HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 47, 36612-36620, November 24, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/47/36612    most recent M005483200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stewart, R. J. Articles by Weitz, J. I. Search for Related Content PubMed PubMed Citation Articles by Stewart, R. J. Articles by Weitz, J. I. Social Bookmarking                      What's this?

Thrombin-activable Fibrinolysis Inhibitor Attenuates (DD)E-mediated Stimulation of Plasminogen Activation by Reducing the Affinity of (DD)E for Tissue Plasminogen Activator

A POTENTIAL MECHANISM FOR ENHANCING THE FIBRIN SPECIFICITY OF TISSUE PLASMINOGEN ACTIVATOR^*

Ronald J. Stewart, James C. Fredenburgh, Janice A. Rischke, Laszlo Bajzar^§, and Jeffrey I. Weitz^¶

From the Hamilton Civic Hospitals Research Centre and McMaster University, Hamilton, Ontario L8V 1C3, Canada

Received for publication, June 22, 2000, and in revised form, August 4, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A complex of D-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k_cat and increasing K_m for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and D-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intravascular fibrinolysis is initiated when plasminogen (Pg)¹ is converted to plasmin by tissue-type plasminogen activator (t-PA) (1, 2). Plasmin then degrades fibrin, yielding soluble fibrin degradation products. Through a positive feedback mechanism, fibrin enhances its own degradation by stimulating t-PA-mediated Pg activation. To potentiate this reaction, fibrin acts as a template onto which both t-PA and Pg bind (3). The activator and its substrate bind to independent sites on intact fibrin because the t-PA interaction is primarily mediated by its fibronectin finger-like domain, whereas Pg binding is kringle-dependent (3-5). As a functional consequence of t-PA and Pg interaction with fibrin, the catalytic efficiency of t-PA-mediated Pg activation is 2-3 orders of magnitude greater in the presence of fibrin than in its absence (1, 3, 6). In contrast to the potent stimulatory effect of fibrin, fibrinogen (Fg) produces only a 25-fold enhancement in the catalytic efficiency of Pg activation by t-PA (1, 7). Because t-PA preferentially activates Pg in the presence of fibrin rather than Fg, it is designated a fibrin-specific Pg activator.

When cross-linked fibrin is solubilized by plasmin, a major degradation product is (DD)E, a complex of D-dimer (DD) noncovalently associated with fragment E (8, 9). Recently, we demonstrated that (DD)E compromises the fibrin specificity of t-PA, because this soluble fragment is as potent as fibrin at stimulating Pg activation by t-PA (10, 11). Like fibrin, (DD)E binds t-PA and Pg with high affinity (5, 10, 12). In contrast to its predominantly finger-dependent interaction with fibrin, t-PA binds to (DD)E via its second kringle domain (4, 5, 11). Although Pg also binds to (DD)E in a kringle-dependent fashion, the activator and substrate do not compete for (DD)E binding, indicating that they have distinct binding sites (5).

A latent carboxypeptidase B (CPB)-like enzyme, termed thrombin-activable fibrinolysis inhibitor (TAFI), has recently been identified in plasma (13-15). When activated by the thrombin-thrombomodulin complex, activated TAFI (TAFIa) attenuates fibrinolysis, presumably by removing carboxyl-terminal lysine residues on fibrin, thereby removing the newly exposed binding sites for Pg that are generated as fibrin is degraded by plasmin (16, 17). TAFIa prevents the 2.5-fold rate enhancement of Pg activation that occurs during the early stages of fibrinolysis and blocks the conversion of Glu-Pg to the more readily activated Lys-Pg, a process also dependent on exposure of new carboxyl-terminal lysine residues (18, 19). Thus, TAFIa eliminates the enhancement of Pg activation associated with partial degradation of fibrin by plasmin.

Given its mechanism of action, we speculated that TAFIa or CPB would compromise the cofactor activity of (DD)E by abrogating Pg and/or t-PA binding. To explore this possibility, the ability of (DD)E to potentiate Pg activation by t-PA was examined before and after exposure of (DD)E to TAFIa or CPB. Herein we demonstrate that the stimulatory activity of (DD)E is nearly abolished upon exposure to TAFIa or CPB. Because plasmin generation in the presence of TAFIa- or CPB-exposed (DD)E is less than that with native (DD)E, TAFIa or CPB attenuates degradation of (DD)E into its constitutive fragments, E and DD, the latter of which can impair fibrin polymerization. To explore the mechanism responsible for this phenomenon, light scattering spectroscopy was used to compare the affinities of TAFIa- or CPB-exposed (DD)E for Glu-Pg, Lys-Pg, or t-PA with those of native (DD)E. TAFIa or CPB exposure reduces the affinity of (DD)E for Glu-Pg and Lys-Pg 2- and 4-fold, respectively. In contrast, the affinity of (DD)E for t-PA is reduced 160-fold. These data raise the possibility that by attenuating the systemic plasmin generation induced by (DD)E, TAFIa renders t-PA more fibrin-specific.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Plasminogen Activator-- Wild-type recombinant t-PA, kindly provided by Dr. B. Keyt (Genentech Inc., S. San Francisco, CA) was 93% single chain when analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on 4-15% gradient gels (Ready-Gel; Bio-Rad) as determined using an ImageMaster Video Documentation System (Amersham Pharmacia Biotech). Active site blocked t-PA was prepared by incubating the activator with a 5-fold molar excess of D-phenyl-prolyl-arginine chloromethyl ketone (PPACK; Calbiochem) as described previously (5, 20). The molecular weight and extinction coefficient used for t-PA were 65,000 and _1%²⁸⁰ = 20.0, respectively (4).

