©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Single-chain Urokinase and Plasminogen Activator Inhibitor Type 1 (*)

(Received for publication, June 6, 1995; and in revised form, June 15, 1995 )

Naveen Manchanda Bradford S. Schwartz (§)

From the Departments of Medicine and Biomolecular Chemistry, University of Wisconsin and the Medical Service, William A. Middleton VA Hospital, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Urokinase (u-PA) is synthesized and secreted as a single-chain polypeptide (single-chain u-PA, scu-PA), which has such little enzymatic activity in solution that it has been considered essentially enzymatically inert. We found that plasminogen activator inhibitor type 1 (PAI-1), the major PAI in plasma, demonstrated concentration-dependent inhibition of this solution-phase scu-PA enzymatic activity. I-scu-PA formed complexes with PAI-1 in a concentration- and time-dependent manner, as detected by SDS-polyacrylamide gel electrophoresis under reducing conditions. Among a given population of scu-PA molecules, all measurable enzymatic activity was inhibited by a 10-fold molar excess of PAI-1. However, at this stoichiometry, only a minority of I-scu-PA molecules formed SDS-stable complexes with PAI-1 (i.e. complexes that formed a covalent bond upon denaturation), even though the uncomplexed PAI-1 molecules remained competent to inhibit u-PA enzymatic activity. Neither the extent nor the time course of complex formation was altered by using PAI-1 that had been pre-incubated with native human vitronectin, compared with native PAI-1 alone. I-scu-PAbulletPAI-1 complexes that would form a covalent bond if denatured were reversible and existed in equilibrium with either non-complexed or loosely complexed reactants. These data suggest that scu-PA has more enzyme-like properties than previously appreciated and raises the possibility that it resembles single-chain tissue type-plasminogen activator in lacking a complete zymogen conformation.


INTRODUCTION

Urokinase-type plasminogen activator (u-PA) (^1)is involved in a wide variety of biological processes(1, 2, 3) . It is synthesized and secreted as a single-chain molecule (scu-PA), which has little activity in solution (4, 5, 6, 7) but exhibits appreciable plasminogen-activating activity when bound to its specific cell surface receptor (6, 7) or when plasminogen is bound to an appropriate lysine on partially degraded fibrin(8) . The plasmin thus generated cleaves scu-PA to two-chain u-PA (tcu-PA), which has three to four logs more enzymatic activity than soluble scu-PA (4, 7, 8, 9) and is efficiently inhibited by plasminogen activator inhibitors (PAIs), members of the serpin family of protease inhibitors(10) .

There has been debate regarding the enzymatic activity of scu-PA, with the question of whether scu-PA must first be activated to tcu-PA to exhibit meaningful enzymatic activity(5, 6, 7, 11, 12) . Recent data from our laboratory described a reversible noncovalent interaction between scu-PA and plasminogen activator inhibitor type 2 (PAI-2), with resultant complete inhibition of scu-PA enzymatic activity(13) . Such an interaction suggested sufficient enough enzyme-like properties for scu-PA that specific interactions with inhibitors might be important.

Plasminogen activator inhibitor type 1 (PAI-1) is the primary PAI of human plasma, is found in platelets, and is synthesized and secreted by a variety of cells in culture(14) . PAI-1 circulates in plasma and exists in extracellular matrix bound to vitronectin (VN)(15, 16) , in which state its time-dependent loss of inhibitory activity is attenuated as compared with isolated PAI-1(15, 17) . PAI-1 has a high affinity for both tcu-PA and tissue-type plasminogen activator (t-PA), effectively limiting the enzymatic activity of these proteases (18) . The interaction between PAI-1 and tcu-PA follows the reaction paradigm described for serine proteases and their inhibitors. There is an initial reversible interaction between the molecules, followed by the formation of a ``stable intermediate'' complex wherein the active site serine is complexed to the P(1) residue of the inhibitor to yield a complex that is stable to denaturing agents(19) .

In this report, we describe interactions between scu-PA and PAI-1, present findings that support the concept that scu-PA is not an inert proenzyme, and suggest a previously undescribed level of regulation of plasminogen activation.


