn in the fibrinogen solution. GluPgn, however, can be cleaved to form LysPgn during fibrinolysis(37) . Since Glu and LysPgn have different fibrin cofactor requirements for activation(38) , the effect of TAFI on fibrinolysis using either Glu or Lys Pgn as the initial source of plasminogen was investigated. The lysis times for GluPgn in the absence of FXa averaged 50 min, and there was a TAFI-dependent increase in lysis time, in the presence of FXa, from 50 to 72 min. Lysis times for clots produced with LysPgn averaged 47 min in the absence of FXa and 46 min in the presence of FXa over the range of TAFI used. Fig. 9shows the relative prolongation observed for each concentration of TAFI in the presence of Glu or LysPgn. In contrast to the 1.38-fold prolongation in the presence of GluPgn, no prolongation was observed in the presence of LysPgn over the same range of TAFI concentrations. These data indicate that clot lysis by plasmin generated from LysPgn is unaffected by activated TAFI. This observation is consistent with the concept that carboxyl-terminal lysines are less important in the activation of LysPgn than GluPgn(38) , implies that activated TAFI exerts its effect prior to the formation of LysPgn or plasmin, and suggests that activated TAFI inhibits the activation of GluPgn.


Figure 9: Effect of LysPgn on TAFI-induced prolongation of lysis time. Relative lysis times were determined for clots produced from fibrinogen solution containing either GluPgn () or LysPgn () in the presence of TAFI and the presence and absence of FXa.



Activation ofI-GluPgn during Fibrinolysis

Two series of 12 identical clots containing I-plasminogen were formed, one in the presence and one in the absence of FXa. At various times the clots were solubilized and quenched. One clot from each series was allowed to lyse completely. Subsequently, each sample was subjected to acid-urea gel electrophoresis and autoradiography. Fig. 10shows the time course of GluPgn consumption and concomitant formation of plasmin-antiplasmin for clots formed in the presence and absence of FXa, overlaid with their respective turbidometric profiles. The lysis times were determined to be 44 min in the absence of FXa and 87 min in the presence of FXa, indicating an approximate 2-fold prolongation. In both instances the GluPgn consumed equals the sum of the plasmin-antiplasmin complexes and LysPgn accumulated. Consumption of GluPgn in the absence and presence of FXa ceased after the fibrin had been lysed. In the interval from 0 to 20 min, in both instances, rates of plasminogen activation, as inferred by the slopes of plasminogen consumption, were approximately equal and relatively slow. Between 20 and 40 min, however, the rate in the absence of FXa was approximately 10-fold greater than that in the presence of FXa. These data show that activated TAFI suppresses fibrinolysis by attenuating the activation of GluPgn, especially following the lag phase typically characteristic of plasminogen activation(39) .


Figure 10: Activation of I-plasminogen during fibrinolysis. I-Plasminogen was used to follow the activation of plasminogen in clots by autoradiography in the presence of TAFI (140 nM). One series of clots was formed in the absence and one in the presence of FXa. At various times clots were quenched and subjected to acid-urea electrophoresis and autoradiography. Bands on the gel corresponding to radiolabeled GluPgn, LysPgn, and plasmin-antiplasmin complexes were excised, counted, and concentrations of each species were calculated. Shown is the consumption of GluPgn (,), formation of plasmin-antiplasmin complexes (,), and lysis profiles (- -,--) in the absence (,,- -) and presence (,],--) of FXa.




DISCUSSION

TAFI was purified from BAP by differential precipitation with (NH)SO, anion exchange chromatography, gel filtration, and affinity chromatography using Pgn-Sepharose. The purification scheme resulted in a 12.1% recovery of activity with a 14,300-fold purification. Based on amino-terminal sequence data we have identified TAFI as a plasma CPB, as described by Eaton et al.(3) . Utilizing a of 26.4, a M of 48,422, and a recovery of 12.1% we calculate that the concentration of TAFI in plasma is approximately 50 nM.

We also provide evidence that TAFI is cleaved by IIa to produce a 35-kDa fragment that correlates with both a relative increase in lysis time, in a system of defined components, and CPB activity. Furthermore, we show that the specific carboxypeptidase inhibitor GEMSA completely inhibits the prolongation of lysis time in a system of defined components observed in the presence of active porcine CPB and in a similar system in which TAFI is activated by IIa produced in situ. Since complete inhibition of the prolongation observed in BAP supplemented with II and PC/PS vesicles in the presence of FXa occurs with an EC identical to that of TAFI in a system of defined components, we conclude that the prolongation observed in plasma can most likely be attributed to the carboxypeptidase activity generated by the activation of TAFI by IIa.

