©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-type Plasminogen Activator (tPA) Interacts with Urokinase-type Plasminogen Activator (uPA) via tPAs Lysine Binding Site
AN EXPLANATION OF THE POOR FIBRIN AFFINITY OF RECOMBINANT tPA/uPA CHIMERIC MOLECULES (*)

Valery Novokhatny (1), Leonid Medved (1), H. Roger Lijnen (2), Kenneth Ingham (1)(§)

From the (1) Holland Laboratory, American Red Cross, Rockville, Maryland 20855 and the (2) Center for Molecular and Vascular Biology, KU Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Differential scanning calorimetry was used to study the domain structure and intramolecular interactions of tPA/uPA chimeras. A high temperature transition centered near 90 °C was observed upon melting of the tPA/uPA chimera (amino acids 1-274 of tPA and 138-411 of uPA) and its variant lacking the finger and epidermal growth factor-like modules (residues 1-3 and 87-274 of tPA and 138-411 of uPA). Since neither of the two parent plasminogen activators display such a stable structure, one may suggest that a new stabilizing intramolecular interaction occurs in the chimeras. We found that occupation of the lysine binding site of tPA by a lysine or arginine side chain from the urokinase moiety is responsible for the high temperature transition as well as for the failure of the chimeras to exhibit the expected fibrin binding properties. All uPA species, single- and two-chain high molecular weight uPA (Pro-Uk and HMW-Uk) and two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly with tPA and its kringle-containing derivatives. This intermolecular interaction was strongly inhibited by -aminocaproic acid indicating that the lysine binding site of tPA is involved. The binding of uPA with the fluorescein-labeled A-chain of tPA, registered by changes in fluorescence anisotropy, was estimated to have a Krange of 1-7 µ M. The interaction of tPA with uPA determined by solid-phase assays appeared to be tighter, with a Krange of 50-300 n M. Two synthetic peptides, with and without carboxyl-terminal lysine, corresponding to urokinase residues 144-158 and 144-157, were 100-fold more potent than -aminocaproic acid with respect to inhibition of the tPA-uPA interaction, indicating that the tPA binding site on urokinase is located within this sequence, close to the activation site Lys-Ile. The discovered intermolecular interaction may be related to the reported synergistic effect of simultaneous administration of these two plasminogen activators.


INTRODUCTION

Tissue-type plasminogen activator (tPA)() and urokinase-type plasminogen activator (uPA) are the two major physiological activators of plasminogen, the inactive precursor of the main fibrinolytic enzyme plasmin. Both of these proteins are serine proteases that cleave a single Arg-Val peptide bond to convert plasminogen to plasmin. uPA is a 411-amino acid glycoprotein that exists in several molecular forms (1, 2) . High molecular weight single-chain urokinase-type plasminogen activator (Pro-Uk) is a M= 54,000 zymogen with little or no activity (3) . Specific cleavage of the Lys-Ilepeptide bond generates an active two-chain enzyme (HMW-Uk). An active two-chain low molecular weight form (33, 0) starting from Lys(LMW-Uk) can be isolated after cleavage with plasmin (4) . tPA is a 68-kDa glycoprotein that is synthesized as a single chain of 527 amino acids and may be converted by plasmin into a two-chain form (2) . In contrast to uPA and most other serine proteases, the single- and two-chain forms of tPA are both enzymatically active. It is well established that the activation of plasminogen by tPA is significantly stimulated by fibrin (5, 6) . uPA displays fibrin specificity only when its activation peptide bond Lys-Ileis intact (7) and is not dependent on the presence of of the amino-terminal 143 amino acids (4) .

tPA and uPA are mosaic proteins consisting of similar homologous modules that are also found in other plasma proteins. These include an epidermal growth factor-like (E) module, a kringle module (K), and a serine protease module (SP) which are common for the two plasminogen activators. The modular composition of uPA is E-K-SP. tPA has an additional finger module (F) homologous to fibronectin type 1 repeats, and its modular composition can be presented as F-E-K1-K2-SP. While the SP module is responsible for enzymatic activity, all other modules in tPA and uPA are involved in interactions with other proteins and ligands. K2 of tPA carries a so-called lysine binding site (LBS) which is responsible for the interaction with -aminocarboxylic acids and is the major site which mediates binding of tPA with fibrin (8, 9, 10) . It has been reported that in addition to K2, fibrin binding by tPA also involves the finger module (9) . The uPA kringle and K1 of tPA do not possess lysine binding properties or affinity to fibrin (10, 11) . Although the uPA kringle is able to bind heparin (12) , no function for K1 of tPA is known. The E module of uPA is responsible for the interaction with the uPA receptor (13) .