Plasminogen-- Native Glu-Pg was isolated from fresh frozen plasma by lysine-Sepharose affinity chromatography as described previously (5, 21). Isolated Glu-Pg was free of Lys-Pg based on urea/acetic acid PAGE analysis (22) and contained no plasmin as assessed using the plasmin-directed substrate D-valyl-leucyl-lysine p-nitroanilide dihydrochloride (S-2251; Chromogenix, Mississauga, Canada). Glu-Pg concentrations were calculated by measuring absorbances at 280 and 320 nm to distinguish tryptophan and phenylananine absorbance from light scattering, respectively (23), using a molecular weight of 90,000 and _1%²⁸⁰ = 16.1 (21). Lys-Pg was purchased from Enzyme Research Laboratories Inc. (South Bend, IN). Lys-Pg was free of Glu-Pg and contained no plasmin, determined as described above.

Fibrinogen-- Human Pg-depleted Fg was purchased from Enzyme Research Laboratories and rendered fibronectin-free by batch adsorption with gelatin-agarose (Sigma) for 30 min at room temperature followed by centrifugation at 3000 × g for 10 min (11). Fg concentration was calculated by measuring absorbances at 280 and 320 nm and using a molecular weight of 340,000 and _1%²⁸⁰ = 16.0 (24).

(DD)E-- The fibrin degradation product, (DD)E, was prepared by plasmin-mediated lysis of cross-linked fibrin clots as described previously (11). Briefly, 100 mg of Fg was clotted with 64 nM human -thrombin (Enzyme Research Laboratories) and 10 mM CaCl₂ in the presence of 93 nM activated recombinant factor XIII (a generous gift from Dr. P. Bishop, Zymogenetics Inc., Seattle, WA), 0.4 µM Glu-Pg, and 4.3 nM t-PA. Under these conditions, a clot formed within 30 s. After a 20-min incubation, the reaction was terminated by the addition of 5 µM D-valyl-phenyl-lysine chloromethyl ketone (VFKCK; Calbiochem) and 1 µM PPACK to inhibit plasmin and t-PA, respectively. Remaining aggregates were removed by centrifugation at 12,000 × g for 5 min, and fibrin degradation products were separated using a Biosep-Sec-S3000 size exclusion column (Phenomenex, Torrence, CA) fitted to a liquid chromatography system (System Gold; Beckman Instruments Inc., Palo Alto, CA). (DD)E-containing fractions were identified based on their apparent molecular weight and by immunoblot analysis using antibodies against DD and fragment E (12). When subjected to PAGE under nondenaturing conditions, (DD)E migrated as a single band. As expected, upon the addition of 10 mM H-Gly-Pro-Arg-Pro-OH (GPRP, Novabiochem, San Diego, CA) prior to native PAGE, or when subjected to SDS-PAGE, two lower molecular weight bands appeared, corresponding to DD and fragment E, respectively (8, 25). (DD)E concentrations were calculated by measuring absorbances at 280 and 320 nm and using a molecular weight of 250,000 and _1%²⁸⁰ = 16.0 (8).

Carboxypeptidase B and TAFIa-- CPB, potato tuber-derived CPB inhibitor (CPI), and the CPB-directed synthetic substrate hippuryl-L-arginine were purchased from Sigma. CPB activity was assessed by incubating 20 nM CPB with 0.4 mM hippuryl-L-arginine dissolved in 0.02 M Tris-HCl, 0.15 M NaCl, 0.01% Tween 20, pH 7.4 (TBS) in a quartz cuvette. Increases in absorbance at 254 nm were monitored for 20 min at 22 °C using a DU 7400 Spectrophotometer from Beckman (Mississauga, Canada). Under these conditions, CPB has a specific activity of 41 units/mg, where one unit hydrolyzes 1 µmol of hippuryl-L-arginine/min. When the experiment was repeated in the presence of 1 µM CPI, the lowest concentration used to inhibit CPB prior to Pg activation assays, no increase in A₂₅₄ was observed, indicating complete CPB inhibition. TAFI was isolated from fresh frozen human plasma by Pg-Sepharose affinity chromatography and activated with thrombin and soluble thrombomodulin as described elsewhere (15, 26). The specific activity of the resultant TAFIa against hippuryl-L-arginine was similar to that of CPB. Because TAFIa activity is unstable at room temperature (27, 28), TAFIa was used immediately or kept on ice until used. Like CPB, the activity of 20 nM TAFIa was completely inhibited by 1 µM CPI. CPB was used for the majority of experiments because its activity is more stable than that of TAFIa and it does not require preactivation. To demonstrate that TAFIa has effects similar to CPB, however, confirmatory experiments were done using TAFIa.

Methods

Effect of TAFIa or CPB on the Rate of (DD)E-stimulated Glu- and Lys-Pg Activation by t-PA-- To examine the effect of TAFIa or CPB on the ability of (DD)E to stimulate Pg activation by t-PA, 1.0 ml of a 8 µM (DD)E solution was incubated with 20 nM TAFIa or CPB for 40 min at 22 °C. At intervals, 20-µl aliquots were removed, and 2 µl of 40 µM CPI was added to inhibit the TAFIa or CPB. Complete TAFIa or CPB inhibition was achieved because no residual activity was detected using the CPB-directed synthetic substrate hippuryl-L-arginine. The ability of TAFIa- or CPB-treated (DD)E to stimulate Pg activation was then compared with that of the starting material by adding 20 µl of 2 mM S-2251 and 0.5 nM t-PA to wells of a 96-well microtiter plate containing 0.5 µM Glu-Pg or 0.12 µM Lys-Pg and 0.5 µM (DD)E, dissolved in 80 µl of TBS. Plasmin generation was assessed by measuring absorbance at 405 nm at 30-s intervals for 60 min using a Spectramax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) thermostated at 37 °C. Initial rates of plasmin formation were calculated by dividing the slope determined from the linear portion of the plot of A₄₀₅ versus time squared by the specific activity of plasmin (0.017 optical density units s¹ µM¹), which was determined in a separate experiment (5).

