©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Variants of Tissue-type Plasminogen Activator Which Display Substantially Enhanced Stimulation by Fibrin (*)

(Received for publication, April 24, 1995; and in revised form, June 5, 1995)

Kathy Tachias Edwin L. Madison (§)

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Unlike most proteases, tissue-type plasminogen activator (t-PA) is secreted from cells as an active, single chain ``proenzyme'' whose catalytic efficiency is comparable with that of the corresponding mature, two-chain enzyme. We have previously suggested that the absence of the ``zymogen triad'' (Asp-His-Ser; chymotrypsin numbering) contributes to this unusually high enzymatic activity of single chain t-PA. Consistent with this prediction, the single chain form of a variant of t-PA containing the zymogen triad displayed dramatically reduced activity toward synthetic substrates. Activation cleavage of this variant, however, resulted in a mature, two-chain enzyme with full catalytic activity. To further examine the functional significance of the zymogen triad, we used site-specific mutagenesis to construct a variant of t-PA, t-PA/R275E,A292S,F305H, that contained this triad but could not be converted into its two-chain form by plasmin. Characterization of this variant demonstrated that the presence of the zymogen triad specifically suppressed plasminogen activation by single chain t-PA in the absence of fibrin. In addition, these studies indicated that, like wild type t-PA, zymogen activation of this variant could be accomplished by binding to the co-factor fibrin. The combination of full activity in the presence of fibrin and reduced activity in its absence resulted in novel variants of t-PA that displayed dramatically enhanced stimulation by fibrin. While the presence of fibrin increased the catalytic efficiency of t-PA toward plasminogen by a factor of approximately 520, this stimulation factor increased to 130,000 for t-PA/R275E,A292S,F305H. Plasmin-resistant, zymogen-like variants of t-PA, therefore, may represent thrombolytic enzymes with enhanced ``clot selectivity.''


INTRODUCTION

A wide variety of critical biological processes(1, 2, 3) depend on specific cleavage of individual target proteins by serine proteases. Important examples include both the formation and dissolution of blood clots that are catalyzed, respectively, by the blood coagulation cascade and the endogenous fibrinolytic system(4, 5) . While blood coagulation can be initiated by the activity of either Factor VIIa (the extrinsic pathway) or Factor XIIa (the intrinsic pathway), the initiating, and rate-limiting, step of the fibrinolytic system is activation of the circulating zymogen plasminogen by the tissue-type plasminogen activator (t-PA) (^1)or urokinase-type plasminogen activator (u-PA)(4, 5) .

Unlike typical chymotrypsin-like enzymes, the single chain or ``proenzyme'' form of t-PA possesses high catalytic activity(6, 7, 8, 9, 10, 11, 12) . In the absence of the co-factor fibrin, single chain t-PA is approximately 8% as active as two-chain t-PA. In the presence of fibrin, however, single chain and two-chain t-PA display equivalent enzymatic activity. ``Zymogen activation'' of single chain t-PA, therefore, can be accomplished either by activation cleavage or by binding to a specific co-factor.

We have previously demonstrated that introduction of the zymogen triad into t-PA substantially increases the enzyme's ``zymogenicity,'' or ratio of activity of the mature, two-chain enzyme to that of the single chain form, by specifically suppressing the activity of single chain t-PA(12) . These earlier measurements of the zymogenicity of the mutated enzyme t-PA/A292S,F305H (^2)relied upon activity assays that utilized small, synthetic substrates. Measuring the activity of the single chain form of t-PA/A292S,F305H toward the normal substrate plasminogen proved technically difficult because plasmin generated during this assay very rapidly and efficiently converted single chain t-PAs into the corresponding two-chain enzymes by cleaving the Arg-Ile bond of single chain t-PA. To overcome this technical difficulty, we have now added the mutation R275E to the zymogen-like variants of t-PA. This strategy rendered the resulting variants resistant to activation cleavage by plasmin and therefore allowed accurate measurement of the kinetic constants for plasminogen activation by the single chain form of the enzymes. These measurements demonstrated that variants of t-PA that contain the zymogen triad exhibited zymogen-like properties toward not only synthetic substrates but also the normal protein substrate plasminogen and that, as with the wild type enzyme, zymogen activation of these variants could be accomplished by binding to the co-factor fibrin as well as by activation cleavage. The combination of these properties dramatically enhanced the extent of fibrin stimulation of the variants compared with wild type t-PA. While fibrin enhanced the activity of t-PA by a factor of approximately 520, the activity of the variant t-PA/R275E,A292S,F305H was stimulated by fibrin by a factor of approximately 130,000.