tPA and uPA have attracted much attention because their recombinant forms are used as thrombolytic agents. A number of attempts have been made to improve the fibrinolytic properties of these enzymes by constructing chimeric plasminogen activators that combine the best features of both molecules to improve the activating properties and fibrin specificity (2, 14, 15, 16, 17) . But these attempts were not as successful as hoped since the created chimeric molecules did not exhibit appreciable affinity for fibrin. Upon module shuffling, one needs to be aware of possible intramolecular domain-domain interactions which might be important for the function. The domain structures and interactions of tPA and uPA have already been studied by differential scanning calorimetry (18, 19, 20) . It was shown that in uPA all domains are independent and noninteracting, whereas in tPA, the serine protease domain interacts strongly with the amino-terminal F and/or E domains. In order to explain the failure of the tPA/uPA chimeras to bind fibrin effectively, one must first be sure that all modules are folded properly in their new environment and, second, check whether the interactions between them are preserved. In the present paper, we report the results of such a study of the tPA/uPA chimera and its deletion mutant lacking F and E domains. It is shown that uPA interacts with tPA intermolecularly by means of the LBS of K2 of the latter. This interaction is preserved in the tPA/uPA chimeric molecule where the LBS of tPA is occupied intramolecularly by residues within the Leu-Lyssequence of the urokinase moiety. This unnatural filling of the major fibrin binding site explains the poor fibrin affinity of the chimeras.


MATERIALS AND METHODS

Proteins

All urokinase species were kindly provided by Dr. J. Henkin from Abbott Laboratories. Recombinant high molecular weight Pro-Uk and HMW-Uk were expressed and purified from SP2/0 cell culture by Abbott Biotech (Needham, MA) (21) . Low molecular weight urokinase (Abbokinase) was from Abbott Laboratories and was further purified by affinity chromatography on benzamidine-Sepharose (22) . Recombinant single-chain tPA was a Genentech product known under the trade name ``Activase.'' The deletion variant FE-rtPA (23) was obtained from Dr. B. Isaacs of the Genetics Institute (Boston, MA). The 32/35K subtilisin fragment of tPA consisting of F-E-K1-K2 domains was prepared and isolated as described elsewhere (19) . The extended chimeric tPA/uPA protein consisting of amino acids 1-274 of tPA and 138-411 of uPA was expressed in CHO cells as described (24) . The FE-tPA/uPA deletion mutant (residues 1-3, 87-274 of tPA, and 138-411 of uPA) was obtained by a similar approach (25) . The monoclonal antibody directed against tPA K1 (PAM-2) was purchased from American Diagnostica (Product No. 372). The monoclonal antibody directed against the urokinase A-chain was obtained from Dr. J. Henkin, Abbott Laboratories. -Aminocaproic acid (-ACA), Tris, glycine, and the proteolytic enzymes subtilisin, trypsin chymotrypsin were obtained from Sigma. All other chemicals were of reagent grade or higher.

Fluorescence Binding Measurements

Fluorescent 32/35K tPA fragment was prepared by labeling with fluorescein isothiocyanate (FITC). Protein was dialyzed against 0.1 M NaHCO, pH 9.5, mixed with a 10 M excess of FITC (Sigma), and incubated for 2 h at 37 °C. Unreacted dye was removed by chromatography on a Sephadex P-10 prepacked column with subsequent dialysis against 0.02 M Tris, 0.15 M NaCl, pH 7.4 (TBS). The degree of labeling was determined optically as described elsewhere (26) . The protein concentration was measured from the absorbance at 280 nm, correcting for the contribution from covalently attached fluorescein (26) .

