INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Tissue-type plasminogen activator (t-PA)¹ is a naturally occurring serine protease that initiates fibrinolysis by converting plasminogen (Pg) to plasmin (1). Not only is fibrin the target for plasmin attack, but fibrin also stimulates t-PA-mediated Pg activation (2, 3). To accomplish this, fibrin acts as a template to which both t-PA and Pg bind (4). The fibrin-binding properties of t-PA have been ascribed to its finger and second kringle (K₂) domains (5, 6), although recent studies suggest that the protease domain also influences the interaction of t-PA with fibrin (4, 7, 8). The binding of both Glu- and Lys-plasminogen (Glu-Pg and Lys-Pg, respectively) to fibrin is entirely kringle-mediated, with Lys-Pg having higher affinity for fibrin than Glu-Pg (9).

As a functional consequence of t-PA interaction with fibrin, the catalytic efficiency of t-PA-mediated Pg activation is 2-3 orders in magnitude higher in the presence of fibrin than in its absence (3, 10). In contrast to fibrin, fibrinogen (Fg) stimulates Pg activation by t-PA only 25-fold (3, 10). Based on these considerations, t-PA is designated a fibrin-specific plasminogen activator (11). Despite this designation, t-PA causes systemic plasminemia and fibrinogenolysis when given to patients (12). In recent studies, we have demonstrated that t-PA causes systemic plasminemia, because, like intact fibrin, soluble fibrin degradation products stimulate t-PA-mediated Pg activation (13). Furthermore, we have identified the (DD)E complex as the fibrin derivative primarily responsible for this effect (14) and have shown that the stimulatory activity of (DD)E is similar to that of fibrin.² (DD)E, a complex of D-dimer noncovalently bound to fragment E, is the major degradation product of cross-linked fibrin (15). As a potent stimulator of t-PA-mediated activation of Pg, (DD)E generated during thrombus dissolution has the potential to induce systemic plasminemia (12, 15).

The limited fibrin specificity of t-PA has prompted the development of plasminogen activators with greater selectivity for fibrin (16). One such agent is the plasminogen activator isolated from the saliva of vampire bats (Desmodus rotundus) (17). Full-length vampire bat salivary plasminogen activator (designated DSPA₁) has over 72% amino acid sequence identity to t-PA (18). The major structural difference is that vampire bat plasminogen activator (b-PA) contains only one kringle domain, whereas t-PA has two. The single kringle domain of b-PA more closely resembles the first kringle domain of t-PA in that it lacks a lysine-binding site (18, 19).

Although fibrin stimulates Pg activation by b-PA to the same extent as t-PA (10), b-PA causes less ₂-antiplasmin and Fg consumption than t-PA in experimental animals when the two agents are used in concentrations that produce equivalent thrombolysis (20-23). This has been attributed to the fact that Fg potentiates Pg activation by t-PA more than b-PA (10, 24-26). Because our studies demonstrated that (DD)E compromises the fibrin specificity of t-PA, we examined the possibility that the greater fibrin-specificity of b-PA over t-PA reflects less (DD)E-mediated stimulation of Pg activation by b-PA relative to t-PA. Herein, we demonstrate that (DD)E and fibrinogen stimulate plasmin formation by t-PA to a greater extent than b-PA. To explore the possibility that differences in potentiation reflect differences in binding parameters, we measured the affinities of t-PA, b-PA, Glu-Pg, and Lys-Pg to (DD)E as well as to fibrin and Fg. Binding was quantified in the absence and presence of the lysine analogue -amino-n-caproic acid (EACA) to identify kringle-dependent interactions.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials

Plasminogen Activators-- Wild-type recombinant t-PA was kindly provided by Dr. B. Keyt (Genentech Inc., S. San Francisco, CA), and recombinant b-PA (DSPA₁) was a generous gift from Dr. W. Witt (Schering AG., Berlin, Germany). t-PA and b-PA were found to be 93 and 100% single chain, respectively, when analyzed by SDS-polyacrylamide gel electrophoresis (27) on 4-15% gels (Ready-Gel; Bio-Rad, Mississauga, Canada), as determined by laser densitometry (Ultroscan XL; LKB-Pharmacia, Baie d'Urfe, Canada). The chromogenic substrate used in Pg activation studies was the plasmin-directed substrate S-2251 (D-valyl-leucyl-lysine p-nitroanilide dihydrochloride) from Chromogenix (Mississauga, Canada). Active site-blocked, fluorescently labeled derivatives of t-PA or b-PA were prepared by adding 1 ml of 0.05 M sodium pyrophosphate, 0.15 M NaCl, 0.5 M (NH₄)₂SO₄, pH 7.2 to 1 ml of a 2 mg/ml stock enzyme solution followed by incubation with a 5-fold molar excess of dansyl glutamyl-glycyl-arginine chloromethyl ketone (Calbiochem) at 22 °C (28). The residual activity of the active site-blocked plasminogen activators was evaluated by measuring their ability to hydrolyze the chromogenic substrate N-methylsulfonyl-D-Phe-Ala-Gly-Arg-4-nitroanilide acetate (Chromozyme t-PA; Boehringer Mannheim, Laval, Canada). t-PA activity was abolished after a 1-h incubation with dansyl glutamyl-glycyl-arginine chloromethyl ketone, whereas a 3-h incubation was needed to block b-PA activity. Both enzymes were then dialyzed against the pyrophosphate-containing buffer overnight at 4 °C. The protein concentrations were determined by measuring absorbance at 280 and 320 nm. Absorbance at 335 nm was used to distinguish dansyl group absorbance from light scattering, as described previously (29). Based on calculations of protein concentration, 90-95% of the plasminogen activators were recovered after dialysis against pyrophosphate buffer. Active site-blocked, unlabeled derivatives of t-PA or b-PA were prepared by the same procedure, except D-phenyl-prolyl-arginine chloromethyl ketone (PPACK, Calbiochem) was used in place of dansyl glutamyl-glycyl-arginine chloromethyl ketone. Under these conditions, t-PA activity was abolished after a 30-min incubation with PPACK, whereas a 2-h incubation was needed to block b-PA activity. Immediately prior to use, a 1-ml volume of the plasminogen activator was dialyzed against 2 liters of 0.02 M Tris-HCl, 0.15 mM NaCl, 0.01% Tween 20, pH 7.4 (TBS) for 3 h with vigorous stirring and then centrifuged at 12,000 × g for 7 min at 22 °C in a microcentrifuge to remove any aggregated material. Based on these calculations of protein concentration, dialysis against TBS resulted in a 40-60% loss of t-PA and a 30-40% loss of b-PA. The molecular weights and extinction coefficients used were 65,000 and _1%²⁸⁰ = 20.0 for t-PA (29) and 54,500 and _1%²⁸⁰ = 17.1 for b-PA (10).

Fibrinogen-- Human Fg, purchased from Enzyme Research Laboratories Inc. (South Bend, IN), was dissolved in a 0.02 M Tris-HCl, 0.15 M NaCl, pH 7.4. Prior to use, Fg (2 mg/ml) was incubated for 30 min at 22 °C with 10 ml of lysine-Sepharose (Pharmacia Biotech Inc., Baie d'Urfe, Canada) to remove residual Pg. After centrifugation at 3000 × g for 10 min at 22 °C, the supernatant was incubated for 30 min at 22 °C with 6 ml of gelatin-Sepharose (Sigma) to remove fibronectin. After centrifugation at 3000 × g for 10 min at 22 °C, the final Fg concentration in the supernatant was calculated by measuring absorbance at 280 and 320 nm and using a molecular weight of 340,000 and _1%²⁸⁰ = 16.0 (30). Typically, the two batch absorption procedures resulted in losses of Fg ranging from 0 to 20%.

Plasminogen-- Native Glu-Pg was isolated from freshly frozen plasma by lysine-Sepharose affinity chromatography as described previously (31) but in the absence of aprotinin. Subsequently, the column was washed extensively with 0.1 M sodium phosphate, pH 8.0, followed by 20 mM Tris-Cl, pH 8.0. Adsorbed Pg was eluted with 10 mM EACA, 20 mM Tris-Cl, pH 8.0, directly onto a DEAE-Fast Flow column (1 × 20 cm). The DEAE column was washed with 20 mM Tris-Cl, pH 8.0, to remove the EACA, and Glu-Pg was then eluted with a 0-200 mM linear NaCl gradient in TBS, pH 7.4. Glu-Pg was concentrated by ammonium sulfate precipitation with subsequent solubilization and dialysis against TBS, pH 7.4. As determined by urea/acetic acid polyacrylamide gel electrophoresis (32), isolated Glu-Pg was free of Lys-Pg and contained no plasmin chromogenic activity using S-2251. Glu-Pg concentrations were calculated by measuring absorbance at 280 and 320 nm and using a molecular weight of 90,000 and _1%²⁸⁰ = 16.1 (31). Lys-Pg was purchased from Enzyme Research Laboratories.

