Share Common Binding Sites in Limited Plasmin-digested. Fibrin* ... lysines are unique both for the K2 domain of t-PA and the Glu- .... Free ['251]NaI was sites ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry
Vol. 265, No. 23, Issue of August 15, pp. 13547-13552,199O Printed in U.S.A.
and Molecular Biology, Inc.
Tissue-type Plasminogen Share Common Binding
Activator and Its Substrate Glu-plasminogen Sites in Limited Plasmin-digested Fibrin* (Received for publication,
Carlie
de Vries,
Harry
From the Central Laboratory Amsterdam, The Netherlands
Veerman,
Esther
of the Netherlands
Koornneef, Red Cross
The enzyme tissue-type plasminogen activator (tPA) and its substrate Glu-plasminogen can both bind to fibrin. The assembly of these three components results in about a lOOO-fold acceleration of the conversion of Glu-plasminogen into plasmin. Fibrin binding of t-PA is mediated both by its finger (F) domain and its kringle-2 domain. Fibrin binding of Glu-plasminogen involves its kringle structures (Kl-K5). It has been suggested that particular kringles contain lysinebinding sites and/or aminohexyl-binding sites, exhibiting affinity for specific carboxyl-terminal lysines and intrachain lysines, respectively. We investigated the possibility that t-PA and Glu-plasminogen kringles share common binding sites in fibrin, limitedly digested with plasmin. For that purpose we performed competition experiments, using conditions that exclude plasmin formation, with Glu-plasminogen and either t-PA or two deletion mutants, lacking the F domain (tPA de1.F) or lacking the K2 domain (t-PA del.K2). Our data show that fibrin binding of t-PA, mediated by the F domain, is independent of Glu-plasminogen binding. In contrast, partial inhibition by Glu-plasminogen of t-PA K2 domain-mediated fibrin binding is observed that is dependent on carboxyl-terminal lysines, exposed in fibrin upon limited plasmin digestion. Halfmaximal competition of fibrin binding of both t-PA and t-PA de1.F is obtained at 3.3 pM Glu-plasminogen. The difference between this value and the apparent dissociation constant of Glu-plasminogen binding to limitedly digested fibrin (12.1 pM) under these conditions is attributed to multiple, simultaneous interactions, each having a separate affinity. It is concluded that t-PA and Glu-plasminogen can bind to the same carboxyl-terminal lysines in limitedly digested fibrin, whereas binding sites composed of intrachain lysines are unique both for the K2 domain of t-PA and the Gluplasminogen kringles.
The assembly
of the enzyme tissue-type
plasminogen
acti-
* This study was supported by Netherlands Organization for Scientific Research (MEDIGON) Grant 900-526-070 and by a grant from Eli Lilly (Indianapolis, IN). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: c/o Publication Secretariat, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, P. 0. Box 9406, 1006 AK, Amsterdam, The Netherlands. Tel. o-20-5123125; Fax: O-20-5123332.
Blood
and Hans Transfusion
November 27, 1989)
PannekoekS Service,
Department
of Molecular
Biology,
vator (t-PA)’ and the substrate Glu-plasminogen on a fibrin surface is of utmost importance for the generation of the product plasmin (1). On the one hand, such conditions ascertain the presence of plasmin in the close vicinity of its “target” fibrin. Simultaneously, fibrin acts as a cofactor that enhances the affinity of t-PA for Glu-plasminogen and allows an efficient conversion into plasmin. Mechanistically, the assembly of fibrin, t-PA, and Glu-plasminogen has been described in a model, denoted the “cyclic ternary complex” (2, 3). Plasmin cleaves fibrin polymers sequentially at the carboxyl-terminal side of specific lysine and arginine residues, generating defined fibrin degradation products (4). “Early” fibrin degradation products, e.g. fragment X polymers, display a higher specific cofactor activity for plasminogen activation than either intact fibrin or smaller degradation products (5-7). This is reflected by an increased binding of both t-PA and Glu-plasminogen when a fibrin matrix is limitedly degraded by plasmin (5, 8-12). The elucidation of the amino acid sequence of both Gluplasminogen and t-PA has revealed the molecular equipment of these (precursor) serine proteases for their various interactions. The catalytic center is located in the carboxyl-terminal part of the molecules. Furthermore, these proteins are provided with an array of structural domains, located at their respective amino termini. Notably, Glu-plasminogen harbors five kringle structures, Kl-K5 (13), whereas t-PA contains a finger (F) domain, an epidermal growth factor-like domain and two kringle structures, Kl and K2 (14). Three lines of evidence show that these structural domains embody autonomous functions. (a) The characterization of deletion mutants of t-PA, lacking one or more of the structural domains, demonstrates that specific functions can be abolished, while simultaneously other functions are retained (15, 16); (b) the construction of chimeric variants between parts of t-PA and the catalytic region of yet another plasminogen activator, urokinase, has shown the feasibility of “transposing” distinct autonomous functional domains (17-19); (c) both isolated kringle 4 (K4) of plasminogen, obtained upon digestion of the protein with elastase (20), and isolated kringle 2 (K2) of tPA, separately expressed in Escherichiu coli (Zl), display autonomous functions apparently similar to those exhibited by these domains within the intact proteins. The fibrin binding of Glu-plasminogen is mediated predominantly by its kringle structures (1, 13). Particular kringle 1 The abbreviations used are: t-PA, tissue-type plasminogen activator; t-PA del.K2, deletion mutant of t-PA lacking the K2 domain; t-PA del.F, deletion mutant of t-PA lacking the F domain; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; GGACK, dansyl-L-glutamylglycyl-L-arginine chloromethyl ketone; Glu-plasminogen, native human plasminogen with amino-terminal glutamic acid, Lys-plasminogen, plasmin-modified plasminogen, mainly with amino-terminal lysine-78.
