©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substrate Specificity of Tissue Type Plasminogen Activator
CHARACTERIZATION OF THE FIBRIN INDEPENDENT SPECIFICITY OF t-PA FOR PLASMINOGEN (*)

(Received for publication, January 9, 1995)

Edwin L. Madison (2)(§) Gary S. Coombs (1)(¶) David R. Corey (1)(**)(§)

From the  (1)From theHoward Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 and (2)The Scripps Research Institute, Department of Vascular Biology, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tissue-type plasminogen activator (t-PA) is a remarkably specific protease: the only known substrate of this enzyme in vivo is a single peptide bond (Arg-Val) within the proenzyme plasminogen. Part of the substrate specificity of t-PA is due to a ternary interaction between fibrin, t-PA, and plasminogen which reduces the K of t-PA for plasminogen by a factor of 440. However, even in the absence of fibrin, t-PA continues to hydrolyze plasminogen more rapidly than does trypsin, a homologous serine protease. We have measured the extent of the specificity of t-PA for plasminogen by assaying t-PA and trypsin toward substrates modeled after the peptide sequence in plasminogen surrounding Arg-Val. Surprisingly, t-PA hydrolyzes these substrates with k/K values which are 28,000-210,000-fold lower than those obtained using trypsin. Both the high activity toward plasminogen and the low activity toward peptides are also exhibited by the isolated protease domain. This suggests that the protease domain, in spite of its high homology to the nonspecific enzyme trypsin, is inherently specific for recognition of one or more structural features displayed by native plasminogen.


INTRODUCTION

Tissue type plasminogen activator (t-PA) (^1)has been widely and successfully used as a therapeutic agent for the treatment of acute myocardial infarction(1) . t-PA does not, however, dissolve blood clots directly; rather, the enzyme catalyzes conversion of the zymogen, plasminogen, into the active protease, plasmin, which efficiently degrades the fibrin mesh forming the core of a thrombus. Both of these important fibrinolytic enzymes, t-PA and plasmin, are chymotrypsin-like serine proteases(2, 3) . Unlike plasmin, however, t-PA is a remarkably specific enzyme, a single peptide bond of plasminogen (Arg-Val) is the only known substrate of t-PA in vivo. Because such highly restricted substrate specificity is in striking contrast to the broad specificity of well-studied serine proteases such as trypsin, chymotrypsin, and elastase, the molecular basis of the selectivity of t-PA for plasminogen is of considerable interest. Moreover, a detailed understanding of the molecular basis of the extraordinary specificity of t-PA may facilitate eventual development of the ability to tailor proteases with desired substrate and inhibitor specificity, a capability that is very likely to have wide ranging applications.

Mature t-PA is a complex protein that contains 527 amino acids and is organized by 16 disulfide bonds into five distinct structural domains that are homologous to similar domains found in other secreted and cell surface proteins(2, 4, 5) . Residues 4-50 of t-PA form a ``finger'' domain that is closely related to the fibrin binding finger structures of fibronectin(6) . Residues 51-87, the ``growth factor domain,'' share homology with the precursor of epidermal growth factor (7) and with similar domains in a variety of other proteins including urokinase (8) , protein C(9) , and the receptor for low density lipoprotein(10) . Residues 88-175 and 176-263 form two sequential ``kringle'' domains, each built around three characteristic intradomain disulfide bonds(11, 12, 13) . Kringle domains are found in a number of other proteins including plasminogen, thrombin, urokinase, and lipoprotein (a)(5, 14, 15) . The remainder of the protein (residues 276-527) constitutes the catalytic domain which shares homology with other chymotrypsin-like enzymes and contains a typical catalytic triad (Asp, His, and Ser)(2) .