MATERIALS AND METHODS

The following proteins were kindly provided by the individuals noted: recombinant native scu-PA by Dr. Jack Henkin (Abbott Laboratories) and recombinant human PAI-1 by Drs. David Ginsburg and Daniel Lawrence (Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI). Three batches of PAI-1 were used in these studies, exhibiting 47, 53, and >95% active PAI-1 as assessed by inhibition of tcu-PA-mediated hydrolysis of a chromogenic substrate. Human plasma VN was isolated under nondenaturing conditions by Dr. Deane Mosher (University of Wisconsin, Madison, WI), fibrinogen by Dr. Michael Mossesson (University of Wisconsin, Milwaukee, WI), and tcu-PA by Dr. Gene Murano (National Bureau of Standards, Wash., D. C.). Plasminogen was isolated from human plasma as previously described(7, 21) . Chemicals for SDS-polyacrylamide gel electrophoresis (PAGE) (22) were from Bio-Rad. All other chemicals were obtained from Sigma. Proteins were iodinated using the Iodogen method as previously described(7, 13) .

Plasminogen-activating activity was measured using the plasmin-independent I-plasminogen cleavage assays as previously described(13, 23) . Inhibition of scu-PA activity was determined by comparing the generation of I-plasmin light chain, as assessed by SDS-PAGE under reducing conditions and autoradiography, of reactions wherein scu-PA had been preincubated with PAI-1 to scu-PA that had been preincubated with either ovalbumin or buffer alone.

To detect SDS-stable complexes, I-scu-PA was incubated with PAI-1 under conditions specified in the text. The reaction mixture was then boiled in SDS-PAGE buffer with beta-mercaptoethanol and analyzed by SDS-PAGE followed by autoradiography. Alternatively, unlabeled proteins were combined as above and analyzed by SDS-PAGE and silver staining. To determine whether scu-PA and tcu-PA competed with one another for interaction with PAI-1, varying concentrations of scu-PA were incubated with PAI-1 in 10 µl of Tris-buffered saline (0.15 M NaCl, 0.01 M Tris, pH 7.4) for 10-15 min at room temperature. I-tcu-PA in 5 µl of Tris-buffered saline was then added to each reaction for either a further 10 s or 5 min at 4 °C, followed by SDS-PAGE under reducing conditions and autoradiography. Alternatively, to determine whether the association of scu-PA with PAI-1 was reversible, I-scu-PA was incubated with PAI-1 at the indicated concentrations for 2 h at 37 °C, followed by the addition of increasing concentrations of tcu-PA for 3 h at 37 °C. The samples were then boiled in reducing SDS-gel buffer, which contained extra tcu-PA such that all samples had the same quantity of all reactants when analyzed by SDS-PAGE and autoradiography.

To determine the influence of VN on PAI-1 interactions with scu-PA, native human VN was incubated with PAI-1 at room temperature for 2 h; this PAI-1bulletVN mixture was then combined with I-scu-PA as described in the text. Preliminary experiments showed the PAI-1:VN mixture inhibited thrombin activity in a fluorogenic substrate-based assay(24) , whereas PAI-1 alone did not. Hence, PAI-1bulletVN complexes were present in the mixture used in these experiments.


RESULTS AND DISCUSSION

We employed an I-plasminogen cleavage assay carried out in the presence of high concentrations of trasylol to monitor the generation of I-plasmin (13, 23) to demonstrate that scu-PA was inhibited in a concentration-dependent manner by PAI-1 but not by ovalbumin (Fig. 1). We have previously shown that I-scu-PA remains in the single-chain form in this assay(13) . As demonstrated in Fig. 2A, I-scu-PA formed SDS-stable complexes with PAI-1 of molecular mass 98 K. Identical SDS-stable complexes were formed between unlabeled scu-PA and PAI-1 as determined by silver staining of gels (Fig. 2B), indicating that such complex formation was a property of native scu-PA and not an artifact of iodination. tcu-PA on the other hand, formed complexes with a molecular mass of 75 K as expected (see Fig. 3). Hence, the scu-PAbulletPAI-1 complexes were not the result of tcu-PA being generated, then complexing with PAI-1.