MATERIALS AND METHODS

Site-directed Mutagenesis and Construction of Expression Vectors Encoding Variants of t-PA

Oligonucleotide-directed site-specific mutagenesis was performed by the method of Zoller and Smith (13) as modified by Kunkel(14) . Mutations were introduced into the 472-base pair EcoRI fragment of cDNAs encoding t-PA, t-PA/F305H, and t-PA/A292S,F305H that had been previously subcloned into bacteriophage M13 mp18. The mutagenic primer had the following nucleotide sequence: 5`-CAGCCTCAGTTTGAGATCAAAGGAGGG-3`.

Following mutagenesis, single-stranded DNA corresponding to the entire 472-base pair EcoRI fragment was fully sequenced to assure the presence of the desired mutation and the absence of any additional mutations. Replicative form DNA was prepared for appropriate phage, and the mutated 472-base pair EcoRI fragments were recovered after digestion of replicative form DNA with EcoRI and electrophoresis of the digestion products on an agarose gel. The isolated EcoRI fragments were used to reconstruct full-length cDNAs encoding t-PA/R275E,t-PA/R275E,F305H and t-PA/R275E,A292S,F305H.

Expression of Enzymes by Transient Transfection of COS Cells

cDNAs encoding t-PA, t-PA/R275E, t-PA/R275E,F305H and t-PA/R275E,A292S,F305H were ligated into the transient expression vector pSVT7 (15) and then introduced into COS 1 cells by electroporation using a Bio-Rad Gene Pulser. 20 µg of cDNA, 100 µg of carrier DNA, and approximately 10^7 COS cells were placed into a 0.4-cm cuvette, and electroporation was performed at 320 V, 960 microfarads, and = . Following electroporation, cells were incubated overnight at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 5 mM sodium butyrate. Cells were then washed with serum free medium and incubated in Dulbecco's modified Eagle's medium for 48 h at 37 °C. After the incubation with serum-free media, conditioned media were collected, and enzyme concentrations were determined by enzyme-linked immunosorbent assay.

Purification and Quantitation of Enzymes

Conditioned media were dialyzed against 20 mM sodium phosphate (pH 7.0), 100 mM NaCl, 20 mM arginine, and 0.05% Tween 80 and loaded onto a lysine-Sepharose (Pharmacia Biotech Inc.) column. The column was washed with 20 volumes of loading buffer, and t-PA was eluted with buffer containing 20 mM sodium phosphate (pH 7.0), 100 mM NaCl, 200 mM arginine, and 0.05% Tween 80. Enzyme concentrations were measured by enzyme-linked immunosorbent assay.

Kinetic Analysis of Plasminogen Activation Using Indirect Chromogenic Assays

Indirect chromogenic assays of t-PA utilized the substrates Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) and were performed as described previously (15, 16, 17) . Assays were performed both in the presence and absence of the co-factor DESAFIB (American Diagnostica). DESAFIB, a preparation of soluble fibrin monomers, was produced by digesting highly purified human fibrinogen with the protease batroxobin. Batroxobin cleaved the Arg-Gly bond in the Aalpha-chain of fibrinogen and consequently released fibrinopeptide A. The resulting des-AA-fibrinogen or fibrin I monomers are soluble in the presence of the peptide Gly-Pro-Arg-Pro. The concentration of Lys-plasminogen was varied from 0.0125 to 0.2 µM in the presence of DESAFIB and from 0.9 to 15 µM in the absence of the co-factor.

Indirect Chromogenic Assays in the Presence of Various Stimulators

Standard indirect chromogenic assays were performed as described previously(15, 16, 17) . Briefly, 0.25-1 ng of enzyme, 0.2 µM Lys-plasminogen, and 0.62 mM Spectrozyme PL were present in a total volume of 100 µl. Assays were performed either in the presence of buffer, 25 µg/ml DESAFIB, 100 µg/ml cyanogen bromide fragments of fibrinogen (American Diagnostica), or 100 µg/ml of the stimulatory, 13-amino acid peptide P368. P368 was kindly provided by Marshall Runge (University of Texas Medical Center, Galveston, TX). Assays were performed in microtiter plates, and the optical density at 405 nm was read every 30 s for 1 h in a Molecular Devices Thermomax. Reactions were performed at 37 °C.