The chymotrypsin family of serine proteases has evolved to contain many members functioning in the same milieu (e.g. human plasma) that are exclusive in their reactivity toward both substrates and inhibitors. While the role of the ``specificity pocket'' of a serine protease in determining the ``primary'' or P1 specificity of the enzyme has long been appreciated(16, 17, 18, 19) , additional determinants of protease specificity remain largely obscure. Although both the location and mechanism of action of these additional determinants of enzyme specificity remain unclear, their existence and critical importance are well established. For example, t-PA and almost all other proteases that participate in the coagulation cascade, the fibrinolytic cascade, and complement activation possess virtually identical, trypsin-like primary specificity pockets and exhibit identical P1 specificity (i.e. they cleave COOH-terminal to arginine and lysine residues). Nevertheless, most of these enzymes display very different substrate and inhibitor specificity. Previous work from a number of laboratories has shown that, in the absence of fibrin, the isolated protease domain of t-PA is as active as t-PA for cleavage of plasminogen(20, 21, 22, 23, 24) , and a study by Ganu and Shaw (25) suggested that t-PA was inactive toward peptides modeled after the cleavage sequence in plasminogen. We report here the quantitation of the fibrin-independent specificity of t-PA for plasminogen by comparison of the activities of t-PA, t-PA protease domain, and trypsin toward plasminogen and toward linear and cyclic peptides containing the P6-P8` sequence of plasminogen. In contrast to trypsin, t-PA and its isolated protease domain show remarkable specificity for its target sequence in native plasminogen and very little activity toward the related small peptides. The localization and characterization of important determinants of this specificity is essential for understanding the evolution of t-PA as a highly specific protease.


MATERIALS AND METHODS

Enzyme Preparations

Purified t-PA (Activase) was kindly provided by Genentech (San Francisco, CA). This enzyme preparation contained approximately 70% single-chain t-PA and approximately 30% two-chain t-PA. Activase was quantitatively converted to two chain t-PA by treatment with plasmin-Sepharose as described previously(26) . Bovine trypsin was purchased from Boehringer Mannheim (Indianapolis, IN). Plasminogen was purchased from American Diagnostica (Greenwich, CT).

Production and Purification of the Protease Domain of t-PA

20 µg of Lys-plasmin was coupled to 0.5 ml of Affi-Gel 10 beads (Bio-Rad). The beads were washed and suspended in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 4% glycerol buffer to a total volume of 1 ml. 100-µl Affi-Gel-plasmin beads were added to 0.5 ml of 1.01 mg/ml t-PA and shaken overnight at 37 °C to yield two-chain t-PA. Dithiothreitol was added to a final concentration of 3 mM to reduce the exposed disulfide bond connecting the chains. After 30 min at 4 °C, the t-PA heavy chain was removed by 3 passages over 0.5 ml of lysine-Sepharose (Pharmacia Biotech Inc.). The purity of the isolated protease domain was confirmed by SDS-PAGE on a Phast system (Pharmacia) followed by silver staining.

Measurement of Enzyme Concentrations

Concentrations of trypsin, t-PA, and t-PA protease domain were measured by active-site titration with 4-methylumbelliferyl p-guanidinobenzoate (27) using a Perkin-Elmer LS 50B Luminescence Fluorometer. Titrations of trypsin were performed in 100 mM NaCl, 20 mM CaCl(2), 50 mM Tris-HCl (pH 8.0). Because CaCl(2) reduces the solubility of t-PA, titrations of t-PA and the isolated protease domain of t-PA were performed in 150 mM NaCl, 10 mM Tris-HCl (pH 7.5).

Synthesis and Preparation of Peptides

Peptides were synthesized by solid-phase synthesis on an Applied Biosystems model 430A peptide synthesizer using Fmoc (N-(fluorenyl)methoxycarbonyl) chemistry. Each peptide was desalted using a Whatman C-4 cartridge (Fisher) or by C18 reverse-phase HPLC. The molecular weights of the peptides were confirmed by Mass Spectral analysis using a VG (Altrincham, United Kingdom) 30-250 Quadrapole mass spectrometer. Spectrozyme t-PA and Spectrozyme PL were obtained from American Diagnostica. Boc-Leu-Gly-Arg-p-nitroanilide was obtained from Bachem Bioscience (Philadelphia, PA). Peptide (1) was cyclized as described (23) by dissolving 25 mg of reduced, desalted peptide in 50 ml of doubly distilled water. The mixture was stirred for 72 h and the cyclization was monitored by titration with 5,5`-dithiobis(2-nitrobenzoic acid) (Sigma). The cyclization was confirmed by Mass Spectral analysis which revealed a reduction in molecular mass from 1411 to 1409 daltons. Peptide solutions of known concentration were prepared by weighing and dissolving purified lyophilized peptide in a known volume of doubly-distilled water to give concentrated stock solutions.