Isolation of (DD)E-- The soluble fibrin fragment, (DD)E, was prepared by plasmin-mediated lysis of a cross-linked fibrin clot. Briefly, a 12-ml solution of Fg (8.3 mg/ml) in 0.02 M Tris-HCl, 0.15 M NaCl, pH 7.4, was clotted with 64 nM 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 2 pM t-PA. Clotting occurred within 10 min, and the resultant fibrin was completely degraded after 55 h. The reaction was terminated by the addition of 1 µM D-valyl-phenyl-lysine chloromethyl ketone (Calbiochem) to block plasmin activity and 1 µM PPACK to block both t-PA and thrombin activity. The clot lysate was then concentrated to a 2-ml volume by ultrafiltration using a Centriprep 10 concentrator fitted with a M_r 10,000 cut-off membrane (Amicon Inc., Beverly, MA). After removing aggregates by centrifugation at 12,000 × g for 5 min, the fibrin degradation products were isolated by passing the material over a Biosep-Sec-S3000 size exclusion column (Phenomenex, Torrance, CA) fitted to a liquid chromatograph (System Gold; Beckman Instruments, Inc., Palo Alto, CA) equipped with two model 126 solvent delivery systems and a model 506 automatic injector. The presence of protein was determined with a model 167 variable wavelength absorbance detector set at 280 nm. Peak protein-containing fractions were pooled and subjected to polyacrylamide gel electrophoresis on 4-15% nondenaturing gels. (DD)E-containing fractions were identified based on their apparent molecular weight and by immunoblot analysis using antibodies against D-dimer and fragment E (14). (DD)E concentrations were calculated by measuring absorbance at 280 and 320 nm using _1%²⁸⁰ = 16.0. When (DD)E was incubated with 10 mM H-Gly-Pro-Arg-Pro-OH (Calbiochem) prior to nondenaturing polyacrylamide gel electrophoresis analysis, two lower molecular weight bands appeared, corresponding to D-dimer and fragment E, respectively.

Methods

(DD)E or Fg Stimulation of Pg Activation-- The effect of (DD)E or Fg on t-PA- and b-PA-mediated Pg activation was determined by comparing plasmin generation in the absence of these cofactors with that in their presence. 20-µl aliquots containing 2 mM S-2251 and 1 nM t-PA or 5 nM b-PA were added to wells of a 96-well microtiter plate containing 0.4 µM Glu-Pg in the absence or presence of either (DD)E or Fg. Plasmin generation was monitored by measuring absorbance at 405 nm at 30-s intervals for 20-30 min using a Spectramax microplate spectrophotometer (Molecular Devices, Menlo Park, CA). Point-to-point slopes were determined and converted to plasmin concentration based on the specific activity of plasmin with S-2251 (0.017 OD s¹ µM¹), which was determined in a separate experiment. Plots of plasmin concentration versus time were used to calculate the rate of plasmin formation.

Fluorescence and Light Scattering Measurements-- All fluorescence and light scattering intensities were measured in a LS50B luminescence spectrometer (Perkin-Elmer, Etobicoke, Canada) using a cuvette thermostatted at 22 °C. Fluorescence measurements were performed in a 1-ml quartz microcuvette, and right angle light scattering measurements were made in a 3-ml quartz cuvette with stirring. To measure the fluorescence of individual samples, three fluorescence intensity readings, each recorded over a 3-s integration time, were averaged. Scattering intensities were continuously monitored in time drive with the interval time set at 1 or 2 s and the response time at 2 or 3 s. Intensity values were determined by averaging scattering intensities observed over a period of at least 100 s. Thus, each scattering intensity value represents the mean of 50-100 individual readings.

Lysine Affinity of t-PA and b-PA-- To compare their affinities for lysine, fluorescently labeled t-PA and b-PA were subjected to affinity chromatography on a lysine-Sepharose column. The fluorescence intensity of a 500-µl sample of dEGR-t-PA or dEGR-b-PA was quantified with excitation (_ex) and emission (_em) wavelengths set to 280 and 530 nm, respectively, a 515-nm cut-off filter, and excitation and emission slit widths both set to 5 nm. The plasminogen activator was then passed over a lysine-Sepharose column (1 × 5 cm), and, after washing, bound material was eluted with 40 mM EACA, and 500-µl fractions were collected. Fractions containing dansyl fluorescence were pooled, and total I₅₃₀ was determined. The amount of plasminogen activator that bound was then calculated by expressing the I₅₃₀ of the eluted material as a percentage of the total I₅₃₀ loaded onto the column.

Binding to Fibrin-- The binding of dEGR-t-PA or dEGR-b-PA to fibrin was determined by adding increasing concentrations of plasminogen activator to a series of microcentrifuge tubes (Sarstedt catalog number 72.702) containing fixed amounts of Fg in TBS (29). A 10-µl aliquot of thrombin (final concentration, 10 nM) was then added to induce clotting. The final reaction volume was 200 µl. After incubation at 22 °C for 1 h, the clots were vortexed and centrifuged at 12,000 × g for 2.5 min to compact the fibrin into the 10-µl tip of the microtube. The fluorescence intensity of 150 µl of clot supernatant in 350 µl of Tris buffer was measured with _ex = 280 nm, _em = 530 nm, a 515-nm cut-off filter, and 15-nm slit widths. A parallel titration was done in the absence of thrombin to establish a standard curve for each ligand. The binding of Lys-Pg and Glu-Pg to fibrin was determined using the same procedure, except unbound Pg was quantified by measuring tryptophan fluorescence of the unlabeled material, and the standard curve of Pg concentrations was established in the absence of Fg. Because the affinity of Pg for fibrin is lower than that of the plasminogen activators, higher Pg concentrations were used in these experiments, thereby obviating the need to use fluorescently labeled Pg. The conditions for measuring tryptophan fluorescence include _ex = 280 nm, _em = 340 nm, a 290-nm cut-off filter, and slit widths set to 2.5 nm.