Fluorescence anisotropy measurements were performed in TBS at 25 °C with an SLM-8000C fluorometer. Concentrated solutions of uPA species were automatically added with a motor-driven syringe to a stirred cuvette containing the FITC-32/35K tPA fragment at 0.1 µ M while monitoring the anisotropy at 524 nm with excitation at 493 nm. The resulting concentration-dependent increase in anisotropy ( A) was fit to Equation 1: A = A[L]/ K+ [L] where K= dissociation constant and [L] = concentration of free ligand. Awas treated as a fitting parameter because the amount of protein added was not enough to achieve saturation. Since the concentration of labeled protein was small compared to the K, the concentration of free protein was assumed to be equal to the total concentration. Competitive displacement studies were performed by premixing 0.1 µ M FITC-32/35K fragment with a sufficient amount of uPA to provide 75% saturation and titrating with -ACA or the synthetic peptides while measuring the decrease in fluorescence anisotropy.

Solid-phase Binding Assay

Solid-phase binding was performed in plastic microtiter plates using an enzyme-linked immunosorbent assay. Microtiter plate wells were coated with proteins at 3.0 µg/ml in coating buffer (0.1 M NaCO, pH 9.5) for 2 h at 37 °C or overnight at 4 °C. Unbound sites were blocked with 3% nonfat milk in TBS for 1 h at 37 °C. After washing with TBS containing 0.05% Tween 20 (TBS-Tween), tPA or uPA species were added to the wells at concentrations from 0-5 µ M in TBS-Tween. Following a 4-h incubation at 37 °C the wells were washed with TBS-Tween. Bound proteins were reacted with either PAM-2 or Uk monoclonal antibodies (1 µg/ml IgG in TBS-Tween in the presence of 3% nonfat milk) followed by goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad). A 1:1 mixture of 3,3`,5,5`-Tetramethylbenzidine peroxidase substrate with hydroxyperoxide solution (Kirkegaard & Perry Laboratories Inc.) was added, and the absorbance at 650 nm was measured. The data were analyzed using Equation 1.

Differential scanning calorimetry measurements were made as described previously (19, 20) with a DASM-4M or updated DASM-1 calorimeter (27) at a scan rate of 1 °C/min and protein concentrations between 0.4 and 2.2 mg/ml. Deconvolution analysis was performed according to a procedure similar to the recurrent procedure of Freire and Biltonen (28) using software provided by Dr. V. Filimonov (Institute of Protein Research, Pouschino, Russia) as described previously (19, 20) .

The homogeneity of all proteins was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis, size exclusion chromatography on Superdex-75 (Pharmacia), and amino-terminal sequence analysis with an HPG1000A Sequencer (Hewlett Packard). Protein concentrations were determined from absorbance at 280 nm using the molar extinction coefficients ( Mcm, 280 nm) calculated from the amino acid composition (29) . The values are: rtPA, 115,600; FE-rtPA, 90,000; 32/35K tPA fragment, 65,400; Pro-Uk and HMW-Uk, 70,000; LMW-Uk, 44,500; tPA/uPA, 110,000; FE-tPA/uPA, 93,800.

Peptide Synthesis

Peptides corresponding to urokinase sequences Leu-Lysand Leu-Phewere synthesized on a MilliGen 9050 Pep Synthesizer using Fmoc chemistry with pentafluorophenyl amino acid active esters and a polyethylene glycol polystyrene support with a 5-(4`-Fmoc-aminomethyl-3`-5`-dimethoxyphenoxy)valeric acid linker. After synthesis, peptides were purified by reverse-phase high performance liquid chromatography and lyophilized. Concentration of the synthetic peptides was determined by amino acid analysis using a Waters Pico Tag system.