Effect of CPB or TAFIa on (DD)E Degradation during t-PA-mediated Pg Activation-- Plasmin degrades (DD)E to DD and fragment E (8, 9). Because exposure of (DD)E to CPB or TAFIa reduces its ability to potentiate Pg activation, we examined whether CPB or TAFIa indirectly attenuates (DD)E degradation during t-PA-mediated Pg activation. 4 µM (DD)E that had been exposed to 20 nM CPB for various intervals or to 20 nM TAFIa for 40 min was compared with 4 µM native (DD)E in terms of its ability to stimulate 0.4 µM Glu-Pg activation by 1 nM t-PA. Under these conditions, (DD)E concentrations were sufficient to monitor its degradation by PAGE analysis. After 1 h of incubation, the reaction was made to 1 µM VFKCK and 10 nM PPACK to inhibit plasmin and t-PA, respectively, and a 4-µl aliquot of the reaction solution was then subjected to PAGE analysis under nondenaturing conditions on 4-15% gradient gels, on 10% gels in the presence of SDS, and on 15% gels in the presence of SDS and -mercaptoethanol. Gels were stained with Fast Stain (Zoion Research, Shrewsbury, MA), and bands were quantified using an ImageMaster Video Documentation System. For amino acid sequencing, protein bands were transferred onto polyvinylidene difluoride membranes and stained with Ponceau S. Appropriate bands were cut from the membrane and subjected to amino-terminal sequence analysis, which was performed by Biotechnology Service Center (University of Toronto).

Determination of the Release of Free Lysine and Arginine from (DD)E by CPB or TAFIa-- To determine whether CPB or TAFIa releases lysine and/or arginine residues from (DD)E, (DD)E was incubated with 20 nM CPB or TAFIa for intervals up to 60 min, and free lysine and arginine were measured using methods similar to those described by Wang et al. (19). After dialysis of (DD)E into 50 mM HEPES, 150 mM NaCl, pH 7.4, an 8 µM (DD)E solution was incubated with 20 nM CPB for 60 min at 22 °C. At various times, 100-µl aliquots were removed, and 10-µl of 40 µM CPI was added to inactivate the CPB or TAFIa. The samples were deprotonated by bringing the solution to 0.2 M with perchloric acid followed by centrifuging at 12,000 × g for 5 min. After the supernatants were neutralized with potassium hydroxide, the samples were placed on ice, and insoluble potassium perchlorate was removed by centrifugation at 12,000 × g for 5 min. The concentration of lysine and arginine in the supernatants was determined enzymatically by methods established by Nakatani et al. (29) and Gaede et al. (30), respectively. For lysine determination, 50 µl of supernatant was added to 40 µl of 0.5 mM NADH (Roche Diagnostics, Laval, Canada) and 2.5 mM -ketoglutaric acid (Roche Diagnostics) in 50 mM HEPES, 150 mM NaCl, pH 7.0 (HBS). Dehydration of NADH was initiated by the addition of 0.33 units of saccharopine dehydrogenase (Sigma) suspended in 10 µl of HBS. Decreases in fluorescence intensity were monitored over 25 min in a Spectra Max, Gemini XS fluorescent plate reader (Molecular Devices, Sunnyvale, CA), with excitation and emission wavelengths set to 340 and 450 nm, respectively, and fitted with a 435-nm emission cut-off filter. The concentration of lysine was calculated based on a standard curve generated by plotting changes in fluorescence intensity produced by known concentrations of L-lysine (Sigma). Determination of free arginine was accomplished in the same manner using pyruvate (Roche Diagnostics) in place of -ketoglutaric acid and 0.5 units of octopine dehydrogenase (Sigma) in place of saccharopine dehydrogenase. Standard curves for free arginine determination were generated by plotting changes in fluorescence intensity produced by known concentrations of L-arginine (Sigma).

Effect of (DD)E Exposure to TAFIa or CPB on the Kinetics of (DD)E-stimulated Glu- and Lys-Pg Activation by t-PA-- To determine the effects of exposure of (DD)E to TAFIa or CPB on the kinetics of (DD)E-stimulated Pg activation, kinetic parameters for Glu- and Lys-Pg activation by t-PA, measured in the presence of TAFIa- or CPB-exposed (DD)E, were compared with those obtained with native (DD)E or in the absence of a cofactor. After exposure of 8 µM (DD)E to 20 nM TAFIa or CPB for 40 min at 22 °C, the reaction was terminated with CPI. Different concentrations of CPI were used to maintain a concentration of CPI in the Pg activation assay between 0.2 and 0.8 µM. This range of CPI concentrations was maintained to ensure CPI levels were well above the K_i for inhibiting CPB (31) but low enough so as not to inhibit plasmin (5 µM CPI causes a 7% reduction in the activity of 0.01 µM plasmin). 0.8 µM CPI has no measurable effect on the rate of plasmin formation (data not shown). Glu- or Lys-Pg, in concentrations ranging from 0 to 16 µM, was incubated with 0.1 nM t-PA in the absence or presence of native (DD)E or TAFIa- or CPB-exposed (DD)E at concentrations up to 6 µM. Plasmin formation was then monitored using the plasmin-directed substrate S-2251 and rates of Pg activation (), determined as described above, were divided by the concentration of activator (') and subjected to nonlinear regression analysis (Table Curve; Jandel Scientific, San Rafael, CA) according to the Michaelis-Menten equation, ' = ([S] × k_cat)/(K_m + [S]), where [S] is the equilibrium concentration of free substrate, calculated for each input concentration of Pg and cofactor as described (3). Free Glu- and Lys-Pg concentrations were calculated based on their affinities for native (DD)E (K_d values of 5.5 and 0.09, respectively) or CPB-exposed (DD)E (K_d values of 11 and 0.35 µM, respectively), which were determined as described below. Michaelis-Menten analysis yielded the rate constant (k_cat) and Michaelis constant (K_m). -Fold stimulation of Pg activation was calculated by dividing the catalytic efficiency (k_cat/K_m) obtained in the presence of saturating concentrations of native or TAFIa- or CPB-exposed (DD)E with that calculated in its absence. Cofactor concentrations were considered saturating when the catalytic efficiency reached a maximum. For comparison purposes, the effect of fibrin and Fg on Glu- and Lys-Pg activation by t-PA was determined as described previously (11).