Binding of t-PA, b-PA, and Pg to Fg or (DD)E-- The binding of t-PA, b-PA, Glu-Pg, and Lys-Pg to Fg or (DD)E was studied using solution phase titrations. Interactions were monitored using right angle light scattering spectroscopy where the solution was excited at a fixed wavelength ( = 400 or 440 nm), and emission intensities were measured at the same wavelength with both excitation and emission slit widths set to either 8 or 12 nm. In the case of Fg, aliquots (5 or 10 µl) of 15 µM Fg were added to 2 ml of 0.1 µM active site-blocked t-PA or b-PA, or 0.3 µM Glu-Pg or Lys-Pg. Control titrations were done to determine the intensity of light scattering of Fg alone. In the case of (DD)E, aliquots (5 or 10 µl) of 5 µM (DD)E were added to 2 ml of 0.1 µM PPACK-t-PA or PPACK-b-PA. Interactions of Glu-Pg and Lys-Pg with (DD)E were monitored in a similar fashion, except 0.1 µM (DD)E was titrated with 80 µM Glu-Pg or 5 µM Lys-Pg. To ensure that none of the target proteins was undergoing self-association, the light scattering intensity of PPACK-t-PA, PPACK-b-PA, or (DD)E (in concentrations ranging from 0.05 to 0.25 µM) was monitored over a 30-min period under the conditions outlined for the binding experiments. In each case, there was no change in scattering intensity over time, indicating that the target proteins were not aggregating.

Data Analyses-- For analysis of fibrin binding, the fluorescence intensities of the supernatants were used to calculate the concentrations of unbound proteins by comparison with fluorescence intensities of known concentrations of protein. The concentrations of bound proteins were determined by calculating the difference between the total and unbound protein concentrations. These values were divided by the Fg concentration to determine the number of moles of dEGR-t-PA, dEGR-b-PA, Lys-Pg, or Glu-Pg bound per mole of fibrin (). For each point in the titration, these values were then plotted against the concentration of unbound protein. Scatchard plots also were constructed, and if these appeared linear, reflecting a single class of binding sites, the binding isotherm was analyzed by nonlinear regression analysis (Table Curve, Jandel Scientific, San Rafael, CA) of the relationship,

(Eq. 1)

where L represents the concentration of unbound protein, n is the stoichiometry, and K_d is the dissociation constant. All binding isotherms were linear, except for that corresponding to the binding of dEGR-t-PA to fibrin in the absence of EACA, which curved downward. These data were best fit to a two-site model by nonlinear regression analysis (Table Curve, Jandel Scientific) according to the following expression.

(Eq. 2)

(Eq. 3)

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Influence of (DD)E or Fg on t-PA- and b-PA-mediated Activation of Pg-- To compare the effect of (DD)E and Fg on t-PA- and b-PA-mediated Pg activation, 0.4 µM Glu-Pg was incubated with 1 nM t-PA or 5 nM b-PA in the absence or presence of various concentrations of (DD)E or Fg for 10 min at 37 °C, and the rate of plasmin formation was monitored (Fig. 1). In the presence of (DD)E, the rate of t-PA-mediated plasmin formation is increased a maximum of 244-fold (from 2.5 × 10⁴ s¹ to 6.1 × 10² s¹). Fg increases the rate of t-PA-mediated plasmin formation 25-fold (from 2.5 × 10⁴ s¹ to 6.2 × 10³ s¹). In contrast, b-PA-mediated plasmin formation is increased only 20-fold with (DD)E (from 1.3 × 10⁵ s¹ to 2.6 × 10⁴ s¹) and 8-fold with Fg (from 1.3 × 10⁵ s¹ to 1.0 × 10⁴ s¹). Thus, (DD)E and, to a lesser extent, Fg are more potent stimulators of Pg activation by t-PA than b-PA.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Influence of (DD)E or Fg on t-PA- and b-PA-mediated activation of Glu-Pg. Glu-Pg (0.4 µM) was incubated with 1 nM t-PA () or 5 nM b-PA () for 10 min at 37 °C in the absence or presence of (DD)E (A) or Fg (B) in the concentrations indicated, and plasmin activity was monitored by measuring the hydrolysis of 0.4 µM S-2251. Plasmin concentrations were calculated based on the specific activity of plasmin for S-2251 (0.017 OD s1 µM1), and rates of plasmin formation were determined by plotting plasmin concentrations as a function of time.

