Reversible interactions between plasminogen activators and plasminogen activator inhibitor-1

Biochimica et Biophysica Acta, 1160 (1992) 325-334 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

325

BBAPRO 34343

Reversible interactions between plasminogen activators and plasminogen activator inhibitor-1 Jun Mimuro, Munekiyo Kaneko, Toshinobu Murakami, Michio Matsuda and Yoichi Sakata Institute of Hematology and Department of Medicine, Jichi Medical School, Tochigi-Ken (Japan) (Received 27 April 1992) (Revised manuscript received 24 June 1992)

Key words: Plasminogen; Plasminogen activator; Plasminogen activator inhibitor-I; Tissue-type plasminogen activator; Urokinase; Reversible binding

We have shown that the urokinase (UK) kringle domain contains a high-affinity plasminogen activator inhibitor-1 (PAI-1) binding site, responsible for the 10-fold faster complex formation between UK and PAI-1 than between PAI-1 and low-molecular-weight urokinase (LMWUK). Complex formation between UK and PAI-1, but not between LMWUK and PAI-1, was suppressed 10-fold in the presence of peptide U-107 derived from the UK kringle domain. Peptide U-373 derived from the UK catalytic domain slowed complex formation between UK and PAI-1 and also LMWUK and PAI-1. Inactivation of tissue-type plasminogen activator (tPA) by PAI-1 was slowed 10-fold in the presence of peptides derived from the tPA finger and kringle-2 domains. DFP-inactivated (DIP) UK and both forms of DIP-tPA inhibited PAI-1 binding to U-107 and to U-373 whereas single-chain urokinase-type PA (scuPA) was unable to compete with either peptide for PAI-1 binding. These data suggest that the reversible PAI-1 binding site in the UK A-chain plays a role in the rapid association with PAI-1 as important as those that reside in the tPA A-chain and that reversible PAI-1 binding sites are expressed on the surface of UK upon conversion from scuPA, in contrast to tPA.

Introduction

The fibrinolytic system plays an important role in a variety of biological processes, including vascular thrombolysis, tumor invasion, neovascularization, inflammation and wound healing [1]. Plasminogen activator inhibitor-1 (PAI-1) is the physiological inhibitor of tissue-type plasminogen activator (tPA) and urokinasetype plasminogen activator (uPA) and, thus, is a primary regulator of the fibrinolytic system [2,3]. Plasminogen activator inhibitor-2 (PAI-2) also has inhibitory activity towards U K and tPA [4], however, the rates of inactivation of these enzymes, especially single-chain tPA, by PAI-2 are considerably slower than by PAI-1 [2-5]. Kinetic analyses of the interaction of tPA and PAI-1 suggest that the interaction between tPA and PAI-1 is a two-step mechanism [5]. The first

Correspondence to: J. Mimuro, Institute of Hematology and Department of Medicine, Jichi Medical School, Tochigi-Ken 329-04, Japan. Abbreviations: PAl-l, plasminogen activator inhibitor-l; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; UK, urokinase (two-chain form uPA); scuPA, single-chain form uPA: LMWUK, low-molecular-weight urokinase; BSA, bovine serum albumin; MoAb, monoclonal antibody.

reaction is a rapid and reversible association of tPA and PAI-1, which is followed by a slow but irreversible complex formation of these molecules. Recent studies of the functional domains of tPA have suggested that at least three different PAI-1 binding sites reside in the tPA molecule. They seem to be located in the finger domain [6,7], the kringle-2 domain [7-10] and the exosite of the catalytic domain [11-14] and are involved in the reversible interaction between tPA and PAI-1. Studies using synthetic peptides have raised the possibility that a wide variety of tPA domains are involved in the binding with PAI-1 [6]. We have shown previously that the tPA f n g e r domain is involved in the reversible interaction between the single-chain form of tPA and PAI-1 which results in steric hindrance of the tPA fibrin binding site in the finger domain, whereas the kringle-2 domain plays an essential role in the reversible interaction between the two-chain form of tPA and PAI-1 [7,8]. This reversible association is mediated partially by a specific tPA amino-acid sequence, N R R L , in the kringle-2 domain [7,8]. Based on this model, we speculated that the kringle domain of urokinase (UK), the two-chain form of uPA, may also be involved in a reversible interaction between U K and PAI-1, although a kinetic study sug-

326 gested that that the interaction between UK and PAI-1 was a single-step complex formation between the active site of UK and the reactive center of PAI-1 [5]. We performed a homology search in the UK sequence for the NRRL binding sequence and found a related NRRR sequence in the kringle domain of UK. Thus, we designed a series of synthetic peptides of UK to investigate the interaction between uPA and PAI-1. Here, we show that the kringle domain of UK plays an essential role in the rapid and reversible association with PAI-1 and that the UK catalytic domain contains reversible PAI-1 binding sites which may be involved in a second interaction with PAI-1. These reversible PAI-1 binding sites appear to be buried in single-chain uPA (scuPA), analogous to the observation that the tPA finger and kringle-2 domains play distinct roles in the rapid association of either single chain or two-chain form of tPA with PAI-1. Furthermore, we show evidence that that the PAI-1 binding sites in the UK kringle domain and the C-terminal region of the UK catalytic domain may not play a role in complex formation between UK and PAI-2. Materials and Methods