Effect of (DD)E Exposure to TAFIa or CPB on Its Affinity for t-PA, Glu-Pg, or Lys-Pg-- The affinities of native or TAFIa- or CPB-exposed (DD)E for t-PA, Glu-Pg, or Lys-Pg were quantified using right angle light-scattering spectroscopy in a LS50B luminescence spectrometer (PerkinElmer Life Sciences) (5, 11). 15- or 20-µl aliquots of active site-blocked t-PA, Glu-Pg, or Lys-Pg (at concentrations of 6, 100, and 10 µM, respectively) were added to 2 ml of a 0.1 µM solution of native or TAFIa- or CPB-exposed (DD)E in a 3-ml quartz cuvette thermostated at 22 °C with stirring. The solution was excited at 400 or 440 nm, and emission intensities were measured at the same wavelength. Excitation and emission slit widths were set to either 8 or 12 nm, and a 1% attenuation emission filter was used. Changes in scattering intensity were continuously monitored in time drive with the interval time set at 2 s and the response time set at 3 s. Scattering intensities after each addition of ligand were obtained from the time drive profile by averaging scattering intensities observed over a period of at least 100 s. Control titrations were performed to determine the light scattering intensity of t-PA, Glu-Pg, or Lys-Pg alone. The emission intensity (I) of the incident beam after each addition of ligand was corrected for changes due to ligand scattering and dilution. Corrected values were compared with the emission intensity of the target alone (I_o). When TAFIa- or CPB-exposed (DD)E was used, scattering intensities were corrected for scattering due to the presence of CPI and TAFIa or CPB. These data, together with the total ligand concentration (L_o), were fit by nonlinear regression analysis (Table Curve, Jandel Scientific) to Equation 1 (5, 11, 32),

(Eq. 1)

where L_o is the total concentration of ligand added, P_o is the concentration of target protein, K_d is the dissociation constant, n is the stoichiometry, and  is the maximum change in emission intensity. To confirm the binding parameters obtained in this fashion, reverse titrations also were performed wherein 0.1 µM active site-blocked t-PA, Glu-Pg, or Lys-Pg was titrated with native or TAFIa- or CPB-exposed (DD)E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of CPB or TAFIa on the Ability of (DD)E to Stimulate t-PA-mediated Pg Activation-- To determine whether CPB or TAFIa treatment of (DD)E modulates its ability to stimulate Pg activation by t-PA, (DD)E was incubated with CPB or TAFIa for intervals up to 40 min. After inhibiting CPB or TAFIa with CPI, the stimulatory activity of CPB- or TAFIa-treated (DD)E was then compared with that of the starting material. When t-PA-mediated activation of Glu-Pg is monitored, plasmin formation is decreased with increasing exposure of (DD)E to CPB (Fig. 1, inset) or TAFIa (data not shown). From these data, rates of plasmin formation were calculated (Fig. 1). After a 40-min exposure of (DD)E to CPB or TAFIa, the rate of Glu-Pg activation in the presence of CPB-treated (DD)E is similar to that in the absence of a cofactor. Analogous results are obtained when Lys-Pg is substituted for Glu-Pg. Although rates of plasmin formation are higher with Lys-Pg than with Glu-Pg, 40-min exposure of (DD)E to CPB or TAFIa almost completely abolishes its stimulatory activity (Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Effect of CPB or TAFIa on the rate of (DD)E-stimulated activation of Glu-Pg by t-PA. 8 µM (DD)E was incubated with 20 nM CPB or TAFIa for 40 min at 22 °C. At the indicated intervals, aliquots were removed, and CPB or TAFIa was inactivated with CPI. CPB- or TAFIa-exposed (DD)E (0.4 µM) was incubated with 0.4 µM Glu-Pg and 0.1 nM t-PA for 60 min at 37 °C, and plasmin formation was monitored at 405 nm using the plasmin-directed substrate S-2251. The inset shows the raw data for plasmin generation in the presence of CPB-exposed (DD)E. From these data, rates of plasmin formation were calculated. Each point represents the mean ± S.E. of at least three experiments. The rate of Pg activation in the presence of (DD)E incubated for 40 min with CPB () or TAFIa () is similar to that measured in the absence of (DD)E.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   The effect of CPB or TAFIa on the rate of (DD)E-stimulated activation of Glu- or Lys-Pg by t-PA. To determine whether exposure of (DD)E to CPB or TAFIa decreases its ability to stimulate the activation of Lys-Pg to the same extent as Glu-Pg, 8 µM (DD)E was incubated with 20 nM CPB or TAFIa for 40 min at 22 °C. After the addition of CPI, 0.4 µM CPB- or TAFIa-exposed (DD)E was incubated with 0.4 µM Glu-Pg or 0.1 µM Lys-Pg and 0.1 nM t-PA, and rates of plasmin formation were determined. The rates obtained in the presence of 0.4 µM native (DD)E were used to calculate the relative rates for CPB- or TAFIa-exposed (DD)E. Each bar represents the mean ± S.E. of at least three experiments. Exposure of (DD)E to CPB or TAFIa reduces its ability to stimulate Glu- or Lys-Pg activation by t-PA to almost background levels.

Plasmin can degrade (DD)E into DD and fragment E (8, 9). Because DD and fragment E are less potent stimulators of Pg activation than intact (DD)E (12), this phenomenon could influence the kinetics of (DD)E-stimulated Pg activation. However, when analyzed by PAGE, no detectable (DD)E degradation is observed during the first 10 min of Pg activation (data not shown), the time over which the initial rates of plasmin formation are calculated. These observations suggest that degradation of (DD)E does not influence the kinetics of Pg activation in the presence of (DD)E.