Affinities of t-PA and b-PA for EACA-- To begin to explore why (DD)E and Fg are less potent stimulators of Pg activation by b-PA than t-PA, we first compared the lysine-binding properties of the plasminogen activators because the affinity of t-PA for lysine determines, at least in part, its affinity for fibrin (33). To compare their relative affinities for lysine, aliquots containing 0.32 mg/ml dEGR-t-PA or 0.2 mg/ml dEGR-b-PA were subjected to affinity chromatography on a lysine-Sepharose column. Plasminogen activator that bound to the lysine-Sepharose was eluted with 40 mM EACA. Whereas 90% of the t-PA bound to lysine-Sepharose, only 3% of the b-PA bound. The affinities of t-PA and b-PA for the lysine analogue, EACA, were compared by quantifying changes in tryptophan fluorescence when each agent was titrated with EACA. Titration of active site-blocked t-PA with EACA results in a concentration-dependent and saturable increase in its tryptophan fluorescence (Fig. 2). Based on analysis of these data, EACA binds to t-PA with a K_d = 214 µM and n = 0.91 EACA/t-PA. In contrast, there is no change in tryptophan fluorescence when active site-blocked b-PA is titrated with EACA (Fig. 2). This finding is consistent with our observation that unlike t-PA, b-PA does not bind lysine-Sepharose.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Relative intrinsic fluorescence intensity plots for the interactions of EACA with PPACK-t-PA and PPACK-b-PA. 0.3 µM PPACK-t-PA () or PPACK-b-PA () was titrated with EACA. The intrinsic fluorescence intensities throughout the titrations are shown relative to the intensity of the plasminogen activator alone. Analysis of these results indicates saturable binding of EACA to PPACK-t-PA with a Kd = 214 µM and n = 0.91 EACA/t-PA. The lack of an increase in the intrinsic fluorescence intensity of PPACK-b-PA when titrated with EACA indicates that EACA does not bind to PPACK-b-PA.

Interactions of t-PA, b-PA, Glu-Pg, and Lys-Pg with Fibrin-- Since fibrin has been reported to stimulate Pg activation by t-PA and b-PA to a similar extent (10), we quantified the binding of the plasminogen activators and Pg to fibrin. The Scatchard plot for the binding of dEGR-t-PA is nonlinear (Fig. 3A), indicating heterogeneous binding sites or negative cooperativity (34). A plot of the double reciprocal (1/B versus 1/F) yields a straight line, whereas a plot of B²/F versus B yields a sigmoidal curve, where B and F represent the amount of bound and free t-PA, respectively (data not shown). These findings are indicative of binding site heterogeneity (34). Accordingly, the data were fit to a two-site model (Equation 2) by nonlinear regression analysis, and the resulting binding parameters are K_d1 = 0.053 µM (n₁ = 0.25 t-PA/fibrin) and K_d2 = 2.6 µM (n₂ = 1.4 t-PA/fibrin). When fibrin is titrated with dEGR-t-PA in the presence of 20 mM EACA (Fig. 3B), Scatchard analysis yields a straight line, indicating a single class of binding sites (K_d = 0.47 µM (n = 0.25 t-PA/fibrin)) that more closely resembles the high affinity interaction of t-PA with fibrin seen in the absence of EACA. Like other investigators (29), we interpret this as indicating that EACA blocks the interaction of the K₂ domain of t-PA with fibrin, while finger-dependent binding is maintained.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Scatchard plots of the binding of dEGR-t-PA or dEGR-b-PA to fibrin in the absence and presence of EACA. Fg (1 µM) was clotted with 10 nM thrombin in the presence of various concentrations of dEGR-t-PA or dEGR-b-PA. The concentration of plasminogen activator that bound to fibrin was calculated by comparing the dansyl fluorescence of the clot supernatant with that in control titrations containing all reagents except thrombin. Bound (horizontal axis) refers to the moles of plasminogen activator bound to fibrin per mole of input Fg. Free refers to the moles of unbound plasminogen activator. Fig. 3A illustrates the binding of dEGR-t-PA to fibrin in the absence of EACA. The solid line represents nonlinear regression analysis of the indicated data that best fit a two-site model with Kd1 = 0.053 µM, Kd2 = 2.6 µM, and n1 = 0.25 t-PA/fibrin, n2 = 1.4 t-PA/fibrin, respectively. The dashed lines represent the theoretical Scatchard lines for the binding of t-PA to these two classes of sites. Fig. 3B shows the binding of dEGR-t-PA to fibrin in the presence of 20 mM EACA. The solid line represents linear regression of these data and indicates that, under these conditions, dEGR-t-PA binds to fibrin through a single class of sites with a Kd = 0.47 µM and n = 0.25 t-PA/fibrin. Fig. 3C represents the binding of dEGR-b-PA to fibrin in the absence () or presence () of 20 mM EACA. Linear regression analyses of these data indicate that dEGR-b-PA binds to fibrin through a single class of sites with a Kd = 0.15 µM and n = 1.0 b-PA/fibrin in the absence of EACA and a Kd = 0.14 µM and n = 0.9 b-PA/fibrin in the presence of EACA.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Plasminogen activator (PA) competition for fibrin binding sites. Clots made with 2 µM Fg, 10 nM thrombin, and either 0.8 µM dEGR-t-PA or 0.8 µM dEGR-b-PA were titrated with PPACK-b-PA or PPACK-t-PA, respectively. The amount of fluorescently labeled plasminogen activator that remained bound to fibrin was calculated by comparing the dansyl fluorescence of the supernatant with that in control titrations containing all reagents except thrombin. These values were then expressed as a percentage of the amount of fluorescently labeled plasminogen activator that bound in the absence of competing unlabeled, active site-blocked plasminogen activator. t-PA competes for almost all of the b-PA binding sites on fibrin (), whereas b-PA only competes for 50% of the t-PA binding sites on fibrin (). The combination of saturating concentrations of b-PA and 20 mM EACA competes for all of the t-PA binding sites on fibrin ().