Kinetics of Cleavage of Synthetic Peptides by Trypsin, t-PA, and the Protease Domain of t-PA

Kinetic data for cleavage of synthetic peptides by bovine trypsin was obtained by incubating each peptide at concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 mM at 37 °C in the presence of 3.6 nM trypsin. Peptide concentrations used to assay t-PA and t-PA protease domain activity were 0.5, 1, 2, 4, 8, and 12 mM. The linear peptide was also assayed at 20 mM. t-PA concentration for each assay was 640 nM. t-PA protease domain concentrations used were 980 nM for assays of the cyclic peptide and 1.96 µM for assays of the linear peptide. Assays were performed in duplicate and errors were determined as described previously(28) . Reactions were stopped at between 5 and 20% cleavage by addition of 0.33% trifluoroacetic acid. Cleavage of the peptides by trypsin, t-PA, or the protease domain of t-PA was monitored at 220 nm by reverse-phase HPLC (Rainin, Woburn, MA) using a Microsorb 5-µm 300-Å reverse phase column (4.6 mm times 25 cm) (Rainin) using a 0-95% gradient of 0.1% trifluoroacetic acid in doubly distilled water and 0.08% trifluoroacetic acid in 95% acetonitrile, 5% doubly distilled water(25) . The percentage of proteolyzed peptide was evaluated by comparing the calculated area beneath the product peaks with the total area beneath both the product peaks and the peaks representing residual starting material. Data were interpreted by Eadie-Hofstee analysis. The identity of the hydrolyzed peptide fragments was determined by Mass Spectral analysis.

Activity Assays Using the Substrate Plasminogen

In the coupled or ``indirect'' assay the chromogenic substrate Spectrozyme PL is cleaved by plasmin which is generated by the action of t-PA on plasminogen. The release of free p-nitroanilide from the chromogenic substrate is measured spectrophotometrically at 405 nm. Enzyme concentrations and other reaction conditions were as described previously(29, 30) . Briefly, 0.5-1.0 ng of t-PA were assayed in a total reaction volume of 100 µl. Reactions were performed at 37 °C in the presence of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 0.01% Tween 80. The concentration of Lys-plasminogen was varied from 0.9 to 15 µM while Spectrozyme PL was present at a concentration of 0.62 mM. The indirect assay does not proceed by Michaelis-Menten kinetics; K(m) and k values were calculated as described previously(31, 32, 33) . Kinetic constants for the cleavage of Plg by trypsin were determined using 7 concentrations of Plg ranging from 0.5 to 11 µM and 3.3 nM trypsin at 37 °C for times ranging from 2.5 to 6 h. Reactions were stopped with 0.3% trifluoroacetic acid and cleavage products were separated by SDS-PAGE using 12% polyacrylamide. Gels were stained with Coomassie Brilliant Blue for 1 h and then destained overnight in 10% acetic acid, 5% methanol, in water. Intensity of substrate and product bands was quantitated on a model 300A scanning densitometer from Molecular Dynamics which was operated with Image Quant 3.0 software. The product bands had molecular sizes of approximately 66, 64, and 28 kDa according to their mobility in the gels and were identified by amino-terminal amino acid sequencing. The 66- and 64-kDa fragments were the amino-terminal portion of the protein and began at lysine 20 and lysine 78, respectively. The NH(2)-terminal of the 28-kDa fragment was valine 561. The fraction of Lys-plasminogen cleaved at each substrate concentration was determined by calculating P/(S + P), where P was the intensity of the product band and S was the intensity of the substrate bands. These ratios were used to calculate initial velocities of cleavage. k and K(m) were derived from Eadie-Hofstee analysis.