Effect of CPB or TAFIa on the Degree to Which (DD)E Is Degraded during t-PA-mediated Pg Activation-- Fig. 3, which illustrates the formation of (DD)E from fibrin and its subsequent degradation by plasmin, helps to describe the gels presented in Fig. 4. (DD)E, which is formed when plasmin degrades two-stranded fibrin protofibrils between adjacent D and E domains, consists of two cross-linked D domains noncovalently associated with an E domain (Fig. 3). The E domain remains bound to DD provided that the amino-terminal portion of at least one  and  chain within the E domain (_E and _E, respectively) remains intact (25, 33). Plasmin first cleaves _E at Lys⁵³ and then _E at Arg¹⁹. The E moiety is characterized by changes in molecular weight that occur upon _E cleavage (33, 34). Fragment E₁, which has a molecular mass of ~60 kDa, has the Lys⁵³-Lys⁵⁴ peptide bonds of both _E chains intact, whereas fragment E₂, with a molecular mass of 55 kDa, has one _E chain cleaved at Lys⁵³. Both fragments E₁ and E₂ remain associated with DD. In contrast, fragment E₃, which has a molecular mass of ~50 kDa, no longer binds DD, because both _E chains are cleaved at Lys⁵³.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Model of (DD)E formation and degradation. Thrombin releases fibrinopeptide A (A1-16) and fibrinopeptide B (B1-14) from Fg, thereby generating fibrin monomers. These aggregate to form two-stranded protofibrils that are arranged in a half-staggered fashion, whereby the E domain of a fibrin monomer of one strand is noncovalently associated with D domains of two adjacent fibrin monomers on the opposite strand. Factor XIIIa catalyzes the cross-linking of  chains of adjacent D domains. Plasmin cleaves the , , and  chains between E and D domains to yield (DD)E1, wherein fragment E1 remains noncovalently associated with DD via two "knob and hole" interactions on each D domain. These interactions only occur when the amino termini of the  and  chains of fragment E are intact. Exposure of (DD)E1 to plasmin results in progressive cleavage of the  and  chains of fragment E. The E moiety is characterized by cleavage of the Lys53-Lys54 peptide bonds on the  chains. Fragment E2, which has one  chain cleaved, remains noncovalently associated with DD via one knob and hole interaction. In contrast, fragment E3, which has both  chains cleaved at position 53, no longer interacts with DD. Cleavage of both  chains at position 19 within fragment E occurs subsequent to cleavage of the  chains.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Effect of CPB on (DD)E degradation during t-PA-mediated Pg activation. 8 µM (DD)E was incubated with 20 nM CPB for 40 min at 22 °C. At intervals, aliquots were removed, and CPB was inhibited with CPI. 4 µM of CPB-treated (DD)E or native (DD)E was used to stimulate the activation of 0.4 µM Glu-Pg by 1 nM t-PA for 1 h, at which time plasmin and t-PA were inhibited with VFKCK and PPACK, respectively. The reaction mixture was then subjected to PAGE analysis under nondenaturing conditions on a 4-15% gradient gel (A), denaturing conditions on a 10% gel (B), or denaturing and reducing conditions on a 15% gel (C). Lane numbers are indicated above each gel, and the time (DD)E was exposed to CPB prior to Pg activation is indicated above the gel marked A. Native (DD)E is shown in lanes 1 and 9, and molecular weight markers, when present, are shown in lane 10. Native (DD)E consists only of DD and fragment E1. When native (DD)E is used to stimulate Pg activation, the E chain is completely degraded, fragment E1 is degraded to E3, and the E moiety no longer remains associated with DD. In contrast, when CPB-exposed (DD)E is used, the E chain is not degraded, and fragment E1 remains intact and associated with DD. Thus, by limiting plasmin formation, CPB indirectly preserves the structural integrity of (DD)E.

When (DD)E is exposed to plasmin, it is degraded to DD and fragment E (8, 9). Since TAFIa attenuates fibrin degradation by limiting plasmin formation, we explored the possibility that CPB or TAFIa modulates (DD)E degradation (15, 16). (DD)E was incubated with CPB for 40 min. At intervals, aliquots were removed, and CPB was inhibited with CPI. Each (DD)E aliquot was incubated with t-PA and Pg for 1 h to promote Pg activation. Using PAGE analysis, the extent to which the various (DD)E samples were degraded was examined. Under nondenaturing conditions, native (DD)E migrates as a single band (Fig. 4A, lanes 1 and 9). When Pg is activated by t-PA in the presence of native (DD)E, (DD)E is degraded to two lower molecular weight species, corresponding to DD and fragment E, respectively (Fig. 4A, lane 2). In contrast, when (DD)E is preincubated with CPB for increasing amounts of time before incubation with t-PA and Pg (Fig. 4A, lanes 3-8), there is a noticeable decrease in the mobility of fragment E and a progressive increase in the amount of intact (DD)E. Under denaturing conditions, native (DD)E migrates as two bands corresponding to DD and fragment E, respectively (Fig. 4B, lane 1). Fragment E from (DD)E is a single species corresponding to fragment E₁, characterized by  chains with amino-terminal Gly residues at position 15 (33). Following a 1-h incubation in the presence of t-PA and Pg, DD mobility is unchanged, but fragment E₁ is degraded to E₃ (Fig. 4B, lane 2), a species characterized by amino-terminal Lys residues at position 54 on both  chains (33). With increasing exposure of (DD)E to CPB, there is less degradation of fragment E, until E₁ is the only fragment E species in (DD)E (Fig. 4B, lanes 3-8). Under reducing and denaturing conditions, the six chains of native (DD)E were identified based on their apparent molecular weight and amino-terminal sequence analysis. These are the  and  chains and - dimer from DD (_D, _D, and -_D, respectively) and , , and  chains from fragment E (_E, _E, and _E, respectively) (Fig. 4C, lanes 1 and 9). When native (DD)E stimulates Pg activation for 1 h, the _E chain is degraded, and, based on amino-terminal sequence analysis, three amino acids are removed from the amino-terminal portion of the _E chain, although there is no noticeable shift in mobility of the _E chain (Fig. 4C, lane 2). All other chains remain intact. These data are consistent with previously reported plasmin cleavage sites (33). When CPB-exposed (DD)E is used to stimulate Pg activation, the _E chain is not degraded (Fig. 4C, lanes 3-8). The _E chain also remains intact, as determined by its amino-terminal sequence.