Interactions of t-PA, b-PA Glu-Pg, and Lys-Pg with Fg-- The relative scatter plots for the interactions of t-PA and b-PA with Fg are shown in Fig. 5. Under the conditions outlined under "Methods" (_ex, _em = 400 nm, slit widths = 12 nm), the scattering intensity of 0.1 µM PPACK-t-PA is 1.0 (I_o). At saturating levels of Fg, the maximum relative scattering intensity (I/I_o) is 42, a value in good agreement with a calculated maximum relative scattering intensity of 39 if the stoichiometry is 1:1 (35). The solid line represents the fit of the data to Equation 3 by nonlinear regression analysis. Based on this analysis, t-PA binds to Fg with a K_d = 0.76 µM and n = 0.59 t-PA/Fg. When t-PA is titrated with Fg in the presence of 20 mM EACA, there is no increase in the scattering intensity relative to Fg alone. Thus, the binding of t-PA to Fg is entirely kringle-dependent. The scattering intensity of 0.1 µM PPACK-b-PA is 0.8, and the relative scattering intensity does not change when Fg is added. Therefore, in contrast to the findings with t-PA, b-PA does not interact with Fg.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   The binding of PPACK-t-PA or PPACK-b-PA to Fg. 0.1 µM PPACK-t-PA () or PPACK-b-PA () was titrated with Fg at the concentrations indicated. Using ex and em = 400 nm, the relative increases in scattering intensities when the plasminogen activator was titrated with Fg (I) were compared with the scattering intensity of the plasminogen activator alone (Io). Io values for PPACK-t-PA and PPACK-b-PA were 1.0 and 0.8, respectively. In the case of t-PA, the titration was repeated in the presence of 20 mM EACA (). t-PA binds saturably to Fg with a Kd = 0.76 µM and n = 0.6 t-PA/Fg, where the curved solid line represents the best fit to Equation 3 by nonlinear regression analysis. No increases in the relative scattering intensities were detected when Fg was titrated with t-PA in the presence of EACA, indicating that EACA completely abolishes t-PA binding to Fg. Similarly, no interaction was detected between b-PA and Fg.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   The binding of Glu-Pg or Lys-Pg to Fg. 0.3 µM Lys-Pg () or Glu-Pg () was titrated with Fg at the concentrations indicated, and light scattering was monitored at 440 nm (I). Since Glu-Pg was titrated with high concentrations of Fg, excitation and emission slit widths were both narrowed to 8 nm; in contrast, interactions with Lys-Pg were monitored with slit widths of 12 nm. Under these conditions, Io values for Glu- and Lys-Pg were 1.6 and 2.7, respectively. Increases in the scattering intensities when Lys-Pg is titrated with Fg indicate saturable binding of Lys-Pg to Fg with a Kd = 0.23 µM and n = 0.64 Lys-Pg/Fg. The solid line represents the best fit to Equation 3. In contrast, when compared with the scattering caused by Glu-Pg alone, Fg does not increase the relative scattering intensity, indicating that Glu-Pg does not bind to Fg.

