Characterization of the Interactions of Plasminogen and Tissue and Vampire Bat Plasminogen Activators with Fibrinogen, Fibrin, and the Complex of d-Dimer Noncovalently Linked to Fragment E*

Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex ofd-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Lys-Pg bind fibrin withK d values of 13 and 0.13 μm, respectively. t-PA binds fibrin through two classes of sites withK d values of 0.05 and 2.6 μm, respectively. The second kringle (K2) of t-PA mediates the low affinity binding that is eliminated with ε-amino-n-caproic acid. In contrast, b-PA binds fibrin through a single kringle-independent site with a K d of 0.15 μm. t-PA competes with b-PA for fibrin binding, indicating that both activators share the same finger-dependent site on fibrin. Glu-Pg binds (DD)E with aK d of 5.4 μm. Lys-Pg binds to (DD)E and Fg with K d values of 0.03 and 0.23 μm, respectively. t-PA binds to (DD)E and Fg withK d values of 0.02 and 0.76 μm, respectively; interactions were eliminated with ε-amino-n-caproic acid, consistent with K2-dependent binding. Because it lacks a K2-domain, b-PA does not bind to either (DD)E or Fg, thereby explaining why b-PA is more fibrin-specific than t-PA.

Tissue-type plasminogen activator (t-PA) 1 is a naturally oc-curring 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 (K 2 ) 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 DSPA␣ 1 ) 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 * This work was supported by operating grants from the Heart and Stroke Foundation of Ontario (T-3768) and the Medical Research Council of Canada (MT-3992). The costs of publication of this article were defrayed in part by the payment of page charges. This 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.
§ The recipient of a fellowship award from the Heart and Stroke Foundation of Canada.
¶ A Career Investigator of the Heart and Stroke Foundation of Ontario. To whom correspondence should be addressed: Hamilton Civic Hospitals Research Centre 711 Concession Street, Hamilton, Ontario L8V 1C3 Canada. Tel.: 905-574-8550; Fax: 905-575-2646; E-mail: weitzj@fhs.mcmaster.ca. 1 The abbreviations used are: t-PA, tissue-type plasminogen activator; b-PA, vampire bat plasminogen activator; (DD)E, complex of Ddimer noncovalently linked to fragment E; EACA, ⑀-amino-n-caproic acid; Pg, plasminogen; Glu-Pg, native plasminogen with N-terminal Glu; Lys-Pg, plasmin-modified plasminogen with N-terminal Lys; Fg, fibrinogen; K 2 , second kringle domain of t-PA; PPACK, D-phenyl-prolylarginine chloromethyl ketone; TBS, Tris-buffered saline. 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.

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 (DSPA␣ 1 ) 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 (NH 4 ) 2 SO 4 , 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 siteblocked 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 ϫ 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 ϭ 20.0 for t-PA (29) and 54,500 and ⑀ 1% 280 ϭ 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 ϫ 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 ϫ 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 ϭ 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 ϫ 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 ϭ 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 CaCl 2 in the presence of 93 nM activated recombinant factor XIII (a generous gift from Dr. P. Bishop, Zymogenetics, Inc., Seattle, WA), 0.4 M 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 M D-valyl-phenyl-lysine chloromethyl ketone (Calbiochem) to block plasmin activity and 1 M 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 ϫ 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 ϭ 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-l 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 M 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 s Ϫ1 M Ϫ1 ), 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-l 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 ϫ 5 cm), and, after washing, bound material was eluted with 40 mM EACA, and 500-l fractions were collected. Fractions containing dansyl fluorescence were pooled, and total I 530 was determined. The amount of plasminogen activator that bound was then calculated by expressing the I 530 of the eluted material as a percentage of the total I 530 loaded onto the column.
As another method of comparing the relative affinities of t-PA and b-PA for lysine, changes in tryptophan fluorescence were monitored as each plasminogen activator was titrated with the lysine analogue, EACA. Additions of 20 -40 l of 20 mM EACA were made to a 2-ml solution containing 0.3 M PPACK-t-PA or PPACK-b-PA. Tryptophan fluorescence was monitored with ex ϭ 280 nm, em ϭ 340 nm, a 290-nm cut-off filter, and slit widths set to 5 nm.
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-l aliquot of thrombin (final concentration, 10 nM) was then added to induce clotting. The final reaction volume was 200 l. After incubation at 22°C for 1 h, the clots were vortexed and centrifuged at 12,000 ϫ g for 2.5 min to compact the fibrin into the 10-l tip of the microtube. The fluorescence intensity of 150 l of clot supernatant in 350 l of Tris buffer was measured with ex ϭ 280 nm, em ϭ 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 ϭ 280 nm, em ϭ 340 nm, a 290-nm cut-off filter, and slit widths set to 2.5 nm.
The effect of EACA on the binding of dEGR-t-PA, dEGR-b-PA, Glu-Pg, or Lys-Pg to fibrin was determined by repeating the same titrations in the presence of 20 mM EACA. In addition, clots formed by incubating 2 M Fg with 10 nM thrombin in the presence of 0.8 M dEGR-t-PA, dEGR-b-PA, Glu-Pg, or Lys-Pg were titrated with EACA (in concentrations ranging from 0 to 20 mM), and the amount of ligand displaced was determined by measuring the concentration of unbound protein in the clot supernatant as described above.
To determine whether t-PA and b-PA compete for the same fibrin binding sites, various concentrations of unlabeled, active site-blocked b-PA or t-PA, with or without 20 mM EACA, were added to a series of microcentrifuge tubes charged with 2 M Fg and 0.8 M dEGR-t-PA or dEGR-b-PA. Thrombin (10 nM) was added, and after incubation for 60 min at 22°C, fibrin was pelleted by centrifugation. The amount of unbound fluorescently labeled enzyme in the supernatant was then compared with that found in control samples prepared in the absence of thrombin.
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 ( ϭ 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   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.
For analysis of solution phase binding of PPACK-t-PA, PPACK-b-PA, Lys-Pg, or Glu-Pg to Fg or (DD)E, the emission intensity (I) of the incident beam after each addition of ligand was corrected for changes due to dilution and ligand scattering. Corrected values were compared with the emission intensity before the addition of ligand (I o ), and these data, together with the total ligand concentration (L o ), were fit by nonlinear regression analysis (Table Curve, Jandel Scientific) to the equation, where L o is the concentration of ligand added, P o is the concentration of target protein, and ␣ is the maximum change in emission intensity.
Using ␣ as a measure of 100% ligand bound, the amount of unbound ligand was determined after each addition of ligand, and Scatchard analysis was used to confirm the binding parameters derived from Equation 3.