Reagents. All chemical reagents were of the highest analytical grade commercially available and were purchased as follows: diisopropylfluorophosphate (DFP), Tris-HCl, bovine serum albumin (BSA), Tween-80, phorbol 12-myristate 13-acetate were from Sigma (St. Louis, MO, USA); sodium phosphate was from Seikagaku Kogyo (Tokyo, Japan); Iodo-beads was from Pierce (Rockford, IL, USA); fetal calf serum and reagents for cell culture were from Gibco (New York, NY, USA). Plastic wares were purchased from Corning (New York, NY, USA). Peptides and proteins. Peptides, 8-26 amino acids in length, were synthesized using a peptide synthesizer model 430A (Applied Biosystems, Foster City, CA, USA). These peptides have sequences derived from uPA or tPA. The amino-acid sequences of the peptides are shown in Tables II and III with each amino acid designated by the standard one-letter code. Cys-groups were protected by acetoamidomethylation. Peptide P-48 was reported elsewhere [7,8]. After purification by reverse-phase HPLC, each peptide preparation was shown to be homogeneous upon' analysis on reversephase HPLC and the N-terminal amino-acid sequence was confirmed by amino-acid sequencing on a sequencer model 900 A (Applied Biosystems). PAI-1 was isolated from the conditioned medium of cultured human HT 1080 cells as described previously [15,16]. PAI-1 was labeled with Na125I (2575 C i / mmol, Du Pont-New England Nuclear) using Iodobeads according to the manufacturer's directions as described previously [7]. The labeled PAI-1 had spe-

cific radioactivity of 1.84.109 cpm/mg. Radiolabeled and unlabeled PAI-1 was activated as described previously [5,15,16]. After activation, radiolabeled and unlabeled PAI-1 activity was titrated agaist UK using the synthetic substrate S-2444. One UK inhinbitory unit represents the amount of PAL1 required to inhibit S-2444 hydrolysis activity of one international unit of UK at 23°C in 10 min. The specific activity of unlabeled PAI-1 was (6.2 _+ 0.30). 104 U/rag (n = 5). The specific UK inhibitory activity of radiolabeled PAI-1 was 101 _+ 7.8% (n = 5) of the unlabeled PAI-I activity. Radiolabeled PAl-1 was shown to be able to form SDS-stable complexes as descrived previously [8]. Single-chain and two-chain tPA was purified from conditioned medium of melanoma cells as described previously [7,8,15,16]. PAI-2 was purified from the conditioned medium of U-937 cells cultured in the presence of phorbol 12-myristate 13-acetate (10 ng/ml) by affinity chromatography on monocional antibody (MAI-21, Biopool, Ume~, Sweden)-coupled Sepharose. UK and scuPA were generous gifts from Mochida Pharmaceutical (Tokyo, Japan). Low-molecular-weight urokinase (LMWUK) was purchased from Protogen (Switzerland). To prepare enzymatically inactive UK, LMWUK and tPA, these enzymes were incubated in phosphate buffer containing 10 mM DFP at 23°C for 30 min followed by dialysis against 50 mM Tris, 0.15 M NaCI (pH 7.5) containing 0.01% Tween-80 (TBS/Tween). The residual activities of DFP-inactivated (DIP) enzymes were less than 0.04% of the untreated enzymes. Monoclonal antibodies to urokinase. B A L B / c mice were immunized with purified human UK and murine monoclonal antibodies to human UK were selected and cloned according to the method of K6hler and Milstein as described previously [17,18]. Monoclonal antibodies (MoAbs) were isolated from mice ascites using protein A-coupled Sepharose as described previously [17,18]. MoAbs were analyzed for binding to UK functional domains by Western blotting. UK was separated by SDS-PAGE under non-reducing and reducing conditions. After electrophoresis, the samples were transferred to a nitrocellulose membrane and incubated with the appropriate MoAb. Bound antibody was detected with enzyme-conjugated anti mouse IgG antibodies as described previously [17,18]. By western blotting, the MoAb JTU-C3 bound to the UK B-chain which contains the catalytic domain, while MoAb JTUA3 bound to the UK A-chain which contains the growth factor and kringle domains (not shown).

Isolation of the N-terminal UK fragment containing the kringle domain. UK (760/zg) was incubated in 800 #1 of phosphate buffered saline (pH 7.4) in the presence of plasmin (70 p.g/ml) at 37°C for 120 min. Limited proteolysis of UK by plasmin was judged by SDS-PAGE to confirm the conversion of UK to LMWUK and the UK N-terminal fragment containing

327 the growth factor and kringle domains. The reaction mixture was applied to a lysine-coupled Sepharose column in the presence of aprotinin (100 U / m l ) to remove plasmin. The unbound fraction was applied to a monoclonal antibody JTU-C3-coupled Sepharose column to remove LMWUK and the uncleaved UK (JTUC3 binds to the UK catalytic domain). The unbound fraction was collected and analyzed by SDS-PAGE and Western blotting using MoAb JTU-A3.