Interaction of t-PA, b-PA, Glu-Pg, and Lys-Pg with (DD)E-- The interactions of t-PA and b-PA with (DD)E are illustrated in Fig. 7. Titrations with (DD)E were performed under the same conditions as titrations with Fg titrations, and I_o values for the plasminogen activators were identical to those previously determined (1.0 for t-PA and 0.8 for b-PA). When t-PA is titrated with (DD)E, the maximum I/I_o observed is 22; a value identical to theoretical I/I_o for a 1:1 t-PA/(DD)E interaction. Based on analysis of the binding data, t-PA binds to (DD)E with a K_d = 0.023 µM and n = 0.8 t-PA/(DD)E. No increase in scattering intensity was detected when t-PA was titrated with (DD)E in the presence of 20 mM EACA, indicating that the interaction of t-PA with (DD)E is entirely kringle-dependent. In contrast to the findings with t-PA, no increase in scattering occurred when b-PA was titrated with (DD)E, indicating that b-PA does not interact with (DD)E.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   The binding of PPACK-t-PA or PPACK-b-PA to (DD)E. 0.1 µM PPACK-t-PA () or PPACK-b-PA () was titrated with (DD)E at the concentrations indicated. Using ex and em = 400 nm, scattering intensities obtained in the presence of (DD)E (I) were compared with those obtained with plasminogen activator alone (Io). PPACK-t-PA binds saturably to (DD)E with a Kd = 0.023 µM and n = 0.8 t-PA/(DD)E. When the titration is repeated in the presence of EACA (), there is no increase in the relative scattering intensity, indicating that EACA completely blocks the interaction of PPACK-t-PA with (DD)E. In contrast to t-PA, b-PA does not interact with (DD)E.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   The binding of Glu-Pg or Lys-Pg to (DD)E. Glu- and Lys-Pg interactions with (DD)E were analyzed by titrating 0.1 µM (DD)E with Glu-Pg (A) or Lys-Pg (B) at the concentrations indicated and monitoring the light scattering intensity at 440 nm. Under these conditions, Io for (DD)E was 7.4. Analysis of these data indicates Glu-Pg binds to (DD)E with a Kd = 5.4 µM and n = 1.2 Glu-Pg/(DD)E, whereas Lys-Pg binds to (DD)E with a Kd = 0.03 µM and n = 1.1 Lys-Pg/(DD)E.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Previously, we demonstrated that t-PA causes systemic plasminemia and subsequent fibrinogenolysis because (DD)E generated during the thrombolytic process stimulates t-PA-mediated Pg activation (13, 14).² We and others (20-23) have shown that t-PA produces more Fg consumption than b-PA in experimental animals. Fig. 1 provides a plausible explanation for the greater fibrin specificity of b-PA over t-PA. Thus, (DD)E and Fg are less potent stimulators of Pg activation by b-PA than t-PA. To explore the possibility that this reflects differences in the affinities of the plasminogen activators for (DD)E and Fg, we compared the binding interactions of t-PA and b-PA with (DD)E and Fg. Since efficient Pg activation requires the formation of a ternary enzyme-cofactor-substrate complex (4), the affinity of both native Glu-Pg and plasmin-derived Lys-Pg for (DD)E and Fg also were quantified. For comparative purposes, we also measured the affinities of the activators and substrates for fibrin.

The binding parameters for the interactions of the plasminogen activators (t-PA and b-PA) and substrates (Glu-Pg and Lys-Pg) with the cofactors (Fg, fibrin, and (DD)E) are listed in Table I, and the structural domains responsible for these interactions are summarized in Table II. Interactions of t-PA and b-PA with (DD)E and Fg elucidate the principal differences between the two activators. t-PA binds to both Fg and (DD)E via its K₂ domain. In contrast, b-PA does not bind Fg or (DD)E because it lacks a functional lysine-binding site. Thus, the presence of a lysine-binding kringle, in addition to its finger domain, gives t-PA a wider binding repertoire than b-PA.

Both the finger and K₂ domains of t-PA independently contribute to its interaction with fibrin (Fig. 3A). Binding is reduced by EACA (Fig. 3B), and we interpret these results as indicating that EACA blocks the low affinity, K₂-dependent interaction of t-PA with fibrin. Although the stoichiometry of the high affinity site is unchanged in the presence of EACA, its affinity decreases from a K_d of 0.053 µM to 0.47 µM. Nesheim et al. (29) also reported that EACA increases the K_d of the high affinity interaction of t-PA with fibrin. The reduced affinity attributed to finger-mediated binding may reflect the conformational changes in t-PA that occur when its K₂ domain is occupied by EACA, a concept supported by our observation that EACA induces changes in the tryptophan fluorescence of t-PA (Fig. 2), and the report that the fluorescence of eosin-t-PA changes when it is titrated with poly-L-lysine (36).

In contrast to t-PA, b-PA binds to fibrin through a single class of high affinity sites (Fig. 3C). Similar results were obtained by Bringmann et al. (10). Since EACA has no effect on binding (Fig. 3C), the interaction is kringle-independent. The finger domain of both b-PA and t-PA recognize the same high affinity binding site on fibrin, because t-PA inhibits b-PA binding to fibrin in a concentration-dependent fashion. In contrast, b-PA partially inhibits t-PA binding by competing only with those t-PA molecules that are bound via their finger domains (Fig. 4). This concept is supported by the observation that complete inhibition of t-PA binding to fibrin occurs with a combination of b-PA and EACA (Fig. 4). Thus, t-PA and b-PA demonstrate comparable high affinity, finger-mediated binding to intact fibrin, whereas t-PA binds additionally to fibrin through a distinct low affinity, kringle-dependent binding site. The observation that the finger domain of t-PA binds fibrin with a stoichiometry of 0.25 mol of t-PA/mol of fibrin both in the absence and presence of EACA, whereas the finger domain of b-PA binds fibrin with 1:1 stoichiometry (Table I), suggests that the kringle domain of t-PA sterically limits the access of its finger domain to fibrin binding sites.