RESULTS

The melting properties of tPA and uPA were well characterized earlier at low pH where both proteins were soluble and the denaturation process was highly reversible (19, 20) . These conditions were selected for comparison of the melting behavior of chimeric molecules with that of tPA and uPA. Original endotherms of the tPA/uPA chimera and its FE deletion variant are presented in Fig. 1 . The dotted lines are simulated curves which were created by the superposition of the melting transitions of the Uk serine protease domain and domains from the A-chain of tPA, taken from the deconvolution analyses presented in Fig. 2 B. Experimental endotherms of the chimeras would be expected to match the simulated curves if no new domain-domain interactions were introduced. However, the experimental curves differ from the simulated ones in the high temperature range. The most prominent difference is the appearance of a new heat-absorption peak centered near 90 °C in both chimeric molecules. This becomes especially evident when the deconvoluted excess heat capacity curves for the chimeras (Fig. 2 A) are compared with the corresponding curves for tPA and LMW-Uk (Fig. 2 B). The new transition reflects melting of a single domain since it is well described by one two-state transition. Since neither of the two parent molecules display such a stable structure, one may conclude that one of the domains is stabilized, i.e. we are dealing with a newly acquired interaction unique to the chimeric molecule.


Figure 1: Original differential scanning calorimetry curves of tPA/uPA ( A) and FE-tPA/uPA ( B) chimeras ( solid lines) in 50 m M glycine, pH 3.4. The dotted lines next to each original curve are simulated profiles expected for these proteins in the case of the absence of the domain-domain interactions.




Figure 2: Deconvolution of excess heat capacity functions obtained with tPA/uPA and FE-tPA/uPA chimeras ( A) and tPA and LMW-Uk ( B) in 50 m M glycine, pH 3.4. The uneven curves represent the experimental curves, and the smooth curves represent the best fits, i.e. the sums of the component transitions obtained by deconvolution. Individual transitions on the tPA curve were assigned as in Ref. 19. A schematic diagram illustrating the modular composition of each species is presented in each panel. The tPA domains are shaded, and the uPA serine protease module is unshaded.



The total enthalpies of denaturation of the full-length chimera and its deletion variant were found to be equal to 299 and 245 kcal/mol. They are in good agreement with the expected values of 310 and 245 kcal/mol, respectively, obtained by summing up the enthalpies of the constituent transitions in Fig. 2B. The number of transitions in both chimeric molecules revealed by deconvolution analysis correlates with their modular composition. Recall that the SP module actually comprises two independently folded domains and that the transition corresponding to melting of the finger module is not present on these curves since it melts at very high temperature and requires addition of denaturants to be detected (19) . Thus, the calorimetric data strongly suggest that all domains are folded properly in the chimeric molecules.

Which domain in the chimeric molecule is responsible for the high temperature transition? It is unlikely that the finger or epidermal growth factor-like domains are involved since the high temperature transition occurs in the FE derivative as well (Fig. 2 A). Kringle domains are known to be stabilized when their lysine binding sites (LBS) are occupied with lysine or related ligands (30, 31, 32) . In particular, the isolated recombinant K2 of tPA, when loaded with -ACA, melts at a temperature of 86.5 °C, close to that of the high temperature transition found in the chimera molecules (33) . We therefore propose the following hypothesis: in the tPA/uPA chimera the lysine binding site of K2 is occupied intramolecularly by the lysine or arginine residues coming from the urokinase moiety resulting in a strong thermal stabilization of its structure. The most direct way to test this hypothesis is to test directly for binding of LMW-Uk to the A-chain of tPA in solution. For this purpose, the 32/35K A-chain fragment of tPA was labeled with fluorescein and titrated with LMW-Uk while monitoring the change in fluorescence anisotropy. As seen from Fig. 3 A, LMW-Uk indeed interacts with the tPA fragment judging by the hyperbolic increase of anisotropy upon titration. A similar hyperbolic increase in anisotropy of FITC-labeled tPA A-chain was observed upon addition of Pro-Uk (Fig. 3 B). The solid lines on these graphs represent the best fits of the experimental data to Equation 1 to obtain the dissociation constants of 7 µ M for LMW-Uk and 2 µ M for Pro-Uk. No changes in anisotropy of FITC-labeled 32/35K tPA fragment were observed upon titration with bovine serum albumin and several active site-blocked serine proteases such as thrombin, trypsin, and chymotrypsin used as controls. Thus, fluorescence titration data unequivocally indicate that tPA interacts with uPA in solution.