The amount of _E chain in each lane in Fig. 4C was quantified by measuring its optical density and comparing this value to the total optical density of all chains in the same lane. As illustrated in Fig. 5, the extent of _E chain degradation is inversely related to the rate of plasmin formation measured prior to PAGE analysis (r = 0.986).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Correlation between E chain degradation and the ability of (DD)E to stimulate Pg activation. The relative amounts of E in each lane of Fig. 4C (), determined by densitometry, are plotted against the time that (DD)E was exposed to CPB prior to Pg activation. The rate of Pg activation by CPB-exposed (DD)E (), measured prior to PAGE analysis, is plotted against time of CPB exposure on the secondary y axis. Both of these values are compared with those obtained with native (DD)E. These data demonstrate that the integrity of the E chain is inversely correlated (r = 0.986) with the extent to which (DD)E stimulates Pg activation.

To determine whether TAFIa-mediated attenuation of plasmin formation also limits the degradation of (DD)E, (DD)E was incubated with 20 nM TAFIa for 40 min. At the end of the incubation period, CPI was added to stop the reaction. Like the results with CPB, when TAFIa-exposed (DD)E is used to stimulate Pg activation, the _E and _E chains are not degraded, and the structural integrity of (DD)E is preserved (data not shown).

CPB- or TAFIa-mediated Release of Lysine and Arginine from (DD)E-- To explore the mechanism by which CPB or TAFIa attenuates the stimulatory activity of (DD)E, we determined whether CPB or TAFIa induces the release of free lysine and arginine residues from (DD)E. Under the conditions outlined under "Methods," CPB or TAFIa causes the release of approximately 4 mol of lysine/mol of (DD)E within 10 and 2 min, respectively (Fig. 6). By 60 min, an additional 4 mol of lysine/mol of (DD)E are released by both enzymes. The biphasic lysine release from (DD)E is not the result of reduction in the activity of CPB, because CPB activity against hippuryl-L-arginine remains constant over the 60-min incubation period. Although the activity of TAFIa against hippuryl-L-arginine is 25% lower at 60 min than it is to start, there is no detectable change in TAFIa activity at 10 min, a point well beyond the transition from slow to rapid lysine release. Thus, the carboxyl-terminal lysine residues on (DD)E exhibit different susceptibilities to CPB or TAFIa. Neither CPB nor TAFIa causes the release of arginine from (DD)E (Fig. 6).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   CPB- or TAFIa-mediated lysine and arginine release from (DD)E. 8 µM (DD)E was incubated with 20 nM CPB or TAFIa for various times at 22 °C. After the addition of CPI, the amount of free lysine or arginine release was determined by monitoring dehydration of NADH using saccharopine or octopine dehydrogenase, respectively. The total concentration of lysine or arginine was divided by the concentration of (DD)E to calculate mol of free amino acid released/mol of (DD)E. CPB () or TAFIa () causes the release of 8 mol of lysine/mol of (DD)E. Under the conditions employed here, ~4 mol of lysine are released within the first 10 and 2 min of CPB and TAFIa incubation, respectively. The remaining 4 mol of lysine are released within 60 min of incubation with either CPB or TAFIa. Neither CPB () nor TAFIa () releases arginine residues from (DD)E.

Effect of (DD)E Exposure to TAFIa or CPB on the Kinetics of (DD)E-Stimulated Glu- and Lys-Pg Activation by t-PA-- The kinetic parameters for (DD)E-stimulated Glu- or Lys-Pg activation by t-PA were measured in the absence or presence of native (DD)E, fibrin or Fg. These results were then compared with those obtained with TAFIa- or CPB-exposed (DD)E. Glu- or Lys-Pg was activated with a constant amount of t-PA in the presence of varying concentrations of Pg and cofactor. Data obtained with each cofactor concentration were fit to the Michaelis-Menten equation by nonlinear regression to determine the values of k_cat and K_m (Fig. 7). Cofactor concentrations were considered saturating when the catalytic efficiency (k_cat/K_m) reached a maximum. Maximum catalytic efficiencies were achieved with 0.4 µM native (DD)E and 5 µM (DD)E previously exposed to TAFIa or CPB.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Effect of CPB on the ability of (DD)E to potentiate t-PA-mediated activation of Glu-Pg. 0.1 nM t-PA was incubated with various concentrations of Glu-Pg in the presence of 0.4 µM native (DD)E () or 5 µM CPB-treated (DD)E () or in the absence of (DD)E (). Plasmin formation was monitored using the plasmin-directed substrate S-2251, and rates of plasmin formation were calculated. CPB markedly reduces the rate of (DD)E-stimulated Pg activation at all Pg concentrations. These data were fit to the Michaelis-Menten equation by nonlinear regression (solid lines) to determine kcat and Km (Table I).

As summarized in Table I, native (DD)E is as potent as fibrin at potentiating both Glu- and Lys-Pg activation by t-PA, stimulating these reactions 360- and 450-fold, respectively. In contrast, after CPB or TAFIa exposure, (DD)E-stimulation of both Glu- and Lys-Pg activation is reduced by 96% (Table I). These reductions reflect decreases in k_cat values and increases in K_m values. The k_cat values for Glu- and Lys-Pg activation with CPB-exposed (DD)E are 3- and 2-fold lower, respectively, than those with native (DD)E, whereas K_m values for these reactions are increased 10- and 12-fold, respectively. Consequently, TAFIa- or CPB-treated (DD)E stimulates Glu- and Lys-Pg activation only 12- and 18-fold, respectively, stimulatory activity similar to that of Fg.