It is evident from Table I that kringle-dependent affinities of t-PA and Pg vary depending on the fibrin(ogen) derivative. Kringle-dependent interactions with Fg and fibrin are weak, whereas (DD)E binding is much stronger. The affinity of the site on (DD)E that binds the K₂ domain of t-PA is 112-fold higher than its counterpart on fibrin. Consequently, t-PA binds to (DD)E via its K₂ domain with an affinity similar to that of its finger domain for fibrin. These findings indicate that when fibrin is solubilized by plasmin to form (DD)E, the binding site for the finger domain is lost, whereas the binding site for the K₂ domain is modified such that its affinity increases. These findings are consistent with previous studies reporting increased binding of t-PA to fibrin that was partially degraded by plasmin or to fibrin formed from Fg that was plasmin-cleaved (37, 38). The observation that (DD)E retains high affinity for t-PA may explain its fibrin-like ability to stimulate t-PA-mediated activation of Pg.

Both Glu-and Lys-Pg bind to intact fibrin, although the affinity of Lys-Pg is much higher than that of Glu-Pg (Table I), a finding consistent with previous reports (9). Both forms of Pg bind via their kringle domains and share the same binding site on fibrin, as evidenced by competition studies (not shown). Plasmin-mediated exposure of new carboxyl-terminal lysine residues may explain why the affinities of Glu-Pg and Lys-Pg for (DD)E are higher than those for fibrin. In support of this concept, fibrin exposed to limited plasmin digestion has been reported to exhibit higher affinity for both forms of Pg (39).

Three lines of evidence indicate that (DD)E and Fg serve as templates onto which the enzyme and substrate assemble. First, near unity stoichiometries for the interactions of t-PA, Glu-, and Lys-Pg with (DD)E and Fg were obtained by nonlinear regression analysis of the binding data. Second, as an independent assessment of stoichiometry, increases in right angle light scattering intensities were compared with those predicted by 1:1 interactions, based on the observation that right angle scattering intensity is related to the square of the molecular mass (35). In all cases, the observed increase was similar to that predicted for simple binary interactions. Third, t-PA and Lys-Pg bind to distinct sites on (DD)E and Fg because high concentrations of Lys-Pg have no effect on t-PA binding to these derivatives (not shown), a finding similar to that observed with intact fibrin (29). Taken together, these data suggest that the cofactor serves as a template onto which one enzyme and one substrate molecule assemble. This hypothesis is supported by the recent observation that t-PA-mediated stimulation of Pg activation by fibrin requires binding of both t-PA and Pg to fibrin (4).

Our results suggest that the affinity of the plasminogen activator for fibrin(ogen) derivatives determines the stimulatory activity of the cofactor. Thus, we have shown that high affinity plasminogen activator-cofactor interactions (b-PA/fibrin, t-PA/fibrin, and t-PA/(DD)E) result in marked stimulation of Pg activation, whereas weaker interactions (t-PA/Fg, b-PA/Fg, and b-PA/(DD)E) elicit modest to poor stimulation. A correlation between a cofactor's affinity for t-PA and its ability to stimulate Pg activation is supported by kinetic models that predict increased stimulation with increasing cofactor-t-PA affinity (4) and the observation that the affinity of t-PA mutants for fibrin corresponds with their ability to degrade plasma clots (40). Furthermore, our findings suggest that, as a determinant of stimulatory activity, the affinity of the cofactor for the activator is more important than the mode of binding. Thus, high affinity, kringle-dependent interactions (t-PA/(DD)E) stimulate Pg activation to the same extent as high affinity, finger-dependent interactions (b-PA/fibrin and t-PA/fibrin), thereby challenging the concept that the K₂ domain of t-PA serves only a docking function that facilitates finger-dependent stimulation (41, 42).

Our studies give considerable insight into the biochemical differences between t-PA and b-PA and provide direction for further study. Although t-PA-mediated Pg activation is stimulated in the presence of fibrin, t-PA has only modest fibrin specificity, because it binds to (DD)E and fibrin with equally high affinity and displays moderate affinity for Fg. These data explain why (DD)E is almost as potent as fibrin at stimulating t-PA-mediated Pg activation² and why Fg is a weaker stimulator. In contrast, b-PA is more fibrin-specific than t-PA (20-23), because it only has affinity for fibrin. Since it is the K₂ domain of t-PA that limits its fibrin specificity by mediating t-PA binding to (DD)E and Fg, our studies also suggest that targeted removal of the lysine binding properties within this domain would render t-PA as fibrin-specific as b-PA.