Figure 3: Titration of fluorescein-labeled 32/35K subtilisin fragment of tPA with diisopropyl fluorophosphate-inhibited LMW-Uk ( A) and Pro-Uk ( B). FITC-32/35K fragment at a concentration of 0.1 µ M was titrated with the indicated proteins in TBS at 25 °C while monitoring the anisotropy at 524 nm. Curves represent a best fit to Equation 1 to yield the K values indicated on the panels.



To localize the Uk binding site on tPA, a solid-phase binding assay was utilized. Results are shown on Fig. 4 , and the obtained dissociation constants are listed in Table I. Whole tPA, its FE deletion mutant, and its A-chain all bind to LMW-Uk coated plates. The continuous best-fit curves in the figure indicate that in all cases the binding is close to saturation and consistent with a single class of binding sites. Since the kringle domains are the only structures common for all three tPA species, they should be responsible for the interaction with uPA. To prove that the LBS is involved in this interaction, a competition assay with -ACA was performed. In this assay, a constant amount of tPA (1 µ M) was added to the LMW-Uk coated wells containing -ACA at varying concentrations. The displacement curve presented in the inset to Fig. 4reflects the fact that the described reaction could be inhibited by -ACA. This strongly suggests that the LBS of K2 is responsible for the interaction with urokinase. Solid-phase binding was also demonstrated in the opposite direction, i.e. with tPA adsorbed on the plate. As seen in Fig. 5 , both Pro-Uk and HMW-Uk bind to solid-phase tPA, and, again, this interaction was strongly inhibited by the presence of -ACA. Data in the inset show that this process is also inhibited by fluid-phase tPA. Thus, the solid-phase data confirm the fluorescence results that tPA and uPA interact with each other and allow us to conclude that the LBS of tPA mediates this intermolecular interaction.


Figure 4: Enzyme-linked immunosorbent binding assay. Increasing concentrations of tPA (), FE-tPA (), and 32/35K subtilisin fragment of tPA () were incubated with microtiter wells coated with LMW-Uk or BSA () as a control. Bound tPA species were detected with monoclonal antibody PAM2. The data are representative of four experiments, each performed in duplicate. The inset shows the displacement of tPA from the LMW-Uk coated plate by -ACA.




Figure 5: Enzyme-linked immunosorbent binding assay. Increasing concentrations of Pro-Uk (), HMW-Uk (), and HMW-Uk in the presence of 0.1 M -ACA (▾) were incubated with microtiter wells coated with tPA or BSA () as a control. Bound uPA species were detected with monoclonal antibody directed against urokinase A-chain. The data are representative of two experiments, each performed in duplicate. The inset shows competition of the binding by fluid-phase tPA.



Having established a role for the LBS in binding of tPA with uPA, it is necessary to address the question of what part of the uPA molecule is reacting with LBS. A clue can be found in the fact that the isolated A-chain of the chimera, in contrast to the A-chain of tPA, did not express fibrin affinity (24) . In other words, the LBS is not available for binding even when separated from the bulk of the urokinase moiety. The only difference between these two molecules is the presence of a short residual peptide from Uk in the A-chain of the chimera. This peptide, comprising residues Leu-Lys, has a carboxyl-terminal lysine residue which might interact with the LBS of K2. It was also recently reported that a similar peptide isolated from reduced LMW-UK was able to inhibit the fibrin-stimulated activation of plasminogen by tPA (34) . Therefore, this peptide was synthesized in two versions, with and without the carboxyl-terminal lysine, in order to check its ability to inhibit the tPA-Uk interaction. Fig. 6 presents competitive displacement data which were obtained by premixing of Pro-Uk with FITC-labeled 32/35K tPA fragment and titrating the complex with synthetic peptides or -ACA. One can see that both peptides displaced the FITC-labeled A-chain of tPA from its complex with Pro-Uk. The Kfor inhibition by these peptides agreed within a factor of 2, precluding any significant role of the carboxyl-terminal lysine residue. In addition, the synthetic peptides are 100-fold more potent then -ACA in disrupting the complex proving that the inhibition is very specific. This specificity was also demonstrated in a solid-phase assay in which Leu-Pheat a concentration of 1 m M increased the apparent Kfor binding of tPA to solid-phase uPA more than 100-fold whereas an irrelevant cationic peptide NVSPPRRARVTDA at 4 m M had no significant effect (data not shown). Thus, the results indicate that the tPA binding site on Uk is located within the sequence Leu-Phe.