Analysis of binding of PAI-1 to the UK N-terminal fragment and to synthetic peptides. Flat-bottomed microtiter plates (96 well, polystyrene) were coated with 100 /zl of phosphate buffered saline (20 mM sodium phosphate, 0.14 M NaC1 (pH 7.4)) containing the UK N-terminal fragment (1 /xg/ml) or 10/xg/ml peptides at 4°C for 16 h. After blocking with BSA (3%) and washing three times with phosphate buffered saline (pH 7.4), containing Tween-80 (0.01%), BSA (1 mg/ml) and poly(ethylene glycol) 6000 (4% (w/v); binding buffer), the plates were incubated in the presence of increasing concentrations of [t25I]PAI-1 in binding buffer and were allowed to stand for 30 min at 23°C. After washing three times with binding buffer, radioactivity in each well was measured with a y-counter (Aroka, Tokyo, Japan). Dissociation constants were calculated after Scatchard analysis as described previously [8]. PAI-1 binding to the UK N-terminal fragment was investigated in the presence of synthetic UK peptides. Microtiter plates coated with the UK N-terminal flag-

ment were incubated in binding buffer containing [125I]PAI-1 (1 nM) in the presence of increasing concentrations of competitors. After 30 min incubation at 23°C, the plates were washed and subjected to y-counting to quantify the amount of [~25I]PAI-1 remaining on the plates. Microtiter plates were incubated with peptides (10 /xM) at 4°C for 16 h. After blocking and washing, the plates were incubated with [~25I]PAI-1 as above. After washing, bound [125I]PAI-1 was quantified. Binding of [~25I]PAI-1 to the UK peptide-coated plates in the presence of increasing concentrations of DIP-UK, DIP-LMWUK, scuPA, single-chain DIP-tPA, two-chain DIP-tPA, or tPA derived peptide were also investigated.

Analysis of the effect of synthetic peptides on UK / PAI-1 complex formation. UK (1 nM) or LMWUK (1 nM) and PAL1 (1 nM) were incubated in the presence or absence of increasing concentrations of synthetic peptides in TBS-Tween at 37°C. After 10 min incubation, the reaction was terminated by the addition of 10 mM p-APMSF. The concentration of UK/PAI-1 complex was determined by an enzyme immunoassay using a monocional antibody against PAI-1 and affinity purified polyclonal antibodies raised against UK as described [15,16]. Second-order rate constants were calculated under pseudo-first-order conditions as described [5,7,8]. Equimolar concentrations of UK and PAI-1 or LMWUK and PAI-1 were incubated at 37°C for various periods of time in the presence or absence of 100/xM peptides. After termination of the reaction

Kringle S ~~ ~ C ~ ~omain !

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k~~,~

~"

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NH2

Fig. 1. Schematic representation of the primary structure of uPA. The primary structure of uPA, adapted from Ref. 22, was drawn and each circle represents an amino-acid residue. Open circles represent amino-acid residues that are not related to this study. The active site residues His-204 (H), Asp-255 (D) and Ser-356 (S) are represented by single-letter symbols in large circles. Arrows indicate two plasmin cleavage sites for conversion of scuPA to U K (Lys-158 and I1e-159) and for conversion of U K to L M W U K (Lys-135 and Lys-136). Residues covered by peptides in this study are indicated by hatched circles. The reversible PAI-1 binding sites demonstrated in this study are represented by closed circles while other putative inhibitor-binding segments adopted from the chymotrypsin model [19] are indicated by dotted circles. The first inhibitor-binding segment from the N-terminus contains a peptide insertion that was covered by peptide U-180.

328 TABLE I

by the a d d i t i o n of 10 m M p - A P M S F , t h e c o n c e n t r a t i o n of U K / P A I - 1 c o m p l e x f o r m e d was d e t e r m i n e d as described above. T h e synthetic s u b s t r a t e S-2444 (Kabi, Stockholm, S w e d e n ) was u s e d to m o n i t o r the inhibition of U K hydrolysis activity by P A I - 1 in the p r e s e n c e a n d a b s e n c e of p e p t i d e to c o n f i r m t h e inhibitory effect o f p e p t i d e s . U K (5 n M ) or L M W U K (5 n M ) was incub a t e d with PAI-1 (2.5 n M ) in the p r e s e n c e or a b s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s of p e p t i d e s at 37°C for 10 rain. T h e residual activity of U K or L M W U K was q u a n t i f i e d by m e a s u r i n g the r a t e of S-2444 hydrolysis. Analysis o f effect o f peptides on complex f o r m a t i o n between UK and PAl-2. A n e n z y m e i m m u n o a s s a y was used to m o n i t o r U K / P A I - 2 complex formation. E q u i m o l a r c o n c e n t r a t i o n s o f U K and P A I - 2 w e r e incub a t e d in the p r e s e n c e or a b s e n c e of p e p t i d e s (100 # M ) at 37°C. A f t e r various p e r i o d s of time, aliquots w e r e r e m o v e d a n d the r e a c t i o n was t e r m i n a t e d by the a d d i tion of p - A P M S F as d e s c r i b e d above. T h e s a m p l e s were i n c u b a t e d in m i c r o t i t e r p a t e s c o a t e d with antiPAI-2 monocional antibody (MAI-21) and the U K / P A I - 2 c o m p l e x e s were d e t e c t e d by e n z y m e - c o n j u g a t e d polyclonal a n t i b o d i e s against UK.