Activity Assays Utilizing Activated Substrates

Kinetic assays of t-PA protease domain, two-chain t-PA, and trypsin using the activated substrates methylsulfonyl-D-cyclohexyltyrosyl-glycyl-arginine-p-nitroanilide (Spectrozyme t-PA) and Boc-leucyl-glycyl-argininyl-p-nitroanilide (Boc-LGR-pNA) were performed with each enzyme present at concentrations between 6 and 8 nM (t-PA and protease domain of t-PA) or between 1.6 and 2.6 nM (trypsin). The concentration of Boc-LGR-pNA was varied from 0.1 to 0.4 mM in assays of all three proteases. The concentration of Spec t-PA was varied from 12.5 to 75 µM in assays of trypsin, from 25 to 150 µM in assays of two-chain t-PA, and from 25 to 200 µM in assays of the protease domain of t-PA. Reactions were performed in 1-ml quartz cuvettes (Fisher) and reaction rates were assessed by continuous measurement of absorbance at 405 nm for 10-20 min using a Hewlett-Packard 8452 diode array spectrophotometer. Data was interpreted by Eadie Hofstee analysis.


RESULTS

Kinetic Analysis of Plasminogen Cleavage

Kinetic analysis of the activation of plasminogen by t-PA was accomplished by performing coupled or ``indirect assays'' that contained mixtures of t-PA, plasminogen, and the chromogenic substrate Spectrozyme PL. These assays were performed both in the presence and absence of the co-factor fibrin (Table 1) and indicated, in accord with earlier results (34) , that fibrin stimulated the activity of t-PA by reducing the K(m) of the enzyme for plasminogen by a factor of approximately 440. Even in the absence of fibrin, however, t-PA retained a relatively high catalytic efficiency toward plasminogen.



Two properties of t-PA greatly simplified analysis of data resulting from the indirect assays: (i) t-PA cleaves plasminogen at only one position (Arg-Val) and (ii) unlike plasmin, t-PA does not hydrolyze Spectrozyme PL. Trypsin shares neither of these properties with t-PA, complicating the application of this method to hydrolysis of plasminogen by trypsin. As a result of this limitation, direct comparison of plasminogen hydrolysis by trypsin and t-PA was performed by SDS-PAGE analysis of the cleavage products (Fig. 1). Amino-terminal sequencing of the isolated polypeptide products of these reactions confirmed that the observed cleavage of plasminogen by both t-PA and trypsin occurred between Arg and Val. These experiments also indicated that t-PA (Fig. 1, lanes 8-13) was a more efficient activator of plasminogen than trypsin (Fig. 1, lanes 2-7). For example, during incubation of 3.3 µM plasminogen with 7.65 nM t-PA or trypsin for 4 h, t-PA converted almost all the plasminogen (>95%) into plasmin while trypsin activated <10% of the plasminogen. Additional experiments of this type were performed, and kinetic parameters for cleavage of plasminogen by trypsin were evaluated by densitometry of the bands on SDS-PAGE gels corresponding to residual plasminogen and cleaved product. The calculated K(m) and k values were very similar to those previously reported by Robbins and co-workers (35) and confirmed that the catalytic efficiency of cleavage, k/K(m), of plasminogen by trypsin was lower than for t-PA (Table 1). The observed 2.4-4.9-fold lower efficiency was entirely the result of a 9-19-fold reduction in k, as trypsin hydrolysis of plasminogen actually exhibited a 4-fold lower K(m) for plasminogen than did t-PA.


Figure 1: Cleavage of plasminogen by trypsin or tissue plasminogen activator examined by SDS-PAGE. Lane 1 contains uncut plasminogen. Lanes 2-7 contain digests of 3300 nM plasminogen with 7.7 nM trypsin at 20, 40, 60, 120, 180, and 240 min. Lanes 8-13 contain digests of 3300 nM plasminogen with 7.7 nM t-PA.