Measurement of Second Order Rate Constants for Inhibition by PAI-1

Second order rate constants for the inhibition of wild type and mutated t-PA were measured under pseudo-first order conditions as described previously(16, 17, 18, 19) . Briefly, enzyme and inhibitor were preincubated at 23 °C for periods of time varying from 0 to 30 min. Following preincubation, the mixtures were diluted, and the residual enzymatic activity was measured in a standard indirect chromogenic assay. For each enzyme, the concentrations of enzyme and inhibitor and the times of preincubation were chosen to yield several data points for which the residual enzymatic activity varied between 20 and 80% of the initial activity. Data were analyzed by plotting ln (residual activity/initial activity) versus time of preincubation and measuring the resulting slopes. Division of this slope by -[I] yielded the second order rate constants shown.


RESULTS AND DISCUSSION

We have previously reported that the enzymes t-PA/F305H and t-PA/A292S,F305H display zymogen-like properties when assayed with synthetic substrates(12) . Kinetic constants for activation of plasminogen by the single chain form of these variants, however, have not been reported. To facilitate accurate measurement of these kinetic constants, we utilized oligonucleotide-directed site-specific mutagenesis to construct the enzymes t-PA/R275E, t-PA/R275E,F305H and t-PA/R275E,A292S,F305H.

The results of a kinetic analysis of the activation of plasminogen, in the absence of fibrin, by t-PA, t-PA/R275E, t-PA/R275E,F305H, and t-PA/R275E,A292S,F305H are depicted in Table 1. Even before significant hydrolysis of the chromogenic substrate Spectrozyme PL occurs in this assay, single chain wild type t-PA is converted into a two-chain enzyme ((9) , data not shown). By contrast, enzymes containing the R275E mutation remain in the single chain form for the duration of the assay ((9) , data not shown). Comparison of k/K values for wild type t-PA and t-PA/R275E, therefore, indicate that, in accord with previous reports(6, 7, 8, 9, 10, 11, 12) , single chain t-PA has approximately 8% of the activity of two-chain t-PA in this assay. Compared with that of t-PA/R275E, the catalytic efficiency of t-PA/R275E,F305H is reduced by a factor of 4.4, while that of t-PA/R275E,A292S,F305H is reduced 20-fold. Compared with the activity of wild type t-PA, the activity of t-PA/R275E,F305H is reduced approximately 58-fold, while that of t-PA/R275E,A292S,F305H is reduced by a factor of approximately 264. These data demonstrate that the mutation F305H suppresses the activity of the single chain form of t-PA toward not only small, synthetic substrates but also the natural protein substrate plasminogen. Moreover, the addition of a second mutation, A292S, further suppresses the activity of single chain t-PA toward plasminogen.



Table 2presents the results of a kinetic analysis of plasminogen activation, in the presence of the fibrin monomer DESAFIB, by the same four enzymes described above. In striking contrast to their reduced activity in the absence of a co-factor, all three mutated enzymes exhibit high catalytic activity in this assay. In the presence of DESAFIB the k/K value for plasminogen activation by all three mutated enzymes varies by less than 6% from that of wild type t-PA. These data demonstrate that, as with single chain wild type t-PA, zymogen activation of the variants t-PA/R275E, t-PA/R275E,F305H, and t-PA/R275E,A292S,F305H can be accomplished by binding to the co-factor fibrin.



By contrast to the other human plasminogen activator u-PA, t-PA both binds to and is substantially stimulated by fibrin(20, 21, 22) . Table 3lists the fibrin stimulation factor, defined as the ratio of k/K in the presence of fibrin to that in the absence of fibrin, for t-PA, t-PA/R275E, t-PA/R275E,F305H, and t-PA/R275E,A292S,F305H. As indicated in Table 3, fibrin enhances the activity of the wild type enzyme by a factor of approximately 520. Fibrin stimulation of all three variants, however, is dramatically enhanced compared with the wild type enzyme. The fibrin stimulation factor for t-PA/R275E, t-PA/R275E,F305H, and t-PA/R275E,A292S,F305H is, respectively, 6900, 30,000, and 130,000. Enhanced fibrin stimulation of all three variants results from the combination of diminished activity in the absence of co-factor and high activity in its presence.