Second-order rate constants (k ~ss) for inactivation of UK by PAL1 and LMWUK by PAL1 in the presence or absence of peptides The second-order rate constants for complex formation between UK and PAL1 and LMWUK and PAL1 in the presence of 100 /xM peptide or absence of peptide were determined as described in Materials and Methods. Each value represents the mean _+S.D. (n = 4). peptide

none 860+39011570+00 UK/PAI- 1

1

U- 107

8.60 + 0.60

5.50 + 0.90

U-373

8.33 + 0.57

0.40 + 0.05

U-107 + U-373

0.41 + 0.02

0.4l) + 0.02

b i n d i n g site in UK. Thus, the U K N - t e r m i n a l f r a g m e n t g e n e r a t e d by p l a s m i n - t r e a t m e n t was p u r i f i e d a n d its ability to b i n d P A I - I was studied. PAI-1 b o u n d to the M r 21000 U K f r a g m e n t in a d o s e - d e p e n d e n t m a n n e r with an a p p a r e n t dissociation c o n s t a n t ( K ~ ) o f 0.812 + 0.086 n M ( m e a n + S.D., n = 4) (Fig. 2A), indicating that a reversible P A I - 1 b i n d i n g site was l o c a t e d in the r e s i d u e s b e t w e e n Ser-1 a n d Lys-135 in UK. A l t h o u g h L M W U K lacks this s e g m e n t , the i n t e r a c t i o n b e t w e e n L M W U K a n d PAI-1 is still c o n s i d e r a b l y rapid, indicating that t h e r e m a y be o t h e r discrete PAI-1 b i n d i n g sites in the L M W U K molecule. Thus, we p e r f o r m e d a

T h e influence of the U K a m i n o - t e r m i n a l d o m a i n on b i n d i n g i n t e r a c t i o n s with PAI-1 was investigated. T h e s e c o n d - o r d e r rate c o n s t a n t (kas ~) for P A I - 1 inactivation of L M W U K was approx. 10-fold s m a l l e r t h a n that for inactivation o f U K ( T a b l e I). Since L M W U K lacks the a m i n o - t e r m i n a l r e s i d u e s Ser-1 t h r o u g h Lys-135 (Fig. 1), this suggests that these r e s i d u e s c o n t r i b u t e to a P A L 1 i

LMWUK/PAI- 1

* Statistically significant (P < 0.0l, Student's t-test).

Results

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Fig. 2. Binding of PAI-1 to the UK N-terminal fragment containing the growth factor and kringle domains. (A), increasing concentrations of [t25I]PAI-1 were incubated on plates coated with the UK fragment containing the kringle domain. After washing, the amount of [~251]PAI-I bound to the plates were quantified (mean+S.D., n = 4). An inset shows the Scatchard analysis of the binding. The other inset shows SDS-PAGE and Western blotting analysis, using MoAb JTU-A3 of UK (lane 1) and the M r 21000 UK fragment containing the kringle domain (lane 2). Arrows indicate the positions and molecular size of the proteins in the blot. (B), [1251]PAI-1 (1 nM) was incubated on plates coated with the UK fragment in the presence of increasing concentrations of peptides (©, U-99; e, U-103; [], U-107; I , U-373). After washing the plates bound [125I]PAI-1 was quantified. The amount of PAI-I bound to the plates in the presence of peptide was expressed as the percentage of that bound in the absence of peptide (mean _+S.D., n = 4).

329 TABLE II

Synthetic peptides derived from uPA Binding of radiolabeled PAI-1 (1 nM) to peptide-coated plates was determined as described in Materials and Methods. Each value represents the mean +_S.D. (n = 4). UK peptide

sequence

domain

[125I]PAI-1 bound (cpm)

U-99 (99-106) U- 103 (103-110) U-107 (107-114) U-180 (180-187) U-373 (373-398)

HNYCRNPD RNPDNRRR NRRRPWCY HRGGSVTY IVSWGRGCALKDKPGVYTRVSHFLPW

kringle kringle kringle catalytic catalytic

n.d. 8 263 + 230 14 718 +_344 n.d. 1510_+ 95

n.d., not detected.