The inhibition of prolongation of lysis time by GEMSA and the formation of the 35-kDa fragment of TAFI during fibrinolysis indicate that carboxypeptidase activity not only prolongs lysis time but also can be generated in situ. The relatively low abundance of the 35-kDa fragment, in comparison to the fragments migrating between 14 and 21 kDa (Fig. 8), suggests that a steady-state level of activated TAFI is attained during fibrinolysis. Degradation of activated TAFI, yielding the small fragments in higher abundance, may involve any one or a combination of the proteases IIa, plasmin, FXa, or t-PA which are present in the system. Furthermore, it is unknown if these or any of the other cleavage products are either partially processed precursors or degradation products with or without CPB activity.

The mechanism by which activated TAFI prolongs lysis time is unclear; however, it is able to modulate t-PA-induced activation of GluPgn. Since activated TAFI is unable to modulate fibrinolysis when GluPgn is replaced by LysPgn, it likely does not effectively inhibit either activation of LysPgn by t-PA or plasmin activity directly. Furthermore, the substrate specificity of activated TAFI (i.e. plasma CPB) appears to be limited to basic amino acids (3, 40). Thus, a reasonable hypothesis is that activated TAFI modifies the fibrin cofactor by removal of carboxyl-terminal lysines, thereby inhibiting the activation of GluPgn. An alternative hypothesis is that the released lysine is antifibrinolytic. This is unlikely, however, in view of the results with LysPgn. The lack of an observed prolongation when LysPgn is the substrate for t-PA is likely a result of its activation being less dependent on the presence of a cofactor with carboxyl-terminal lysines(38) . The results with LysPgn also suggest that the Glu to LysPgn conversion may contribute to the regulation of fibrinolysis by attenuating the effects of activated TAFI.

Several previous studies indicated that APC influences fibrinolysis both in vivo and in vitro(1, 13, 14, 15, 16, 41) . Furthermore, we have shown previously that II activation during fibrinolysis in vitro is antifibrinolytic(1) . Since APC is an anticoagulant and is therefore able to inhibit the activation of II we concluded that APC appears profibrinolytic by virtue of its ability to prevent the antifibrinolytic effect generated by II activation(1) . In addition, the species specificity of the anticoagulant effect of APC, which is due to the species specificity of the cofactor protein S, is also exhibited in the profibrinolytic effect of APC, as shown by Weinstein and Walker(17) . The results of this study rationalize the apparent antifibrinolytic effect of II activation by thrombin's ability to activate TAFI, which subsequently exhibits CPB-like activity and inhibits fibrinolysis. Thus, the rationale for the profibrinolytic effect of APC lies with its anticoagulant properties. By inhibiting the activation of II, APC prevents the subsequent activation of TAFI resulting in a reduced lysis time when compared with that observed when II activation occurs.

  
Table: Isolation of TAFI

Aliquots from each step of the purification were dialyzed against HBS. Activity per unit volume of a sample added to the assay is defined by the expression activity/µl sample = ((Lt + Xa/Lt - Xa) - 1)/volume sample (µl). A unit of activity is defined as the activity in 1.0 ml of BAP.



FOOTNOTES

*
This work was supported by Medical Research Council of Canada Grant MT112144, Heart and Stroke Foundation of Ontario Grant T-2631, and by a Heart and Stroke Foundation of Canada Research Traineeship (to L. B.). 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: Dept. of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6. Tel.: 613-545-2957; Fax: 613-545-2987.

The abbreviations used are: FXa, FVa, FX, FV, factors Xa, Va, X, and V, respectively; Pgn, plasminogen; t-PA, tissue-type plasminogen activator; AT, antithrombin; APC, activated protein C; TAFI, thrombin-activatable fibrinolysis inhibitor; CPB, carboxypeptidase B; GEMSA, 2-guanidinoethylmercaptosuccinic acid; DMEM, Dulbecco's modified Eagle's medium; PC/PS, phosphatidylcholine/phosphatidylserine; BAP, barium-adsorbed plasma; PAGE, polyacrylamide gel electrophoresis; Lt+FXa, lysis time with FXa; Lt-FXa, lysis time without FXa; ACA, epsilon-amino caproic acid.


ACKNOWLEDGEMENTS

We acknowledge gratefully numerous fruitful discussions with Dr. Anton Horrevoets.


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