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-PAand b-PA-mediated Pg activation, 0.4 M 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 ϫ 10 Ϫ4 s Ϫ1 to 6.1 ϫ 10 Ϫ2 s Ϫ1 ). Fg increases the rate of t-PA-mediated plasmin formation 25-fold (from 2.5 ϫ 10 Ϫ4 s Ϫ1 to 6.2 ϫ 10 Ϫ3 s Ϫ1 ). In contrast, b-PA-mediated plasmin formation is increased only 20-fold with (DD)E (from 1.3 ϫ 10 Ϫ5 s Ϫ1 to 2.6 ϫ 10 Ϫ4 s Ϫ1 ) and 8-fold with Fg (from 1.3 ϫ 10 Ϫ5 s Ϫ1 to 1.0 ϫ 10 Ϫ4 s Ϫ1 ). Thus, (DD)E and, to a lesser extent, Fg are more potent stimulators of Pg activation by t-PA than b-PA.
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 ϭ 214 M and n ϭ 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.
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 B 2 /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 d 1 ϭ 0.053 M (n 1 ϭ 0.25 t-PA/fibrin) and K d 2 ϭ 2.6 M (n 2 ϭ 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 ϭ 0.47 M (n ϭ 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 K 2 domain of t-PA with fibrin, while finger-dependent binding is maintained.
In contrast to the results obtained with t-PA, the Scatchard plot of b-PA binding to fibrin is linear (Fig. 3C), indicating a single class of binding sites. Based on analysis of these data, b-PA binds fibrin with a K d ϭ 0.15 M (n ϭ 1.0 b-PA/fibrin). Virtually identical results are obtained in the presence of 20 mM EACA (K d ϭ 0.14 M (n ϭ 0.9 b-PA/fibrin)), consistent with the concept that the interaction of b-PA with fibrin is lysineindependent and reflects the binding of its finger domain to fibrin.
When fibrin clots charged with a fixed concentration of either dEGR-t-PA or dEGR-b-PA were titrated with increasing concentrations of EACA, the EACA competed for approximately 50% of the t-PA binding to fibrin but had no effect on b-PA binding to fibrin (not shown). These findings were taken as further evidence that t-PA binds to fibrin through two classes of sites: a high affinity, finger-independent site and a low affinity, kringle-dependent site. In contrast, b-PA binds to fibrin through a single class of high affinity, kringle-independent sites.
The ability of t-PA and b-PA to compete for the same fibrin binding sites was assessed by titrating fibrin clots containing fixed amounts of either dEGR-t-PA or dEGR-b-PA with increasing concentrations of PPACK-b-PA or PPACK-t-PA, respectively. As illustrated in Fig. 4, t-PA competes for virtually all of the b-PA binding sites on fibrin. In contrast, b-PA is only able to compete for about 50% of the t-PA binding to fibrin. However, the combination of excess b-PA and EACA competes for almost all of the t-PA binding sites on fibrin (Fig. 4). These data support the concept that t-PA and b-PA share a high affinity, lysine-independent class of binding sites on fibrin and that t-PA binds fibrin through a second class of low affinity sites that are lysine-dependent.
The Scatchard plots for the binding of Glu-Pg and Lys-Pg to fibrin are linear (data not shown), indicating that both Glu-Pg and Lys-Pg interact with fibrin through a single class of binding sites. Glu-Pg binds to fibrin with a K d ϭ 13 M and n ϭ 0.72 Glu-Pg/fibrin, whereas Lys-Pg binds to fibrin with a K d ϭ 0.13 M and n ϭ 0.71 Lys-Pg/fibrin. No binding of either Glu-Pg or Lys-Pg to fibrin was detected when the experiments were repeated in the presence of 20 mM EACA, indicating that their interaction with fibrin is entirely kringle-dependent.
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 ϭ 400 nm, slit widths ϭ 12 nm), the

DISCUSSION
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 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 K 2 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 K 2 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, K 2 -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 M to 0.47 M. 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 K 2 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. 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 K 2 domain of t-PA is 112-fold higher than its counterpart on fibrin. Consequently, t-PA binds to (DD)E via its K 2 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 K 2 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.

It is evident from
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, fingerdependent interactions (b-PA/fibrin and t-PA/fibrin), thereby challenging the concept that the K 2 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 activation 2 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 K 2 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.