Figure 6: Displacement of fluorescein-labeled 32/35K subtilisin fragment of tPA from Pro-Uk by -ACA and synthetic peptide corresponding to Uk sequence 144-158 with ( +K) and without ( -K) carboxyl-terminal lysine. The FITC-labeled tPA fragment was premixed with 4 µ M Pro-Uk in TBS, and increasing amounts of competitors were added continuously while monitoring the anisotropy at 524 nm. Curves represent the best fit of the experimental data to Equation 1.




DISCUSSION

Numerous attempts have been made during the last several years to create an improved thrombolytic agent by constructing chimeric plasminogen activators that consist of domains from the A-chain of tPA and the serine protease portion of uPA in order to combine the mechanisms of fibrin selectivity of both molecules (2, 14, 15, 16, 17) . Some of them were very promising. For example, FE-tPA/uPA exhibited a 3-16-fold enhanced thrombolytic potency in hamster, rabbit, and baboon models of thrombolysis due to a 6-20-fold prolonged circulating half-life in vivo (2) . But in vitro, none of these chimeras demonstrated either the fibrin affinity of tPA or the fibrin selectivity of uPA. Results presented in this paper provide an explanation for this phenomenon: the lysine binding site on kringle two of the tPA part (which is also the major fibrin binding site) is occupied intramolecularly by one or more positively charged side chains coming from that part of the urokinase molecule that is responsible for its fibrin selectivity. Thus, in a unique twist of fate, the advantages of both molecules canceled each other in the chimera.

This new type of interaction was quite surprising since initially it was expected that the poor fibrin binding of the chimeras might be accounted for by their failure to duplicate domain interactions that had been identified in tPA (19) . In that molecule, the serine protease domain interacts strongly with the amino-terminal F and/or E domains. If this interaction were important for fibrin binding, its absence in the tPA/uPA chimera could have explained the poor fibrin binding. An alternative explanation would be that the fibrin binding domains in the amino-terminal region of tPA do not fold autonomously in the chimeric molecules (24) . However, calorimetric data presented in Figs. 1 and 2 indicate that the total melting enthalpies of the two chimeras are close to what is expected and that the number of transitions revealed by deconvolution analysis is consistent with the modular composition of these proteins. In this study we did not attempt to observe the unfolding status of the superstable finger module in the chimera since in tPA it melts off scale under the conditions employed. To observe its melting would require the presence of denaturants and a very large quantity of the sample (19) . But the fact that the chimeric molecules exhibited detectable fibrin affinity (24, 35) , which could be mediated by the second fibrin binding site located within the finger structure, suggests that this module is also properly folded. Thus, one can conclude that all domains in the chimeric molecules preserve their autonomous folding status in their new environment. The diminished fibrin affinity is attributed to the fact that the LBS, instead of mediating the interaction with fibrin, is utilized for the intramolecular domain-domain interaction.

This is not the first time that an interaction between a kringle domain and a serine protease has been reported. An interaction of similar affinity was observed between thrombin and the second kringle of prothrombin (36, 37) . Although we are not aware of any report of lysine binding by K2 of prothrombin, a recent crystallographic study revealed that multiple positively charged group(s) on thrombin form contacts with an enlarged anionic center in the same region of K2 where lysine analogues bind to other kringles (38) . A similar extended interface could also occur between urokinase and K2 of tPA. This would explain why the interaction persists at low pH where the high temperature transition was observed since the participating carboxyl groups of the LBS would tend to be inaccessible to solvent.

Although the interaction definitely occurs in solution, the apparent affinity was one or two orders higher when one or the other of the components was adsorbed to a solid phase (). This discrepancy may arise from several factors. One is that immobilization of one of the interacting components on the plastic would decrease its translational and rotational degrees of freedom, lowering the entropic cost of complex formation. A second factor is that the high local concentration of one component on the surface will tend to reduce the off rate compared to that in solution with a corresponding decrease in the equilibrium dissociation constant. A third possibility is that if the fluid-phase component has even a weak tendency to self-associate, the oligomeric forms could bind through multivalent attachment to clusters of the solid-phase component. It is possible that the solid-phase model is more relevant to the biology of the system since binding of uPA to its receptor or of tPA to a fibrin clot could mimic the solid-phase conditions used here.