Kinetic Analysis of Peptide Cleavage

The striking specificity of t-PA for plasminogen might reflect stringent recognition of the primary structure at the cleavage site in a manner analogous to the recognition of substrates by Factor X(36) , TEV protease(37) , or enteropeptidase(38) . To facilitate testing of this hypothesis we synthesized peptides containing the sequence surrounding the Arg-Val bond in plasminogen. Peptide (1) contained the exact P6-P8` wild-type sequence containing two cysteines. These were allowed to form an intramolecular disulfide to yield a constrained loop which might mimic aspects of plasminogen secondary structure. We also prepared peptide (2), which substituted a serine residue for each cysteine in (1) and which had the freedom to mimic any conformation this sequence might adopt in the folded protein. The peptides were incubated with trypsin and t-PA (Table 2), and the reactions were followed by monitoring product appearance by HPLC (Fig. 2). HPLC analysis of the digestion of the cyclic peptide (1) by trypsin, t-PA, or the protease domain of t-PA revealed only one product while similar digestion of the linear peptide (2) yielded two products (Fig. 2). Mass spectral analysis confirmed that both peptides were cleaved between their arginine and valine residues.




Figure 2: HPLC analysis of partial tryptic digests of linear and cyclic peptides. The organic solvent gradient rose from 0% at 3 min to 100% at 13.5 min and returned to 0% by 16 min. The traces shown extend from 8 to 16 min. Traces prior to 8 min contained no relevant information and were omitted to remove a peak produced by the N,N-dimethyl formamide in the peptide stock solution of the cyclic peptide. Total amount of peptide loaded was 80 (cyclic) and 40 nmol (linear). Identities of each peak are as labeled.



Trypsin catalyzed the hydrolysis of both (1) and (2) with relatively low k values and relatively high K(m) values as compared to those noted for other peptide substrates of similar lengths(25) . However, even though the catalytic efficiency of trypsin toward (1) and (2) was relatively low, its hydrolysis of these substrates was 28,000-210,000-fold more efficient than that of t-PA. This was reflected both in k, which was 5,000-51,000-fold lower for t-PA than for trypsin, and in K(m), which was 4.1-5.4-fold higher. The protease domain of t-PA exhibited similarly low catalytic efficiencies toward (1) and (2) relative to trypsin, and the addition of fibrin did not accelerate the hydrolysis of either extended peptide substrate (1) or (2) by t-PA or the protease domain of t-PA. The introduction of a disulfide in substrate (1) increased hydrolysis relative to (2), but only by 4-12-fold.

By contrast to studies with trypsin, kinetic analysis of the cleavage of peptide substrates by thrombin demonstrated a significant role for residues at P9 and P10 which are relatively distant in the primary structure from the scissile bond(39, 40) . To test the hypothesis that a similar requirement by t-PA could account for the low activity of t-PA toward peptide (1) (P8-P6`), we synthesized a 24-residue peptide consisting of the P13-P11` residues of plasminogen. Hydrolysis of the extended peptide by t-PA, however, occurred at a similar catalytic efficiency to that of peptide (1) (k = 0.0012 s; K(m) = 5500 µM; k/K(m) = 0.22 M s). The molecular basis of specificity toward peptide substrates appears, therefore, to differ in important ways among trypsin, thrombin, and t-PA.