With a molecular mass of almost 400 kDa, fibrin is a very large protein whose interaction with t-PA appears to be complex and to involve multiple points of contact with the enzyme (for review, see (20, 21, 22) ). It is therefore noteworthy, and perhaps surprising, that the introduction of only three mutations into t-PA, R275E, A292S, and F305H, can enhance fibrin stimulation of the enzyme to the extent that it becomes comparable with that of Desmodusrotundis plasminogen activator, by far the most fibrin-responsive plasminogen activator that has been reported (23, 24, 25) . Discounting the absence of a kringle 2 domain in the Desmodus enzyme, the primary structures of human t-PA and Desmodusrotundis plasminogen activator still differ at 123 positions. Moreover, the primary structures of the variant of human t-PA and the Desmodus enzyme differ at all three sites of mutation in the human enzyme although both enzymes do contain mutations that prevent normal maturation into a mature, two-chain enzyme.

Data in Table 1Table 2, and Table 3establish that the variants are more fibrin-stimulated than wild type t-PA; data in Fig. 1, on the other hand, indicate that the variants are also more ``fibrin-selective'' than the wild type enzyme. While the activity of t-PA is almost equally responsive to fibrin monomers, cyanogen bromide fragments of fibrinogen, or a stimulatory 13-amino acid peptide, the three variants show a striking preference for the fibrin monomers over either of the nonphysiological co-factors. In fact, again reminiscent of the Desmodus plasminogen activator, both t-PA/R275E,F305H and t-PA/R275E,A292S,F305H maintain high activity in the presence of fibrin monomers but are virtually nonresponsive to the nonphysiological stimulators.


Figure 1: Standard indirect chromogenic assay of plasminogen activation in the presence of buffer () DESAFIB (), cyanogen bromide fragments of fibrinogen (), or the stimulatory peptide P368 (&cjs2134;).



We have previously reported that the single chain form of the zymogen-like variants of t-PA, t-PA/F305H, and t-PA/A292S,F305H exhibit resistance to inhibition by PAI-1(12) . Compared with that of single chain t-PA, the second order rate constant for inhibition by PAI-1 of single chain t-PA/F305H or t-PA/A292S,F305H is reduced by a factor of approximately 5 or 25, respectively. Second order rate constants for inhibition of single chain t-PA, t-PA/R275E, t-PA/R275E,F305H and t-PA/R275E,A292S,F305H are listed in Table 4. These data indicate that t-PA/R275E,F305H and t-PA/R275E,A292S,F305H also exhibit resistance to inhibition by PAI-1; the second order rate constant for inhibition of these enzymes by PAI-1 is reduced by a factor of approximately 9 or 69, respectively, compared with t-PA/R275E.



The major findings of this study are that variants of t-PA containing the zymogen triad exhibit zymogen-like properties when assayed with the normal substrate plasminogen and that zymogen activation of these variants can be accomplished by binding to the co-factor fibrin. While details of the mechanism by which a zymogen is activated upon binding to a specific co-factor remain completely unclear, it is clear that this property is not unique to t-PA. Other examples of this phenomenon include the activation of plasminogen by streptokinase(26, 27, 28, 29, 30, 31) and the activation of prothrombin upon binding to staphylocoagulase(32, 33) . Since several chymotrypsin-like enzymes have this property, it is tempting to speculate that this mechanism of activation of zymogens might provide the initiation event in (a) protease cascade(s) in vivo. Although there is no current evidence to support the idea, this strategy has the attractive feature that it avoids the difficulty of generating proteolytic activity to activate the first zymogen in a cascade.

The mutated enzymes t-PA/R275E,A292S,F305H and t-PA/R275E,F305H are substantially more fibrin-stimulated, and also more fibrin-selective, than wild type t-PA. In vivo, therefore, it is possible that, compared with wild type t-PA, these enzymes would display significantly enhanced ``clot selectivity'' due to reduced activity in the circulation but full activity at a site of fibrin deposition. Whether enhanced clot selectivity would improve the enzyme as a thrombolytic agent remains extremely controversial, although this property may assume increased importance as variants of t-PA with a prolonged half-life are administered as a single bolus.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1 HL52475 and P01 HL31950 (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: Dept. of Vascular Biology, Scripps Research Inst., 10666 N. Torrey Pines, La Jolla, CA. 92037. Fax: 619-554-6402.

^1
The abbreviations used are: t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminogen activator.

^2
Positions 275, 292, and 305 in t-PA correspond to positions 15, 32, and 40 of chymotrypsin.


ACKNOWLEDGEMENTS

We thank Guy Salvesen, Betsy Goldsmith, Jeff Smith, Martin Schwartz and Dave Loskutoff for critical review of this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.