Based on the observed Kin fluid phase, the concentrations of tPA and uPA in blood are seldom high enough for significant complex formation. However, even the fluid-phase interaction could be important in certain situations where the two plasminogen activators are administered simultaneously in high concentration. During such treatment, a synergistic effect was demonstrated in several animal models of thrombosis (39, 40) as well as in studies of patients with acute myocardial infarction (41, 42) . The mechanism of this phenomenon is poorly understood, more so because of inconsistent observations in vitro (43, 44) . The interaction observed here may shed new light on this phenomenon since it would tend to facilitate a local increase of uPA near the fibrin clot.

Data obtained with synthetic peptides allowed us to narrow down the tPA binding site on urokinase within the sequence 144-157. It is clear that the carboxyl-terminal lysine 158 is not important for this interaction. The latter conclusion is deduced not only from the data with peptides but also from the fact that single-chain Pro-Uk binds equally well to tPA. Although carboxyl-terminal lysine is usually considered to be a primary candidate for binding to the LBS, that is not the case with tPA since it was shown to have similar affinity for lysine-Sepharose and aminohexyl-Sepharose (10) . The question of which of the 4 positively charged residues within the 144-157 sequence is crucial remains to be answered. Determination of this residue would allow it to be mutated in the chimera, liberating the lysine binding site to interact with fibrin.

It is important to stress that the synthetic peptides are 100-fold more potent then -ACA with respect to inhibition of the interaction between tPA and urokinase (Fig. 6). This fact is in agreement with Song et al. (34) who showed that the corresponding region of the urokinase A-chain, when isolated from reduced LMW-Uk, was 3500-fold more effective than -ACA as an inhibitor of fibrin-stimulated activation of plasminogen by tPA. This also suggests that the peptide binds with much higher affinity than -ACA to the LBS on tPA. Better understanding of this interaction, in addition to potentially improving the chimera, could also lead to an improved antifibrinolytic agent.

The enhanced fibrin selectivity of single-chain uPA versus the two-chain form is well established although the mechanism is not (3, 7) . This selectivity was also diminished in the single-chain chimera (24) . Conversion of single-chain uPA to the two-chain form with lower fibrin selectivity involves cleavage at the Lys-Ilebond immediately adjacent to the tPA binding site identified here. This suggests that this region is somehow involved in the fibrin selectivity. Since in the chimera this region is already engaged intramolecularly with the LBS on K2 of tPA, it might be less available to confer fibrin selectivity. One may further speculate that this region mediates interaction not only with tPA but with other proteins that possess kringle structures with lysine binding properties. Recent data showing an interaction of uPA with plasminogen (45) are consistent with this idea and indicate that possible LBS-mediated interactions with uPA should always be taken into account.

  
Table: Dissociation constants for the interaction between tPA and uPA species obtained by enzyme-linked immunosorbent assay or fluorescence anisotropy



FOOTNOTES

*
This work was supported by a grant from the Mathers Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0731; Fax: 301-738-0794.

The abbreviations used are: tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; Pro-Uk, high molecular weight single-chain uPA, residues 1-411; HMW-Uk, high molecular weight two-chain uPA, residues 1-158, 159-411; LMW-Uk, low molecular weight two-chain uPA, residues 144-411; tPA/uPA chimera, recombinant protein containing residues 1-274 of tPA and 138-411 of uPA; FE-tPA/uPA chimera, recombinant protein consisting of amino acids 1-3 and 87-274 of tPA and 138-411 of uPA; F, finger module; E, epidermal growth factor-like module; K, kringle module; SP, serine protease module; -ACA, -aminocaproic acid; FITC, fluorescein isothiocyanate; LBS, lysine binding site.


ACKNOWLEDGEMENTS

We thank Dr. Jack Henkin of Abbott Laboratories for provision of uPA species and monoclonal antibodies.


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