Kinetic Analysis of Hydrolysis of p-Nitroanilide-containing Amide Substrates

The extremely low activity of t-PA toward extended peptide chains in which peptide amide bonds were hydrolyzed is in contrast to results with short, activated substrates containing p-nitroanilide leaving groups. Short peptides containing p-nitroanilide leaving groups are more labile than extended peptides, because of the superior ability of the p-nitroanilide leaving group to delocalize the developing negative charge in the transition state. This delocalization reduces the half-life of amide bond cleavage from greater than 7 years (41) to less than 100 h(42) . We assayed the activities of t-PA, the isolated protease domain of t-PA, and trypsin toward Spectrozyme t-PA, a paradigm t-PA substrate, and Boc-Leu-Gly-Arg-p-nitroanilide, a typical trypsin substrate. t-PA exhibited a catalytic efficiency of 2.9 times 10^5M s for Spectrozyme t-PA and 1.1 times 10^4M s for Boc-Leu-Gly-Arg-p-nitroanilide (Table 3). The kinetic parameters for the isolated protease domain were similar to those obtained with t-PA. Although trypsin was more active than t-PA toward both these substrates, the differences in activities between the two enzymes were much less with activated substrates (6.5-18-fold) than with the peptide substrates (>10^4-fold). Substrates which contain p-nitroanilide moieties differ from extended peptide substrates (1) and (2) both by the possession of a more chemically labile leaving group and by the lack of residues at the P2` through P8` positions. It is likely, however, that the enhanced activity toward p-nitroanilide-containing substrates is due to the labile nature of the p-nitroanilide leaving group rather than the lack of interactions on the P` side of the labile bond. t-PA hydrolyzes peptides which only occupy P1` very poorly, and exhibits an increase in activity as the P` sites are filled by peptides of increasing length. (^2)




DISCUSSION

Several investigators have reported that the catalytic efficiency toward plasminogen of t-PA and of the isolated protease domain of t-PA are very similar in the absence of fibrin(20, 21, 22, 23, 24) . Important determinants of the fibrin-independent catalysis of plasminogen by t-PA, therefore, appear to map exclusively to the protease domain. Assays of t-PA and the isolated protease domain of t-PA reported here confirm these previous studies. In contrast to the maintenance of high activity toward plasminogen, our assays indicate that the activity of t-PA and isolated protease domain is strikingly lower than trypsin for peptides containing the P6-P8` target sequence in plasminogen. These results extend earlier work by Ganu and Shaw (25) which suggested that t-PA had no activity toward peptides modeled after the cleavage sequence in plasminogen. However, in contrast to this earlier study, we do observe catalysis of peptides by t-PA. The catalytic efficiencies of t-PA toward these peptide substrates were comparable to those reported for variants of trypsin that lack one member of the catalytic triad, suggesting the possibility that, as the free enzyme exists in solution, catalytic and/or substrate binding structures in t-PA are not properly formed or aligned to perform the energetically demanding task of cleaving normal peptide bonds. As indicated in Table 3, however, t-PA and the protease domain of t-PA are able to hydrolyze the more labile amide bonds of p-nitroanilide-containing substrates with kinetic parameters similar to those of trypsin, suggesting that there is no gross disruption of the catalytic machinery.

Comparison of data in Table 1and Table 2reveals that, even in the absence of fibrin, t-PA is approximately 19,000-87,000 times more active toward native Lys-plasminogen than toward peptides containing the cleavage site from plasminogen. In the presence of fibrin this ratio increases to values varying from 8 times 10^6 to 3.6 times 10^7. Data in Table 1and Fig. 1also demonstrate that the protease domain of t-PA is approximately 3-fold more catalytically efficient toward plasminogen than is trypsin and that trypsin is 29,000-550,000 times more efficient than the protease domain of t-PA for catalysis of the same cleavage sequence within short peptides. The structural context of the labile amide linkage, therefore, results in a 87,000-1,650,000-fold difference in the relative reactivities of trypsin and the protease domain of t-PA toward the same primary structure. While proper folding of a substrate is a hindrance to most proteases, it seems to be required for efficient cleavage by t-PA. The equally low activities of t-PA and the isolated protease domain excludes the possibility that the low activity toward peptides was due to the inhibition of the protease domain by one of the non-protease domains. Conversely, the similarity in the fibrin-independent activities of t-PA and the isolated protease domain toward plasminogen indicates that the non-protease domains do not substantially contribute to the fibrin-independent activation of native plasminogen by t-PA.