Figure 1: PAI-1 inhibits the enzymatic activity of native scu-PA. Scu-PA (1 µg) was incubated for 30 min at 37 °C with the indicated amount of PAI-1 or ovalbumin and then combined with I-plasminogen (Pg) and trasylol for 16 h at 37 °C as described under ``Materials and Methods.'' Reaction mixtures were analyzed by SDS-PAGE under reducing conditions (12% gel), followed by autoradiography to detect the formation of I-plasmin. Indicated on the left of the gel are I-plasminogen, plasmin heavy chain (PnHC), and plasmin light chain (LC), the latter being the best indicator of plasmin generation. The data are representative of three similar experiments.




Figure 2: The formation of complexes between scu-PA and PAI-1 is concentration-dependent and is a property of native as well as iodinated scu-PA. A, a fixed amount of I-scu-PA (75 ng) was incubated with the indicated amounts of PAI-1 for 3 h at 37 °C. Mixtures were analyzed by SDS-PAGE under reducing conditions and autoradiography. The data are representative of five similar experiments. B, a fixed amount of unlabeled scu-PA was incubated with the indicated amounts of PAI-1 as in A, and analyzed by SDS-PAGE under reducing conditions, followed by silver staining.




Figure 3: PAI-1 that does not complex with scu-PA remains active. I-scu-PA (75 ng) was incubated with PAI-1 (750 ng) for 2 h at 37 °C, at which point additional I-scu-PA (75 ng) or I-tcu-PA (75 ng) was added to the mixtures as noted and incubated for 30 min at 37 °C, followed by SDS-PAGE under reducing conditions and autoradiography. The lower molecular weight bands in lanes containing I-scu-PA are the result of degradation of the labeled protein during storage and do not represent I-tcu-PA (note lack of 75-kDa complexes with PAI-1 unless exogenous I-tcu-PA was added). The data are representative of two similar experiments.



The presence of SDS-stable complexes between a serine protease and serpin probably reflects reaction of the tetrahedral protease-serpin intermediate upon denaturation such that a covalent ester bond between the two molecules is formed(19) . Scu-PA apparently forms such an intermediate with PAI-1, which results in covalent bond formation upon denaturation; no such intermediate seems to form between scu-PA and PAI-2, even though scu-PA enzymatic activity is inhibited by PAI-2(13) .

Interestingly, the I-scu-PA was not quantitatively complexed to PAI-1 in an SDS-stable manner. Even a 100-fold molar excess of PAI-1 over I-scu-PA did not yield SDS-stable complexes with all scu-PA molecules (Fig. 2), despite complete inhibition of enzymatic activity. This is in contrast to the interaction of PAI-1 with tcu-PA, wherein a 10-fold excess of PAI-1 results in all tcu-PA becoming complexed with the inhibitor in an SDS-stable manner (data not shown). However, incubation of I-scu-PA with a 10-fold excess of PAI-1, followed by immunoprecipitation of the PAI-1, resulted in coprecipitation of more than 96% of the I-scu-PA. Therefore, all of the scu-PA seems to complex with PAI-1, but only a fraction of the interactions results in the formation of a stable intermediate.

To determine whether a given population of PAI-1 molecules was unable to form more complexes than we observed, I-scu-PA was incubated for 3 h at 37 °C with 10-fold molar excess PAI-1. I-scu-PA, I-tcu-PA, or buffer were then added to the incubation mixtures for a further 30 min, followed by SDS-PAGE under reducing conditions and autoradiography. As seen in Fig. 3, addition of excess I-scu-PA did not result in the formation of more detectable complexes with PAI-1 compared with the reaction wherein buffer was added for the second incubation. However, addition of I-tcu-PA for the second incubation resulted in the formation of I-tcu-PAbulletPAI-1 complexes, with no decrement in the preformed I-scu-PAbulletPAI-1 complexes (i.e. the data do not suggest a single population of active PAI-1 molecules that complexed to either scu-PA or tcu-PA). Hence, the previously uncomplexed PAI-1 molecules were competent to form complexes if the reaction conditions were suitable.

To determine whether prolonged incubation of I-scu-PA with PAI-1 would alter SDS-stable complex formation, I-scu-PA was incubated with buffer or PAI-1 at either 4 or 37 °C for increasing times, followed by SDS-PAGE and autoradiography. As seen in Fig. 4, the formation of SDS-stable complexes was both time- and temperature-dependent, but once a given proportion of I-scu-PA molecules was complexed with PAI-1, no further complex formation could be detected. The use of a 10-fold molar excess of PAI-1bulletVN did not result in more rapid, or quantitatively greater complex formation with I-scu-PA than found with the same molar quantity of PAI-1 alone.