Effect of TAFIa or CPB on the Binding of t-PA, Glu-Pg, and Lys-Pg to (DD)E-- To determine whether the reduced stimulatory activity of TAFIa- or CPB-treated (DD)E reflects changes in its affinity for t-PA and/or Pg, light scattering spectroscopy was used to compare the affinities of t-PA or Pg for native (DD)E with those for TAFIa- or CPB-exposed (DD)E. The relative scatter plots for the interactions of t-PA or Glu-Pg with (DD)E before and after CPB treatment are illustrated in Fig. 8, A and B, respectively. Under the conditions outlined under "Methods," the scattering intensity of 0.1 µM (DD)E is 15 (I_o). At saturating levels of t-PA, the maximum relative scattering intensity (I/I_o) in the presence of untreated (DD)E is 1.5 (Fig. 8A), a value in good agreement with a calculated maximum relative scattering intensity of 1.6 if the stoichiometry is 1:1 (5, 35). The maximum I/I_o when (DD)E was titrated with Glu-Pg was 2.1 (Fig. 8B), a value similar to a predicted I/I_o of 1.9 for a 1:1 substrate interaction with (DD)E (5, 35). Data were fit to Equation 1 by nonlinear regression analysis to determine dissociation constants, and these results are summarized in Table II.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Effect of CPB on the affinity of (DD)E for t-PA or Glu-Pg. 0.1 µM native (DD)E () or CPB-exposed (DD)E () was titrated with active site blocked t-PA (A) or Glu-Pg (B), and scattering intensities obtained in the presence of titrant (I) were compared with those obtained in its absence (Io). Solid lines represent nonlinear regression analysis of the data to determine Kd values, which are summarized in Table II. Whereas exposure of (DD)E to CPB causes a modest reduction in its affinity for Glu-Pg, CPB markedly reduces the affinity of (DD)E for t-PA.

t-PA binds to native (DD)E with a K_d of 0.04 µM. In contrast, t-PA binds to CPB-exposed (DD)E with a K_d of 6.5 µM, an affinity 160-fold lower than that of the activator for native (DD)E. Glu-Pg binds to native and CPB-exposed (DD)E with K_d values of 5.5 and 11 µM, respectively. Thus, CPB treatment produces only a 2-fold decrease in the affinity of (DD)E for Glu-Pg. Similar results are obtained with Lys-Pg, which binds to CPB-exposed (DD)E with a 4-fold lower affinity than that for native (DD)E (K_d = 0.35 and 0.09 µM, respectively). The affinities of TAFIa-exposed (DD)E for t-PA, Glu-Pg, or Lys-Pg are similar to those of CPB-exposed (DD)E (Table II).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

(DD)E, a soluble degradation product of cross-linked fibrin, compromises the fibrin specificity of t-PA by promoting systemic activation of Pg (5, 8, 12). The stimulatory activity of (DD)E, like that of fibrin, reflects its capacity to bind t-PA and Pg (5, 10, 12). In contrast to its predominantly finger-mediated interaction with fibrin, t-PA binds to (DD)E via its second kringle domain (5, 11). Although Pg binding also is kringle-dependent, Pg does not compete with t-PA for (DD)E binding, indicating that Pg and t-PA bind to distinct lysine residues on (DD)E (5). By releasing carboxyl-terminal lysine residues that promote Pg binding to fibrin, TAFIa, a CPB-like enzyme, attenuates fibrin degradation (15, 17). In this study, we explored the possibility that TAFIa modulates the stimulatory activity of (DD)E via a similar mechanism.

(DD)E is as potent as fibrin at stimulating t-PA-mediated Pg activation. When (DD)E is incubated with TAFIa or CPB, its ability to stimulate the activation of either Glu- or Lys-Pg by t-PA is reduced by 96% and becomes comparable with that of Fg. Reduced stimulation by (DD)E reflects both a decrease in k_cat and an increase in K_m for both Glu- and Lys-Pg. The larger effect is on K_m, however, suggesting that exposure of (DD)E to TAFIa or CPB serves to destabilize the ternary t-PA-plasminogen-(DD)E complex.

Lysine residues are released in parallel with the loss of stimulatory activity that occurs as (DD)E is incubated with TAFIa or CPB. A total of 8 mol of lysine/mol of (DD)E is generated. Based on the molecular weight and amino-terminal sequence of the six individual chains within (DD)E as well as the known plasmin cleavage sites on fibrin, the 8 lysine residues released correspond to the 6 carboxyl-terminal lysine residues on the , , and  chains of fragment E at positions 78, 133, and 62, respectively, as well as the 2 carboxyl-terminal lysine residues on the  chains of each D-domain at position 206 (33, 34, 36). Under the conditions employed in this study, most of the stimulatory activity of (DD)E is lost after 2- or 10-min incubation with TAFIa or CPB, respectively, times at which ~4 mol of lysine/mol of (DD)E are released. These findings suggest that some lysine residues on (DD)E are more susceptible to TAFIa- or CPB-mediated release than others and that those most susceptible to release are the lysine residues important for formation of the ternary t-PA-plasminogen-(DD)E complex.

Because plasmin generation in the presence of TAFIa- or CPB-exposed (DD)E is less than that with native (DD)E, TAFIa or CPB indirectly preserves the structural integrity of (DD)E. This may be of physiologic relevance, because DD binds to fibrin with high affinity and inhibits its polymerization, whereas intact (DD)E has no effect on fibrin polymerization (37-39). Limiting DD formation may promote clot stability, because fibrin clots are dynamic structures that undergo continuous formation and degradation. Thus, the anti-fibrinolytic properties of TAFIa may include clot stabilization by the prevention of (DD)E degradation to DD and fragment E.

To explore the mechanism by which TAFIa or CPB eliminates the cofactor activity of (DD)E, we compared the affinities of t-PA and Pg for TAFIa- or CPB-treated (DD)E with those for native (DD)E. Exposure of (DD)E to TAFIa or CPB reduces its affinity for Glu- and Lys-Pg 2- and 4-fold, respectively. In contrast, the affinity of (DD)E for t-PA is reduced 160-fold after TAFIa or CPB exposure. The fact that TAFIa or CPB exposure lowers the affinity of (DD)E for t-PA more than it reduces its affinity for Glu- or Lys-Pg, suggests that TAFIa or CPB modulates the stimulatory activity of (DD)E primarily by decreasing its affinity for the activator rather than for plasminogen.