homology search for the known and theoretical PAI-1binding amino-acid sequences in the UK amino-acid sequence using Hibio-ProsisT M (Hitachi software engineering, Yokohama, Japan) and synthesized peptides (Table II). The criteria for choosing a particular uPA peptide sequence (Fig. 1) for synthesis was based on several observations. First, the UK kringle-domain inner loop is predicted to be a reversible PAI-1 binding site in the UK A-chain, since it contains the NRRR sequence (peptides U-103 and U-107) which is homologous to the PAI-1 binding sequence NRRL in the tPA kringle-2 domain [8]. Second, based upon a model for inhibitor-binding segments in the chymotrypsin catalytic domain, residues 373-391 of UK correspond to two of eight inhibitor-binding segments (212-221 and 225-230) exposed on the chymotrypsin catalytic domain [19]. The amino-acid sequence of this UK segment (peptide U-373) was the most highly conserved among eight chymotrypsin inhibitor-binding segments in the serine proteinase catalytic domain. Third, i

residues around Arg-181 (peptide U-180) correspond to the exosite of tPA catalytic domain that was proposed as a reversible PAI-1 binding site by Madison et al. [13]. In the chymotrypsin model, this polypeptide segment appears to be inserted into one of the inhibitor-binding segments (35-43) in the catalytic domain. We investigated the effect of these peptides on PAI-1 binding to the UK N-terminal fragment which contains the growth factor and the kringle domains. U-103 and U-107, overlapping peptides containing the NRRR sequence derived from the UK kringle domain, solely inhibited the PAI-1 binding to the UK fragment (Fig. 2B). Peptide U-99 derived from the kringle domain but missing the sequence, failed to inhibit the binding. The UK N-terminal fragment appeared to have a single PAI-1 binding site, since more than 90% of PAI-1 binding was inhibited by 1 mM U-107 peptide. We studied the binding of radiolabeled PAI-1 to the |

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Fig. 3. Inhibition of complex formation between U K and PAI-1 or L M W U K and PAl-1 by synthetic peptides. U K (1 nM) and PAl-1 (1 nM) (panel A) or L M W U K (1 nM) and PAI-1 (1 riM) (panel B) were incubated in the presence of increasing concentrations of the peptides (o, U-99; o, U-103; [3, U-107; II, U-180; 4 , U-373) and the amount of U K / P A I - 1 complex was determined as described in Materials and Methods. Each value represents the mean of four determinations and was expressed as a percentage of the complex amount formed in the absence of competitors.

330 various solid-phase uPA peptides. PAI-1 bound to peptide U-103, U-107 and U-373, however, PAI-1 binding to U-99 or U-180 was not observed (Table II). Those peptides that bound to PAI-1 could inhibit complex formation with UK, but inhibition was dependent on the presence or absence of the UK kringle domain (Fig. 3). U-107 and U-373 inhibited U K / P A I - 1 complex formation in a dose-dependent manner, whereas U-103 was about 8-fold less effective (Fig. 3A). Complex formation between L M W U K and PAI-1 was inhibited solely in the presence of U-373 (Fig. 3B). Peptides U-99 and U-180 had no effect on PAI-1 interactions with either UK or LMWUK, consistent with the previous data showing a lack of direct peptide binding to PAI-1. Similar inhibitory effect of peptide U-107 on inactivation of UK by PAI-1 and that of U-373 on intactivation of UK by PAI-1 and L M W U K and PAI-1 were observed in experiments in which residual enzyme activity was monitored by the S-2444 synthetic substrate (not shown). These data also indicate that peptide effects were not mediated by interference with the detection of PAI-1 by monoclonal antibodies. The second-order rate constants for inactivation of UK by PAI-1, and of L M W U K by PAI-1 in the presence of 100 ~ M U-107, 100 # M U-373, or both U-107 (100/xM) and U-373 (100 p~M) are shown in Table I. In the absence of peptides, k~,ss for interaction between L M W U K and PAI-1 was approx. 1/10 of that for complex formation between UK and PAI-1. In the

|

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presence of U-107, whose amino-acid sequence was derived from the uPA kringle domain, the rate of complex formation between UK and PAI-1 was decreased approx. 10-fold, to the rate of complex formation between L M W U K and PAI-1 in the absence of peptides. Peptide U-107 had no effect on the rate of complex formation between L M W U K and PAI-1, indicating that the uPA kringle-domain inner loop is involved in the reversible interaction between UK and PAI-1. Both interactions between UK and PAI-1 and L M W U K and PAI-1 were affected in the presence of U-373, derived from the uPA C-terminal region and the second-order rate constants decreased to 1/10 of the control values. When both peptides were present in the reaction mixture, the inactivation rate of UK by PAI-1 decreased to approx. 1/200 of the control, whereas inactivation rate of L M W U K by PAI-1 did not change significantly from that in the presence of U-373 alone. None of the peptides used in this study inhibited the plasminogen activation activity, S-2444 hydrolysis activity of UK, or the binding of monoclonal antibody JTC-3 to PAI-1 that was utilized in the enzyme immunoassays (not shown). To further characterize the nature of interaction between PAI-1 and uPA, [12SI]PAI-1 binding to solidphase peptide U-107 was determined in the presence of various competitors (Fig. 4A). Binding of PAI-1 to U-107 was inhibited by D I P - U K in a dose-dependent manner while no inhibition by L M W U K was observed (Fig. 4A). ScuPA had relatively little effect, with 20%