The observation that t-PA and the protease domain of t-PA possess similarly high activity toward native plasminogen suggests that, in the absence of fibrin, important interactions between t-PA and plasminogen require only the protease domain of t-PA. In the absence of structural information for either t-PA or plasminogen, understanding of the molecular basis of the substrate specificity of t-PA will necessarily remain inexact. However, the finding that t-PA processes both linear and cyclic peptide mimics of its target sequence extremely inefficiently does suggest that t-PA recognizes complex and/or multiple elements on the surface of native plasminogen whose precise conformation or alignment is likely to be dependent on the tertiary structure of plasminogen. One possible explanation of this observation is that binding of plasminogen induces a subtle alteration in the catalytic machinery of the protease domain of t-PA which enables it to efficiently cleave peptide amide bonds. Alternatively, some aspect of the secondary or tertiary structure of plasminogen may be required to interact with the protease domain to correctly align the labile Arg-Val bond for cleavage.

The recent description of the structure of complement Factor D provides the first evidence that the former mechanism might be utilized by highly specific proteases to restrict substrate specificity. Examination of the 2.0-Å crystal structure of complement Factor D reveals that alterations relative to trypsin at residues 94, 214, and 215 prevent aspartic acid 102 from interacting with histidine 57 in the free enzyme. Narayana and co-workers (43) propose, therefore, that binding of the substrate (a complex of Factor B and the co-factor C3b) to Factor D must cause the catalytic triad and the S1 pocket of Factor D to assume an active conformation and thereby promote rapid catalysis of the target peptide bond. While t-PA and complement Factor D both possess high reactivity toward their specific substrate and low reactivity toward peptides, complement Factor D is functionally different from t-PA because it is relatively unreactive toward ester and activated amide substrates. This functional distinction between the two proteases suggests that the observed disruption of the catalytic machinery of complement Factor D is probably more severe than any disruption which may be present in t-PA. Consequently, the structural basis for the low reactivity of the two proteases toward peptide substrates is not likely to be identical, suggesting that multiple mechanisms may have evolved to enhance the specificity of serine proteases.

We have demonstrated that, even in the absence of fibrin, t-PA is more active than trypsin toward plasminogen and that this efficient catalysis is in contrast to the dramatically lower catalysis by t-PA of other peptide and protein substrates. We also provide the first quantitative treatment of the cleavage by t-PA of linear and cyclic peptides that contain its normal target sequence from plasminogen. These assays indicate that t-PA is 19,000-87,000 times more active toward plasminogen than toward peptides containing the cleavage site from plasminogen. Important determinants of this impressive specificity for cleavage of native plasminogen appear to reside in the protease domain of t-PA. Kinetic measurements reported in this study emphasize, and also quantify, profound differences between the protease domain of t-PA and trypsin, two highly homologous enzymes. This study may facilitate the identification of peptides that mimic the ``recognition surface'' on plasminogen and offers an incentive for homology modeling of the protease domain of t-PA based upon the structure of trypsin. Such efforts may provide important new insights regarding the evolution of the protease domain of t-PA into an exquisitely specific protease and may facilitate the development of more selective thrombolytic agents.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant RO1 HL52475 (to E. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Fax: 619-554-6402 (E. L. M.); 214-648-5095 (D. R. C.).

Supported by National Institutes of Health Biophysics Predoctoral training program 5P32GMO8287.

**
Assistant Investigator with the Howard Hughes Medical Institute.

(^1)
The abbreviations used are: t-PA, tissue type plasminogen activator; Spec t-PA, methylsulfonyl-D-cyclohexyltyrosyl-glycl-arginine-p-nitroanilide; Boc-LGR-pNA, Boc-leucyl-glycyl-argininyl-p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

(^2)
G. S. Coombs, unpublished results.


ACKNOWLEDGEMENTS

We thank Lynn DeOgny, Bikash C. Pramanik, and Dr. Clive Slaughter for peptide synthesis and mass spectral analysis.


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