13547
13548
Fibrin
Binding
of t-PA and Glu-plasminogen
structures contain so-called aminohexyland lysine-binding sites, that display affinity for specific intrachain lysines, and carboxyl-terminal lysines, respectively (22, 23). For t-PA, both its F and K2 domains are involved in fibrin binding (15, 16). Until now, the nature of the interaction of the F domain with fibrin has not been established. In contrast, the K2 domain contains, analogous to Glu-plasminogen, a lysinebinding site and an aminohexyl type of binding site that mediate the interaction with fibrin (21, 24, 25). Experiments using lysine and lysine analogues show that the ligand-binding affinity of the t-PA K2 domain is different from that of the Glu-piasminogen kringles (20, 21). Those data indicate that the difference in affinity for lysine residues embedded in a polypeptide chain and carboxyl-terminal lysines is more pro-
nounced for the Glu-plasminogen kringles than for the K2 domain of t-PA. To better understand the mechanism of plasminogen activation in the presence of fibrin, we addressed the question whether Glu-plasminogen and t-PA share common binding sites in fibrin or, alternatively, whether each component only has unique binding sites. To that end, we performed competition experiments between t-PA and Glu-plasminogen on fibrin matrices predigested with limited amounts of plasmin. In addition, t-PA-deletion mutants, lacking either the F or the K2 domain, were applied to assign specific functions to these particular domains in relation to competition with Gluplasminogen. We present evidence that fibrin binding, mediated either by the F domain of t-PA or by the aminohexylbinding sites of both t-PA and Glu-plasminogen, is accomplished
by unique,
distinct
binding
sites in fibrin.
Further-
more, it is shown that t-PA and Glu-plasminogen can both bind to the same carboxyl-terminal lysines exposed in fibrin upon limited plasmin digestion. EXPERIMENTAL
PROCEDURES
Reagents and Proteins-Trasylol (10,000 kallikrein-inhibiting units/ml) was purchased from Bayer (Leverkusen, FRG). Radioactive materials were obtained from Amersham Corp. Porcine pancreatic carboxypept,idase B (EC 3.4.17.2) was treated with diisofluorophosphate by the supplier (Boehringer Mannheim). Bovine serum albumin (fraction V crystallized, essentially globulin-free) was obtained from Sigma and human fibrinogen (Grade L) was purchased from Kabi Vitrum (Stockholm, Sweden). Fibrinogen (25 mg/ml) was treated with 1 mM diisofluorophosphate for 18 h at room temperature to inactivate potentially contaminating serine proteases and was incubated simultaneously with 1 ml of a lysine-Sepharose suspension (Pharmacia Fine Chemicals AB, Uppsala, Sweden) to remove traces of plasminogen. The Sepharose suspension was removed by centrifugation (2000 rpm) and the fibrinogen solution was dialyzed at room temperature against 50 mM Tris-HCI (pH 7.4), 150 mM NaCl. Aliquots were frozen at -70 “C until use. Glu-plasminogen was purified from fresh human plasma by affinity chromatography on lysineSepharose, followed by Sephacryl S-300 (superfine) gel filtration (Pharmacia) as described before (26). Glu-plasminogen was activated with high molecular weight urokinase (kindly provided by Dr. G. Cassan< Lepetit, Milan, Italy) that was coupled to CNBr-activated Seoharose (Pharmacia). 