Figure 4: Effects of time, temperature, and presence of vitronectin on formation of scu-PAbulletPAI-1 complexes. I-scu-PA (150 ng) was incubated at 4 °C (panelA) or 37 °C (panelB) with PAI-1 (1.5 µg) alone or with PAI-1, which had been preincubated with vitronectin (2.5 µg) for 30 min at 37 °C (the presence of PAI-1bulletVN complexes in these pre-incubations was documented by the presence of thrombin inhibitory activity as outlined under ``Materials and Methods''(24) ). Aliquots were removed at the indicated times (0-20 h) and analyzed by SDS-PAGE under reducing conditions and autoradiography. The data are representative of three similar experiments.



The above data suggested that scu-PAbulletPAI-1 complexes existed in equilibrium between a state that yielded a covalent bond upon denaturation and a state that did not. Implicit in this was the reversibility of scu-PA-PAI-1 interactions, which was tested by incubating increasing concentrations of scu-PA with a constant concentration of PAI-1 for 15 min at 22 °C to allow the formation of scu-PAbulletPAI-1 complexes. A constant concentration of I-tcu-PA was then added to each reaction for another 10 s at 4 °C, followed by SDS-PAGE and autoradiography. As demonstrated in Fig. 5A, the interaction of scu-PA with PAI-1 prevented, in a concentration-dependent manner, subsequent complex formation between I-tcu-PA and PAI-1. Plasminogen (also a serine protease zymogen) demonstrated no such inhibition. However, if the above experiment were repeated, but with the I-tcu-PA added for 5 min instead of 10 s prior to SDS-PAGE (to allow a longer time to compete off the scu-PA), no inhibition of I-tcu-PAbulletPAI-1 complex formation by scu-PA could be demonstrated (Fig. 5B). Hence, the interaction of scu-PA with PAI-1 seems to be reversible, as PAI-1 that had been complexed with scu-PA (Fig. 5A) could dissociate from that scu-PA and subsequently interact with I-tcu-PA (Fig. 5B).


Figure 5: Scu-PA competes with I-tcu-PA for binding to PAI-1. A, PAI-1 (75 ng) was incubated with either scu-PA or plasminogen (Pg) at the indicated molar ratios (PAI-1:scu-PA or PAI-1:Pg) for 15 min at room temperature. Mixtures were cooled for 2 min in an ice slurry, 75 ng I-tcu-PA was added to the mixtures for 10 s, and samples were analyzed by SDS-PAGE under reducing conditions and autoradiography. B, the experiment was performed exactly as in A, except I-tcu-PA was added for 5 min to allow competition for PAI-1 previously bound to scu-PA. The data are representative of three similar experiments.



It is possible that the above reversible scu-PA-PAI-1 interaction involved only those complexes that did not form a covalent bond upon denaturation, with the more stable complexes not dissociating. To determine specifically whether the scu-PAbulletPAI-1 complex, which yields a covalent bond upon denaturation, is reversible, I-scu-PA was incubated 2 h at 37 °C with buffer alone or with a 10-fold molar excess of PAI-1. Increasing concentrations of unlabeled tcu-PA were then added to parallel incubations for 3 h at 37 °C, followed by SDS-PAGE and autoradiography. Fig. 6shows that the more tcu-PA was added to pre-formed I-scu-PAbulletPAI-1 complexes, the fewer of those complexes remained. This implies that I-scu-PAbulletPAI-1 complexes, which would form a covalent bond upon denaturation, do indeed dissociate. If there is only I-scu-PA present to interact with the now free PAI-1, new I-scu-PAbulletPAI-1 complexes form, and the detected level of these complexes does not change, resulting in the observed equilibrium. However, the more tcu-PA is present, the more free PAI-1 becomes complexed to it (tcu-PA), the less PAI-1 is available for complex formation with I-scu-PA, and the fewer I-scu-PAbulletPAI-1 complexes are detected. Hence, I-scu-PAbulletPAI-1 complexes that would form a covalent bond upon denaturation are reversible, albeit slowly (note in Fig. 3, I-tcu-PA was added to I-scu-PAbulletPAI-1 for only 30 min, compared to 3 h in Fig. 6; the shorter time did not yield an observable decrease in I-scu-PAbulletPAI-1 complexes). This is consistent with scu-PA and PAI-1 existing in a tetrahedral intermediate as described for alpha(1)-antitrypsin and elastase(25) .


Figure 6: Reversible interaction between I-scu-PA and PAI-1. I-scu-PA (75 ng) was incubated 2 h at 37 °C with PAI-1 (750 ng). Increasing concentrations of tcu-PA, as noted in the figure, were then added to the pre-formed I-scu-PAbulletPAI-1 complexes in parallel incubations for 3 h at 37 °C, followed by SDS-PAGE under reducing conditions and autoradiography. The relative quantities of I-scu-PAbulletPAI-1 complexes remaining were determined by densitometry and are plotted on the ordinate versus the amount of tcu-PA added (abscissa). The inset shows the autoradiogram. This experiment is representative of five performed.



These data suggest that scu-PA is either more enzyme-like or less zymogen-like than was previously appreciated and that scu-PA interacts with PAI-1 much like a ``conventional'' active serine protease interacts with its cognate serpin. The initial interaction is rapidly reversible and depends on non-active site determinants. The second part of the interaction can involve a ``stable intermediate'' state, which presumably involves the active site serine of scu-PA(19, 25) . Furthermore, it suggests that scu-PA may be more similar to single-chain t-PA than previously appreciated(26) . This would make it more likely that scu-PA is competent to activate plasminogen in the appropriate setting without cleavage to tcu-PA, much as single-chain t-PA can activate plasminogen without cleavage to two-chain t-PA(26, 27) . The mechanisms for initiating plasminogen activation on the surface of either cells or fibrin clots may therefore be mechanistically similar. Each plasminogen activator may lack true zymogen-like properties, as described for t-PA(26) , yet be a poor plasminogen activator in solution. Binding of either enzyme to the appropriate site (cell surface receptor for scu-PA, fibrin for t-PA) in proximity to appropriately bound plasminogen would be the initiating event for the generation of plasmin(7, 8) .

The data presented in this report may also explain some interesting findings in the literature. Lijnen et al.(28) described evidence for reversible inhibition of scu-PA added to plasma; the interaction of scu-PA with PAI-1 may explain some of this inhibition. We would hypothesize that because the protein C inhibitor inhibits tcu-PA(29) , this serpin may also interact in a reversible way with scu-PA and explain much of the other plasma-derived scu-PA inhibition (28) . In addition, Ciambrone and McKeown-Longo (30) observed a 98-kDa complex on reduced SDS-PAGE analysis of supernatants from HT1080 cells that had been cultured with [S]methionine. These 98-kDa complexes were immunoprecipitated with antibodies to either u-PA or PAI-1.

We therefore hypothesize that the reversible interaction of scu-PA with u-PA-specific inhibitors of the serpin class may be found in a wide range of situations and may play a role in controlling the initiation of urokinase-mediated plasminogen activation (consistent with the hypothesis presented in (13) ). The observation that scu-PA and PAI-1 can form stable intermediates implies that a molecular conformation of scu-PA possesses appreciable enzyme-like qualities. A more complete description of this must await appropriate studies.


FOOTNOTES

*
This work was supported by Public Health Service Grant HL043506 and a Merit Review Award from the Department of Veterans Affairs. Part of this work was presented in abstract form at the 12th International Congress on Fibrinolysis (September, 1994). 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: Depts. of Medicine and Biomolecular Chemistry, University of Wisconsin and the Medical Service, William A. Middleton VA Hospital, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-4982; Fax: 608-263-4969.

(^1)
The abbreviations used are: u-PA, urokinase-type plasminogen activator; scu-PA, single-chain u-PA; tcu-PA, two-chain u-PA; t-PA, tissue-type plasminogen activator; PAGE, polyacrylamide gel electrophoresis; VN, vitronectin; PAI-1, plasminogen activator inhibitor type 1; serpin, serine protease inhibitor.


ACKNOWLEDGEMENTS

We thank Ellen Martin for expert technical assistance and Marcy Salmon for superb and patient secretarial support.


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