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(M)

Fig. 4. Inhibition of PAI-I binding to peptides by DIP-UK, DIP-LMWUK, scuPA, DIP-tPA, or tPA peptides. (A), [12SI]PAI-I(1 nM) was incubated on microtiter plates coated with U-107 in the absence or presence of increasing concentrations of competitors (0, DIP-UK; o, DIP-LMWUK; 1:3,scuPA; A, single-chain DIP-tPA; A, two-chain DIP-tPA; v, P-48; v, F-7) and bound PAI-I was quantified after washing. The amount of PAI-1 bound to the plates in the presence of a competitorwas expressed as a percentage of that in the absence of any competitor (mean _+S.D., n = 4). (B), binding of [12SI]PAI-1(10 nM) to microtiter plates coated with U-373 in the presence of increasing concentrations of competitors (0, D1P-UK; o, DIP-LMWUK; A single-chain DIP-tPA; N, two-chain DIP-tPA; O, scuPA) was determined as described in panel A (mean _+S.D., n = 4).

331 inhibition observed only at the highest concentration and this effect could be mediated partly by a trace amount of UK contaminating the scuPA preparation. Two-chain DIP-tPA and peptide P-48, whose aminoacid sequence is derived from the tPA kringle-2 domain, inhibited PAI-1 binding to U-107 in a manner similar to DIP-UK. In contrast, single-chain DIP-tPA was approx. 10-fold less inhibitory relative to DIP-UK or two-chain DIP-tPA. Peptide U-107 inhibited PAI-1 binding to peptide P-48 (not shown). Peptide F-7, whose amino-acid sequence is derived from the tPA finger domain had no effect. Since U-373 bound to PAI-1 and interfered with complex formation between UK and PAI-1 and LMWUK and PAI-1, another set of competition experiments was performed (Fig. 4B). When DIP-UK, DIPLMWUK, single-chain DIP-tPA, or two-chain DIP-tPA was included in the reaction mixture, binding of PAI-1 to U-373 was similarly inhibited in a dose-dependent manner. However, no inhibitory effect of scuPA on the binding was observed. These data indicate that both forms of tPA and UK bind to the same region in the PAI-1 molecule and that the inhibitor-binding site expression in the C-terminal region of the uPA molecule is observed only in UK, whereas the site is expressed similarly in the single-chain and two-chain forms of tPA molecule. The observation that PAI-1 binding sites on uPA are selectively expressed in the two-chain form of the enzyme reflects previous data with tPA and PAI-1. In the latter case, however, there is still a rapid association of PAI-1 with single-chain tPA even in the presence of the inhibitory P-48 peptide derived from the tPA kringle-2 domain [8]. Thus, additional studies were conducted to assess the contribution of the tPA finger domain in reversible PAI-1 binding. The amino-acid sequence of peptide F-7 is derived from the tPA finger domain and is identical with the sequence that inhibits binding of tPA to cultured human umbilical vein endothelial cells [20] and with a part of the peptide which suppresses inactivation of tPA by PAI-1 [6]. PAI-1

1

i . ~

°=

0.81

~'o.6 E

O o

~0.4

a. ,¢

0.2

0 C .... 0

| .... 60 Time

! .... 120 (min)

Fig. 5. Complex formation between UK and PAI-2 in the absence of presence of peptides. UK (1 nM) was incubated with PAI-2 (1 nM) in the absence (©) or presence of 100/zM U-107 (11) or 100/zM U-373 (,x) at 37°C. After incubation, aliquots were harvested at the indicated time and were incubated with p-APMSF to terminate the complex-formation reaction. The amounts of complexes formed between UK and PAI-2 (mean, n = 2) was quantified using an enzyme immunoassay as described in Materials and Methods.

bound to both peptides and P-48 did not inhibit the binding of PAI-1 to F-7 and vice versa (not shown), indicating that they bound to the different regions of the PAI-1 molecule. As shown in Table III, F-7 bound to PAl-1 and inhibited complex formation between single-chain tPA and PAI-1, but had a lesser effect on complex formation between two-chain tPA and PAI-1. P-48 mainly inhibit the interaction between two-chain tPA and PAI-1 (Table III) [8]. When both peptide were present in the reaction mixture containing tPA and PAI-1, the rates of inactivation of both forms of tPA by PAI-1 were slowed equally to approx. 1/10 of the respective control, indicating that these PAI-1 binding sites in the tPA A-chain work together, but in a distinct

TABLE III

Characteristics of synthetic peptides derived from the tPA non-catalytic domains The second-order rate constants (kass) for complex formation between tPA and PAI-1 in the presence of peptide (100 tzM) and PAI-1 binding to peptide-coated plates were determined as described in Materials and Methods. Each value represents the mean_+ S.D. (n = 4). Peptide

None P-48 (248-255) F-7 (7-17) F-7+P-48

Sequence

NRRLTWEY RDEKTQMIYQQ

From Ref. 8. *, P < 0.05; * *, P < 0.01 (Student's t-test)

a

kass (M-1 s 1.10-6) single-chain t P A / P A I - 1

two-chain t P A / P A I - 1

[IZsl]PAi_ 1 bound (cpm)

6.4 _+2.0 q*]-I 4.0 ± 0.4 a j , *1 1.7 ±0.9 J* * 0.66±0.22 J

11.4+0.9 *]*?] 3.1 + 0.3 a j , I 8.0+3.5 * 1.6±0.10 J

18103 +__720 10357+_286 -

332 manner in the two forms of tPA. Taken together, these observations support the notion that PAI-1 has at least two reversible tPA binding sites, one for the tPA finger domain [7,8], that plays an important role in the rapid interaction with single-chain tPA and the other for the tPA kringle-2 domain [7,8], that is involved mainly in the rapid reversible interaction with two-chain tPA. Binding interactions between UK and PAI-2 appear to be mediated by distinctly different sites based on peptide inhibition study (Fig. 5). When 100/~M U-107 or 100 ~,M U-373 was included in reaction mixtures of UK and PAI-2, there was no effect on complex formation between UK and PAI-2, indicating that the UK kringle domain and the C-terminal region of the UK catalytic domain that are involved in the interaction with PAI-1 do not appear to play a role in inactivation of UK by PAI-2. Discussion

Because of the important role of PAI-1 in regulation of the fibrinolytic system, much attention has been paid to interactions between plasminogen activators and PAl-1. Kinetic analyses of the interactions have suggested that the reaction between tPA and PAI-1 is achieved through two steps and that the interaction between UK and PAI-1 is a single-step reaction [5]. However, PAI-1 binds to a n h y d r o - U K - c o u p l e d Sepharose with high affinity [21], indicating that reversible PAl-1 binding site(s) resides in the anhydro-UK molecule. Here, we have shown that a reversible association between UK and PAI-1 occurs between the UK kringle domain and PAI-1. The reaction of UK and PAI-1 is presumably simpler than that between tPA and PAI-1. TPA differs from most serine proteinases in that the single-chain form of the molecule has appreciable enzymatic activity for catalyzing plasminogen activation in the presence of fibrin and makes SDS-stable complexes between PAI-1 (see reviews 2, 3 and 22). In contrast, scuPA is virtually a zymogen of UK that has 1/1000 or less activity than UK [22-25] and no complexes are formed between scuPA and PAI-1 [26]. Furthermore, tPA non-catalytic domains appear to have at least two reversible PAI-1 binding sites, one is in the finger domain [6,7] and another is in the kringle-2 domain [7-10]. The finger domain of single-chain tPA plays an essential role in the interaction with PAI-1 (Table III), while the kringle-2 domain is mainly involved in the interaction between two chain tPA and PAI-1 [7,8]. Although the tPA kringle-2 domain in the single-chain tPA molecule is less effective than the tPA finger domain for PAI-1 binding, it nevertheless plays a role in the interaction. The finger domain of the two-chain tPA molecule is also involved in PAI-1 binding (Table III and Ref. 8, see below).

While the kinetics of UK and PAI-1 interactions may be simpler than the t P A / P A I - 1 system, the binding interactions proved to be as complex to as certain. We have shown that PAI-1 binds reversibly to the UK kringle domain and peptides derived from this domain bind to P A I d . The kringle-derived peptide interfered with complex formation between UK and P A I d , but did not affect binding of LMWUK and PAI-1. In addition, the U-373 peptide derived from the C-terminal region of UK bound to PAI-1 and inhibited complex formation with both UK and LMWUK. Furthermore, PAl-1 binding to the kringle-domain-derived peptide was inhibited solely by DIP-UK, whereas PAI-1 binding to the C-terminal peptide was inhibited by both DIP-UK and DIP-LMWUK. Thus, these results indicate that both domains in the UK molecule are involved in reversible binding to PAI-1. Our observation that peptide U-373, which includes these peptide segments, inhibited complex formation between UK and PAI-1 is consistent with the chymotrypsin model. The UK kringle domain and the tPA kringle-2 domain appear to bind to the same region of the PAI-1 molecule and the C-terminal regions of UK and both forms of tPA may bind to the same peptide segment of PAI-1. ScuPA had virtually no effect on PAI-1 binding to U-107, indicating that the PAI-1 binding site in the kringle domain is buried in the scuPA molecule. ScuPA also did not inhibit the binding of PAI-1 to U-373, suggesting that this C-terminal region in the UK catalytic domain is not exposed on the surface of the scuPA molecule or it is exposed, but in a different conformation that does not support PAI-1 binding. These data indicate that at least these reversible PAI-1 binding sites in the UK molecule are not available in scuPA and that once the peptide bond between Lys-158 and Ile-159 is cleaved, these reversible PAI-1 binding sites are exposed on the surface of the two-chain form of uPA. This model in which the A-chain of a serine proteinase plays a role in the initial interaction with an inhibitor is similar to the complex formation reaction between plasmin and a2-plasmin inhibitor (o~2-antiplasmin), in which the A-chain lysine binding site(s) of plasmin is involved in the initial rapid association with a2-plasmin inhibitor [29]. It may be possible to apply the proposed role of UK A-chain to the complex interaction between tPA and PAI-1, since the expression of the PAI-1 binding site in the uPA kringle domain upon conversion of the single-chain form to the two-chain form is similar to that in the kringle-2 domain of the tPA molecule [8]. The observation that a 10-fold decrease in the inactivation rate of tPA by PAI-1 occurs in the presence of P-48 and F-7 (Table III) was similar to that observed for the inactivation of UK by PAI-1 in the presence of U-107 (Table I). These data support the hypothesis that the A-chain of tPA plays an important role in the

333 rapid interaction with PAI-1, because its high-affinity binding sites in the finger and kringle-2 domains have distinct contributions to PAI-1 binding between the two forms of tPA, as observed in the interaction between UK and PAI-1. The C-terminal regions of tPA and U K share considerable sequence identity and both forms o f DIP-tPA could compete with U-373 for PAI-1 in a manner similar to DIP-UK competition. These data indicate that the inhibitor binding segments in the tPA catalytic domain are exposed similarly on the surface of both forms of tPA, in contrast to the expression of the segment in the U K molecule only upon conversion from its single-chain form. It is still possible that other sites, such as the remainder of eight predicted inhibitor-binding segments in the catalytic domain, are involved in the reversible interaction between PAI-1 and tPA or UK. An exo-site of the tPA catalytic domain consisting of residues around Arg-298 was proposed as a secondary PAI-1 binding site by Madison et al. [11-13], who utilized a series of point and deletion mutations to show that the recombinant tPAs with mutations between residues 296-304 were resistant to inactivation by PAI-1. Thus, it was possible that the similar region in the UK molecule might be involved in the reversible interaction between UK and PAI-1. However, our current observations do not support the prediction, since U-180 failed to bind to PAI-1 or to inhibit the complex formation between U K and PAI-1. Similarly, a previous study of the interaction between tPA and PAI-1 also showed no effect on the inactivation of tPA by PAI-1 of a synthetic peptide covering the exosite of tPA catalytic domain [6]. These conflicting observations may be due to the use of different molecules or reagents in the experiments and the possibility that the recombinant mutant molecules have altered conformations as compared with the wild-type protein. According to previous studies, it appears that a fibrin binding site resides in the tPA finger domain and the lysine binding site resides in the tPA kringle-2 domain [30]. Bennett et al. reported that an increase of fibrin stimulation of tPA activity was achieved by alanine substitutions for residues in the exosite of the catalytic domain and that fibrin binding was decreased when alanine was substituted for residues in the catalytic domain or in the kringle-1 domain [14]. These data suggest that recombinant tPA mutants have unique conformational changes or that the amino-acid mutations affect essential ionic interactions of the functional domain. PAI-2 has plasminogen activator inhibitory activity, however, the rate of inactivation of UK and tPA by PAI-2 is considerably slower than that by PAI-1. Especially, the second-order rate constant for inactivation of single-chain tPA by PAI-2 is 3 - 4 orders of magnitude lower than that by PAI-1, indicating that PAI-2 may not be a physiological tPA inhibitor. This differ-

ence observed between the two PAIs may be accounted for by the observation that PAI-2 probably lacks some of the critical plasminogen activator binding sites which reside in PAI-1. Since the inhibitor-binding segments in the C-terminal region of tPA appear to be similarly expressed on the surface of both forms of tPA, but inhibition by PAI-2 of single-chain tPA is considerably slower than that of two-chain tPA, there may be multiple differences in expression of inhibitorbinding segments between single-chain tPA and twochain tPA. The interaction between UK and PAI-1 fits reasonably to a general model of interaction between a serine proteinase and a serine proteinase inhibitor, since both the U K A-chain and the inhibitor-binding segments in the catalytic domain are involved in the association with PAI-1. It was rather surprising that the theoretical inhibitor-binding segments in the Cterminal region of the UK catalytic domain did not appear to be involved in the complex formation reaction between UK and PAI-2. Thus, it appears that the reaction between UK and PAI-2 does not fit to the general model of interaction between a serine proteinase and its physiological inhibitor.

Acknowledgements The authors are grateful to Ms. F. Muroi and Ms. H. Sunaga for excellent technical assistance and to Ms. M. Takano for secretarial work in preparation of this manuscript. This work was partly supported by Grantsin-Aid for Scientific Research No. 04671532 (to Y.S.) and No. 04671533 (to J.M.), a Grant-in-Aid for Cooperative Research A, No. 03304049 (to Y.S.) from the Ministry of Education, Science and Culture of Japan and a Grant-in-Aid from the Ryoichi Naito Foundation for Medical Research (to J.M.).

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