1 ml of Glu-ulasminoaen (4.5 mg/ml in PBS (0.14 M NaCl, 0.01 M sodium phosphate, pH 7.4)) ias incubated with 100 ~1 of urokinase/Sepharose (250 rg/ml of Sepharose) for 30 min at 37 “C. The generated plasmin activity was determined with the chromogenic substrate H-D-Val-Leu-Lys-p-nitroanilide (S2251) (Kabi Vitrum). The “S2251-assay” (final chromogenic concentration 0.6 mM in 0.1 M Tris-HCl, pH 7.5, 0.1% (v/v) Tween 80) was calibrated with active site-titrated plasmin. Human thrombin was from Sigma, whereas human single-chain recombinant t-PA was obtained from Boehringer Ingelheim (Alkmaar, The Netherlands). Radiolabeling of Proteins-Diisofluorophosphate-treated fibrinogen (1 mg/O.l ml) was diluted with an equal volume of 0.5 M sodium phosphate (pH 7.2). To this mixture, 1 mCi of carrier-free [““I]NaI and 10 pg of chloramine-T (1 mg/ml in PBS) were added for 1 min at room temperature. The reaction was terminated by the addition of 10 pg of sodium bisulfite, and then 50 ~1 of 1% (w/v) KI and 100 ~1
of 0.5 M sodium phosphate (pH 7.2) were added. Free [‘251]NaI was removed by dialysis at room temperature against PBS. Glu-plasminogen (1 mg/200 ~1) was iodinated with one Iodobead (rehydrated in PBS for 5 min) and 10 ~1 of [“Y)NaI (1.1 mCi) for 15 min at room temperature. Free label was removed by Sephadex G-25 gel filtration in PBS, 0.01% (v/v) Tween-80. After labeling, plasminogen entirely retained the Glu-plasminogen form. Fibrin-binding characteristics of Glu-plasminogen were not changed upon radiolabeling (data not shown). As shown in Fig. lB, Glu-plasminogen could be converted mto Lys-plasminogen upon incubation with plasmin. The specific radioactivity of fibrinogen and plasminogen was 135,000 and 500,000 counts/min/pg, respectively. The ‘zSI-labeled proteins were stored at -20
“C
-6o&ruction, Expression, and Purification of t-PA tion Mutants-Plasmid pSV2/t-PA, containing full
and
t-PA-deb-
length human tPA cDNA (15), was used for the construction of the t-PA deletion mutants, t-PA de1.F and t-PA del.KB, as described before (12). The respective t-PA domains were deleted from the cDNA exactly at the intron-exon borders of the corresponding gene, employing the “M13gapped duplex out-looping” mutagenesis procedure (27). For mutant t-PA del.F, base pairs 200-337 were removed from the cDNA, whereas mutant t-PA del.KZ lacks base pairs 716-973. The proteins were expressed by stable cell lines of mouse L cells, transfected with the respective cDNAs, and metabolically labeled using [“HJleucine (12). The recombinant proteins were purified from the conditioned media by immunoaffinity chromatography on immobilized monoclonal antibody ESP2 IgG (Bioscot, Edinburgh, Scotland) (12). This antibody is directed against an antigenic determinant of the t-PA “light” chain present on each of the deletion mutants (28). The (mutant) t-PA preparations were exclusively in the single-chain form as shown by SDS-polyacrylamide gel electrophoresis under reducing conditions (29) followed by fluororadiography (Fig. L4). Inactiuation. of t-PA Wariants) with GGACK-Single-chain t-PA (2.2 mg/ml) was incubated at 37 “C with 0.14 mM GGACK (Calbiochem). After 30 min of incubation GGACK was again added (0.28 mM final concentration) and the incubation was continued for 15 min at 37 “C. The excess of GGACK was removed by dialysis against 0.7 M potassium bicarbonate, pH 8.2, 0.01% (v/v) Tween-80. The metabolically labeled plasminogen activators (approximately 4-30 nM) were incubated with GGACK at a final concentration of 0.04 mM in 50 mM sodium phosphate, pH 7.4, 0.01% (v/v) Tween-80 for 30 min at 37 “C, followed by addition of GGACK (0.05 mM final concentration) for 15 min at 37 “C and, finally, dialyzed against PBS, 0.01% (v/v) Tween-80. Residual plasminogen activator activity was assayed spectrophotometrically using an indirect plasminogen activation assay with the chromogenic substrate S2251 and was shown to be