Because TAFIa- or CPB-exposed (DD)E has little affinity for t-PA, it is a poor stimulator of Glu- or Lys-Pg activation by t-PA. This observation is consistent with our previous work with the Pg activator from the saliva of the vampire bat (b-PA). Lacking a lysine-binding kringle, b-PA does not bind (DD)E (5). Consequently, (DD)E is a poor stimulator of Pg activation by b-PA (5, 40). These data support the concept that only Pg activators that bind to (DD)E are significantly potentiated by this fragment.

Although it does not bind to (DD)E, b-PA binds fibrin with high affinity via its finger domain and is stimulated by fibrin to the same extent as t-PA (5, 41). Because (DD)E and fibrin stimulate t-PA to a similar extent, whereas b-PA is potentiated only by fibrin, t-PA is less fibrin-specific than b-PA. Thus, (DD)E compromises the fibrin specificity of t-PA. Our current findings raise the possibility that by decreasing the capacity of (DD)E to bind t-PA, TAFIa enhances the fibrin specificity of t-PA.

Upon exposure to TAFIa or CPB, the affinity of (DD)E for Glu- or Lys-Pg is only modestly reduced, suggesting that Pg binds to internal lysines as well as carboxyl-terminal lysine residues. In contrast, our data suggest that t-PA predominantly binds to carboxyl-terminal lysine residues on (DD)E because the affinity of t-PA for (DD)E is markedly reduced when (DD)E is exposed to TAFIa or CPB. These findings are consistent with the previously reported noncompetitive binding of t-PA and Pg to (DD)E (5).

Like their effect on (DD)E, TAFIa and CPB also release lysine residues from fibrin partially degraded by plasmin (19, 42). This results in a reduction in the affinity of plasmin-exposed fibrin for Glu- or Lys-Pg and blocks the accumulation of fluorescently labeled Pg on fibrin (16, 43). TAFIa also prevents the conversion of Glu-Pg to Lys-Pg, an early event in the course of t-PA-mediated clot lysis that serves as a positive feedback mechanism because Lys-Pg, which has higher affinity for fibrin than Glu-Pg, is more readily activated by t-PA (18, 19). Although the effects of TAFIa on the affinity of the activator for fibrin have yet to be investigated, CPB blocks the 2-3-fold increase in t-PA binding that occurs when fibrin is exposed to plasmin, suggesting that this increase reflects kringle-dependent binding of t-PA to newly exposed carboxyl-terminal lysine residues (42). The inhibitory effect of TAFIa or CPB on these positive feedback events results in a 3-4-fold prolongation of the rate of t-PA-mediated fibrinolysis (15, 26).

Our results with (DD)E have similarities to those with fibrin. TAFIa or CPB causes the release of free lysine residues from (DD)E, reduces the affinity of the substrate and activator for the cofactor, and attenuates cofactor degradation. However, other features distinguish the effects of TAFIa or CPB on (DD)E from those on fibrin. Although fibrin degradation is attenuated by TAFIa or CPB, it is not inhibited, suggesting that even in the presence of these enzymes, fibrin remains a competent stimulator of Pg activation by t-PA (26). Furthermore, the inhibitory effects of TAFIa on fibrinolysis can be overcome by substituting Lys-Pg for Glu-Pg (18, 19). In contrast, exposure of (DD)E to TAFIa or CPB produces a 96% reduction in its ability to stimulate the activation of either Glu- or Lys-Pg by t-PA. These differences between fibrin and (DD)E are explained by the observation that TAFIa or CPB reduces the affinity of t-PA for (DD)E more than its affinity for fibrin. Whereas t-PA maintains high affinity binding to intact or partially degraded fibrin in the presence of CPB (42, 44), TAFIa or CPB almost abolishes t-PA binding to (DD)E.

(DD)E compromises the fibrin specificity of t-PA because it binds t-PA with high affinity and stimulates Pg activation to the same extent as fibrin. By reducing the affinity of (DD)E for t-PA, TAFIa converts (DD)E from a fibrin-like stimulator to one that has minimal stimulatory activity, much like Fg. Thus, our data suggest a novel role for TAFIa. By reducing the stimulatory activity of (DD)E and attenuating systemic Pg activation, TAFIa may enhance the fibrin specificity of t-PA. Furthermore, since DD can impair fibrin polymerization, limiting (DD)E degradation may augment the anti-fibrinolytic properties of TAFIa.

	FOOTNOTES

^* This work was supported by Heart and Stroke Foundation of Ontario Grants T-3768 and T-2881 and Medical Research Council of Canada Grant MT-3992.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Traineeship award from the Heart and Stroke Foundation of Canada.

^§ Research Scholar of the Heart and Stroke Foundation of Canada.

^¶ A Career Investigator of the Heart and Stroke Foundation of Ontario and recipient of the Heart and Stroke Foundation of Ontario/J. Fraser Mustard Chair in Cardiovascular Research. To whom correspondence should be addressed: Hamilton Civic Hospitals Research Centre, 711 Concession St., Hamilton, Ontario, L8V 1C3, Canada. Tel.: 905-574-8550; Fax: 905-575-2646; E-mail: jweitz@thrombosis.hhscr. org.

Published, JBC Papers in Press, September 1, 2000, DOI 10.1074/jbc.M005483200

	ABBREVIATIONS

The abbreviations used are: Pg, plasminogen; Glu-Pg, native plasminogen with amino-terminal Glu; Lys-Pg, plasmin-modified plasminogen with amino-terminal Lys; t-PA, tissue-type plasminogen activator; b-PA, vampire bat plasminogen activator; Fg, fibrinogen; (DD)E, complex of D-dimer noncovalently associated with fragment E; DD, D-dimer; CPB, carboxypeptidase B; TAFI, thrombin-activable fibrinolysis inhibitor; TAFIa, activated TAFI; CPI, carboxypeptidase inhibitor from potato tuber; PAGE, polyacrylamide gel electrophoresis; PPACK, D-phenyl-prolyl-arginine chloromethyl ketone; VFKCK, D-valyl-phenyl-lysine chloromethyl ketone; S-2251, D-valyl-leucyl-lysine p-nitroanilide dihydrochloride.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES