Conversion of Glu-Plasminogen to Lys-Plasminogen Is Necessary for Optimal Stimulation of Plasminogen Activation on the Endothelial Cell Surface*

Yun Gong, Sun-OK KimDagger, Jordi Felez§, Davida K. Grella, Francis J. Castellino, and Lindsey A. Miles||

From The Scripps Research Institute, La Jolla, California 92037,  The University of Notre Dame, Notre Dame 46556, Indiana, and the § Institut De Recerca Oncologica, 08907 Barcelona, Spain

Received for publication, February 13, 2001, and in revised form, March 21, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

When Glu-plasminogen is bound to cells, plasmin (Pm) formation by plasminogen (Pg) activators is markedly enhanced compared with the reaction in solution. It is not known whether the direct activation of Glu-Pg by Pg activators is promoted on the cell surface or whether plasminolytic conversion of Glu-Pg to the more readily activated Lys-Pg is necessary for enhanced Pm formation on the cell surface. To distinguish between these potential mechanisms, we tested whether Pm formation on the cell surface could be stimulated in the absence of conversion of Glu-Pg to Lys-Pg. Rates of activation of Glu-Pg, Lys-Pg, and a mutant Glu-Pg, [D646E]Glu-Pg, by either tissue Pg activator (t-PA) or urokinase (u-PA) were compared when these Pg forms were either bound to human umbilical vein endothelial cells (HUVEC) or in solution. ([D646E]Glu-Pg can be cleaved at the Arg561-Val562 bond by Pg activators but does not possess Pm activity subsequent to this cleavage because of the mutation of Asp646 of the serine protease catalytic triad.) Glu-Pg activation by t-PA was enhanced on HUVEC compared with the solution phase by 13-fold. In contrast, much less enhancement of Pg activation was observed with [D646E]Glu-Pg (~2-fold). Although the extent of activation of Lys-Pg on cells was similar to that of Glu-Pg, the cells afforded minimal enhancement of Lys-Pg activation compared with the solution phase (1.3-fold). Similar results were obtained when u-PA was used as activator. When Glu-Pg was bound to the cell in the presence of either t-PA or u-PA, conversion to Lys-Pg was observed, but conversion of ([D646E]Glu-Pg to ([D646E]Lys-Pg was not detected, consistent with the conversion of Glu-Pg to Lys-Pg being necessary for optimal enhancement of Pg activation on cell surfaces. Furthermore, we found that conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was markedly enhanced (~20-fold) on the HUVEC surface, suggesting that the stimulation of the conversion of Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

When Glu-plasminogen (Glu-Pg),1 the native circulating form of the zymogen, is bound to cell surfaces, plasmin (Pm) generation by plasminogen (Pg) activators is markedly stimulated compared with the reaction in solution (1-10). This results in arming cell surfaces with the proteolytic activity of Pm. In the case of endothelial cells, Pm becomes localized to sites of thrombus formation and, in the case of leukocytes, the cells become armed with proteolytic activity required for processes in which cells degrade extracellular matrices to migrate. However, a key component of the mechanism of stimulation of Pg activation on the cell surface is not understood. It is not known whether: 1) direct activation of Glu-Pg by Pg activators is promoted on the cell surface or 2) plasminolytic conversion of Glu-Pg to Lys-Pg is necessary to observe enhanced Pm formation on the cell surface. (Pm catalyzes cleavage of Glu-Pg at the carboxyl sides of Lys62, Arg68, Lys77 (11-13) and at additional minor sites (14) to generate new amino termini of Pg, resulting in Pg molecular forms that are collectively termed "Lys-Pg". Lys-Pg is more readily activated by Pg activators (15-17).) In the first mechanism, Glu-Pg remains uncleaved, yet its direct activation is promoted on the cell surface relative to the solution phase, perhaps through conformational changes induced in the molecule upon its interaction with the cell surface. In the second mechanism, conversion of Glu-Pg by Pm to yield the more readily activated Lys-Pg is necessary, leading to increased Pm production on the cell surface. Furthermore, it is not known whether localization of Glu-Pg on the cell surface enhances its conversion to Lys-Pg by Pm. This question has been addressed also in the current study.

In previous studies, using kinetic assays (1-10) it was not possible to distinguish between the two mechanisms listed above to explain the stimulation of activation of Glu-Pg in the presence of cells. In these earlier studies, cell-associated Glu-Pg was converted to Lys-Pg (~50-60% conversion) on the surfaces of both U937 monocytoid cells (5) and HUVEC (18) in the absence of added Pg activators or Pm. Thus, it was not possible to distinguish whether the direct activation of Glu-Pg was enhanced on the cell surface compared with the reaction in solution. In contrast, in our studies, Glu-Pg remained in its native form, without conversion to Lys-Pg when bound to HUVEC. This enabled us to address the role of conversion of Glu-Pg to Lys-Pg in enhancement of activation on the cell surface. In the current investigation, we employed a Pg recombinant variant, [D646E]Glu-Pg, to assess the requirement for conversion of Glu-Pg to Lys-Pg on the cell surface for stimulation of Pg activation. This recombinant Pg/Pm variant can be converted to the molecular form of Pm, but does not possess Pm activity because of the absence of the necessary Asp residue in the serine proteolytic catalytic triad (19). Hence, [D646E]Glu-Pg is not converted to [D646E]Lys-Pg following its cleavage by Pg activators. [D646E]Glu-Pg was used as the inactive mutant to be as conservative as possible in introducing an amino acid substitution into the active site triad. The rates of activation of Glu-Pg, Lys-Pg, and [D646E]Glu-Pg by either t-PA or u-PA were compared when these forms were either bound to HUVEC or in solution. These experiments were designed to distinguish between two potential mechanisms by which Pm formation is enhanced on the cell surface compared with the soluble phase: 1) direct activation of Glu-Pg forms by Pg activators is promoted on the cell surface compared with the reaction in solution, or 2) plasminolytic cleavage is necessary for stimulation of Glu-Pg activation on the cell surface. Furthermore, we examined whether conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was enhanced on the HUVEC surface.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins-- Glu-Pg was purified from fresh human blood collected into 3 mM benzamidine, 3 mM EDTA, 100 units/ml Trasylol (Pentex Miles, Inc., Kankakee, IL), and 100 µg/ml soybean trypsin inhibitor (Sigma). The plasma was subjected to affinity chromatography on lysine-Sepharose (20) in phosphate-buffered saline (0.01 M sodium phosphate pH 7.3, 0.15 M NaCl) with 1 mM benzamidine, 0.02% NaN3, and 3 mM EDTA, followed by molecular exclusion chromatography on Biogel A 1.5 M (Bio-Rad, Hercules, CA). The Pg concentration was determined spectrophotometrically at 280 nm using an extinction coefficient of 16.8. Lys-Pg was from the National Institute for Biological Standards and Control (Holly Hill, Hampstead, London). The Lys-Pm control was prepared by incubating Lys-Pg with 10 nM high molecular weight (hmw) u-PA. (Calbiochem, San Diego, CA) for 30 min at 37 °C. Pm was from Amersham Pharmacia Biotech/Chromogenix (Uppsala, Sweden). [D646E]Glu-Pg was generated by primer-directed mutagenesis of single-stranded p119/HPg (21) as previously described (22) and expressed in baculovirus-infected lepidopteran cells, followed by purification on lysine-Sepharose as described (20). The characteristics of this mutant have been described (19). Pg forms were radiolabeled using a modified chloramine T procedure as described (1). t-PA was from Genentech (South San Francisco, CA). Low molecular weight (lmw) u-PA was from Calbiochem (San Diego, CA).

Cells, Cell Culture, and Ligand Binding Analyses-- HUVEC were purchased from Clonetics/BioWhittaker (Walkersville, MD) and cells of passage four and below were used in these experiments. HUVEC were grown to confluence in MCDB 131 medium containing 2% fetal calf serum, 12 µg/ml bovine brain extract, 50 ng/ml amphotericin B, 50 µg/ml gentamicin, 1 µg/ml hydrocortisone, 1 ng/ml human epidermal growth factor.

The binding of radiolabeled Pg forms to HUVEC was performed as previously described from our laboratory (23). Briefly, HUVEC, grown to confluence in 24-well culture dishes (6-8 × 104 cells/cm2), were washed three times with HBSS. The radiolabeled Pg forms were incubated with the cells in a final total volume of 200 µl at a final concentration of 25 nM. Reactions were terminated by aspirating the fluid from the wells and rapidly washing the cultures twice with HBSS-BSA. The cell-bound radioactivity was extracted with 100 µl of reduced sample buffer (31.2 mM TrisHCl, pH 7.2, 2% SDS, 10% sucrose, 0.002% bromphenol blue, 15 mM dithiothreitol, 10 mM EDTA, 10 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor (Sigma), 0.02% Na azide, 5 units/ml Trasylol (Miles, Inc., Kankakee, IL)). Greater than 90% of the cell-associated ligand was eluted by this procedure as assessed by comparing counts bound to the cells prior to elution, with counts eluted. Samples were subjected to 7% SDS-PAGE under reducing conditions that distinguish two-chain Pm from the single chain Pg and were exposed to Biomax MR film. The autoradiograms were scanned on an Alpha ImagerTM 2000.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Role of the Glu-Pg to Lys-Pg Conversion in Pg Activation by t-PA on the Cell Surface-- To monitor the activation of 125I-Glu-Pg, 125I-[D 646E]Glu-Pg and 125I-Lys-Pg by t-PA, SDS-PAGE was employed because kinetic assays could not be performed on [D646E]Glu-Pg because its Pm form is proteolytically inactive. We found that in the absence of added t-PA, >95% of the added 125I-Glu-Pg and 125I-[D646E]Glu-Pg remained in their Glu-Pg forms, and Pg activation was not detected on these cells (Fig. 1). Activation of the three ligands by 20 nM t-PA was compared on HUVEC with the reaction in the solution phase, in the absence of cells. The cell-bound ligand was recovered and subjected to 7% SDS-PAGE under reducing conditions, which distinguish native Glu-Pg from Lys-Pg and distinguish the heavy chains of Glu- and Lys-Pm (Fig. 1). Greater than 90% of the cell-bound ligand was recovered by the elution procedure. The percent Pm formation was calculated by dividing the sum of the densities of the Glu-Pm heavy chain and Lys-Pm heavy chain bands by the sum of the densities of the Glu-Pg and Lys-Pg bands and the Glu-Pm and Lys-Pm heavy chain bands. (The light chain of Pm does not incorporate 125I in proportion to the heavy chain and was not used in the calculation of 100% cell-associated ligand). The activation of 125I-Glu-Pg was enhanced ~13-fold at 10 min compared with the reaction in the solution phase (Fig. 1, panel A and Fig. 2). (Enhancement was observed at later time points, also, but the extent of enhancement was limited because the percent Pm formation that could be attained was finite.) The predominant form of the Pm heavy chain corresponded to the Lys-Pm heavy chain consistent with production of Pm activity (Fig. 1, panel A). Both Glu-Pg and Lys-Pg were present on the cell surface following t-PA activation, although Lys-Pg formation was not detected when the reaction was carried out in solution. Lys-Pg accounted for 27% of the total counts in cell-bound Pg at 10 min and 40% of the total counts in cell-bound Pg at 20 min. A similar distribution of Glu-Pg and Lys-Pg was observed also at 60 min and at 120 min.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 1.   Role of the Glu-Pg to Lys-Pg conversion in Pg activation on the cell surface. A concentration of 25 nM of either 125I-Glu-Pg (A), 125I-[D646E]Glu-Pg (B), or 125I-Lys-Pg (C) was incubated with buffer (HBSS-0.4% BSA) or HUVEC (5 × 104) in the presence or absence of 20 nM t-PA for the indicated times at 37 °C. Cell-bound ligand was obtained as described under "Experimental Procedures" and subjected to electrophoresis on 7% SDS-PAGE under reducing conditions.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   Comparison of cleavage of Pg forms by t-PA on HUVEC and in solution. The autoradiograms in Fig. 1 were scanned. The percent Pm formation was calculated by dividing the sum of the densities of the Glu-Pm heavy chain and Lys-Pm heavy chain bands by the sum of the densities of the Glu-Pg and Lys-Pg bands and the Glu-Pm and Lys-Pm heavy chain bands. The percent Pm in the starting material was subtracted. Symbols are as follows: black-triangle, 125I-Glu-Pg + t-PA; triangle , 125I-Glu-Pg + t-PA + HUVEC; , 125I-[D646E]Glu-Pg + t-PA; open circle , 125I-[D646E]Glu-Pg + t-PA + HUVEC; black-square, 125I-Lys-Pg + t-PA; , 125I-Lys- Pg + t-PA + HUVEC.

From the foregoing experiments, as in previous studies with Pg activation by u-PA on U937 cells (5), it was not possible to determine whether Glu-Pg or Lys-Pg was the predominant substrate for t-PA. To address this issue, cleavage of 125I-[D646E]Glu-Pg by t-PA was analyzed. Cleavage of cell-associated 125I-[D646E]Glu-Pg by t-PA was only 2-fold greater than the reaction in the absence of cells at 10 min (Fig. 1, panel B and Fig. 2). The predominant form of the Pm heavy chain was the 125I-[D646E]Glu-Pm form, and no conversion of 125I-[D646E]Glu-Pg to 125I-[D646E]Lys-Pg was observed on the cell surface, consistent with the absence of Pm activity. Notably, the extent of Pm formation when 125I-[D646E]Glu-Pg was bound to the cells was less than that observed when 125I-Glu-Pg was bound to the cells (Figs. 1 and 2). The extent of cleavage of cell-associated 125I-Lys-Pg by t-PA was similar to that of 125I-Glu-Pg (Fig. 1, panels A and C and Fig. 2). However, at 10 min, the HUVEC-stimulated activation of Lys-Pg was only 1.3-fold greater than the reaction in solution. The cells afforded only this minimal enhancement of Lys-Pg activation by t-PA, compared with the solution phase, because Lys-Pg was activated more rapidly in solution than was Glu-Pg. (At 10 min, 51% of the Lys-Pg in solution was cleaved to Pm whereas only 3% of the Glu-Pg in solution was cleaved to Pm).

To ensure that the increase in Pm associated with the cells was not because of a higher affinity or capacity of the cells for Pm versus Pg, we compared the number of molecules of 125I-plasmin(ogen) forms bound to the cells in either the presence or absence of t-PA (Table I). The molecules of 125I-Glu-Pg bound to the cells did not increase in the presence compared with the absence of t-PA. Furthermore, in the presence of t-PA, the molecules of ligand bound to the cell were not greater when cell-bound 125I-Glu-Pg was activated by t-PA compared with treatment of cell-bound 125I-[D646E]Glu-Pg with t-PA. These data suggest that the enhanced formation of Pm versus Pg on the cell surface could not be accounted for by enhanced binding of Pm to the cells.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of t-PA on plasminogen binding to HUVEC
Confluent HUVEC in wells of 24-well culture dishes were incubated with 25 nM each of either 125I-Glu-plasminogen, 125I-[D646E]Glu-Pg or 125I-Lys-plasminogen in either the presence or absence of 20 nM t-PA for 120 minutes at 37 °C. Reactions were terminated by aspirating the unbound radiolabeled ligands, rapidly washing the cultures twice with HBSS-BSA, and extracting the cell-bound ligands as described under "Experimental Procedures." Values are mean ± S.E. of two experiments.

Role of the Glu-Pg to Lys-Pg Conversion in Pg Activation by u-PA on the Cell Surface-- We also examined cleavage of the Pg forms by another Pg activator, hmw u-PA. Cleavage of Glu-Pg to Pm by hmw u-PA (10 nM) was stimulated 4-fold at 10 min, when Glu-Pg was bound to the HUVEC surface compared with the reaction in solution (Fig. 3, panels A and B). The predominant form of the Pm heavy chain was Lys-Pm (Fig. 3, panel A). In addition, Lys-Pg accounted for 43% of the uncleaved Pg on the cell surface. In contrast, cleavage of cell-associated [D646E]Pg was still markedly less than cleavage of cell-associated 125I-Glu-Pg and cleavage of cell-associated [D646E]Pg was not enhanced compared with the reaction in solution (Fig. 3). The formation of [D646E]Lys-Pg was not detected on the cell surface. The percentage cleavage of Lys-Pg on the cell surface was similar to that of Glu-Pg, but cleavage of Lys-Pg in solution was markedly greater than that of Glu-Pg. (At 10 min, 71% of the Lys-Pg in solution was cleaved to Pm whereas only 17% of the Glu-Pg in solution was cleaved to Pm). Hence, cleavage of Lys-Pg was enhanced only 1.1-fold.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 3.   Role of the Glu-Pg to Lys-Pg conversion in Pg activation by hmw u-PA on the cell surface. A, a concentration of 25 nM of either 125I-Glu-Pg, 125I-[D646E]Glu-Pg, or 125I-Lys-Pg was incubated with buffer (HBSS-0.4% BSA) or HUVEC (5 × 104) for 10 min at 37 °C in the presence or absence of 10 nM hmw u-PA. Cell-bound ligand was obtained as described under "Experimental Procedures" and subjected to electrophoresis on 7% SDS-PAGE under reducing conditions. B, the autoradiograms in A were scanned. The percent Pm formation was calculated by dividing the sum of the densities of the Glu-Pm heavy chain and Lys-Pm heavy chain bands by the sum of the densities of the Glu-Pg and Lys-Pg bands and the Glu-Pm and Lys-Pm heavy chain bands. The percent Pm in the starting material was subtracted.

The foregoing data showed that cell-associated 125I-[D646E]Glu-Pg was less readily cleaved than either cell-associated 125I-Glu-Pg or 125I-Lys-Pg, which is consistent with less direct cleavage of Glu-Pg forms compared with Lys-Pg. However, the case of 125I-[D646E]Glu-Pg was also distinct from that of the other ligands because Pm was not produced following cleavage by the Pg activators. Pm cleaves single-chain t-PA to two-chain t-PA (24), although the catalytic efficiency of both forms are virtually identical (24). Pm also cleaves hmw u-PA to lmw u-PA, which also retains catalytic activity (25). To exclude other potential effects of cleavage of the Pg activator by Pm as an explanation for our results, we compared cleavage of 125I-Glu-Pg and 125I-[D646E]Glu-Pg by lmw u-PA (which is not cleaved by Pm). The cleavage of these Pg forms by lmw u-PA was compared when the ligands were either bound to the HUVEC or in solution (Fig. 4, panels A and B). The extent of Pm formation when cell-associated Glu-Pg was activated with lmw u-PA was similar to that when cell-associated Glu-Pg was activated with hmw u-PA (compare Figs. 3 and 4). When cell-associated Glu-Pg was activated with lmw u-PA, the predominant form of the Pm heavy chain was Lys-Pm and Lys-Pg accounted for 41% of the uncleaved Pg on the cell surface. Under these conditions, activation of Glu-Pg in solution was not detected. Cleavage of 125I-[D646E]Pg bound to HUVEC was not detected under these conditions. These results suggest that the differences in extent of cleavage of 125I-[D646E]Pg compared with 125I-Glu-Pg could not be ascribed to an effect of Pm (produced in the latter reaction) on the Pg activator.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4.   Role of the Glu-Pg to Lys-Pg conversion in Pg activation by lmw u-PA on the cell surface. A, a concentration of 25 nM of either 125I-Glu-Pg or 125I-[D646E]Glu-Pg was incubated with buffer (HBSS-0.4% BSA) or HUVEC (5 × 104) for 10 min at 37 °C in the presence or absence of 15 nM lmw u-PA. Cell-bound ligand was obtained as described under "Experimental Procedures" and subjected to electrophoresis on 7% SDS-PAGE under reducing conditions. B, the autoradiograms in A were scanned. The percent Pm formation was calculated by dividing the sum of the densities of the Glu-Pm heavy chain band and Lys-Pm heavy chain band by the sum of the densities of the Glu-Pg and Lys-Pg bands and the Glu-Pm and Lys-Pm heavy chain bands. The percent Pm in the starting material was subtracted.

Effect of Cells on Conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by Exogenous Pm-- We examined whether localization of Glu-Pg on the cell surface could enhance its conversion to Lys-Pg by exogenous Pm, compared with the solution phase. 125I-[D646E]Glu-Pg was used in this analysis to avoid any contribution of the added ligand to the conversion. 125I-[D646E]Glu-Pg was incubated with either buffer or adherent HUVEC in the presence of increasing concentrations of Pm for 20 min at 37 °C. The conversion of the cell-bound and solution phase 125I-[D646E]Glu-Pg to [D646E]Lys-Pg was monitored by SDS-PAGE. A marked enhancement in conversion to the Lys-form was observed when the ligand was bound to the HUVEC, compared with the solution phase (Fig. 5). For example, under conditions where 59% of the cell-bound ligand was in the Lys-form, only 3% of the ligand in the solution phase was in the Lys-form.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of HUVEC on conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm. 125I-[D646E]Glu-Pg (25 nM) was incubated with either HUVEC (5 × 104)(black-square) or buffer (black-triangle) in the presence of the indicated concentration of Pm for 20 min at 37 °C. The cell-bound ligand was obtained as described under "Experimental Procedures" and subjected to electrophoresis in parallel with the solution phase samples on 7% SDS-PAGE under reducing conditions (A). The autoradiograms were scanned (B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we provide the first demonstration that activation of cell-bound Glu-Pg is markedly enhanced only when its conversion to Lys-Pg on the cell surface is permitted. This result allowed us to distinguish between two potential mechanisms for stimulation of Pm formation when Glu-Pg is bound to the cell surface (1-10). In the first mechanism, the direct activation of Glu-Pg is promoted on the cell surface. In the second mechanism, initial conversion of Glu-Pg to Lys-Pg is necessary, so that formation of Pm is enhanced because the more readily activated Lys-Pg becomes the predominant substrate for Pg activators. These two potential mechanisms for this key step in cell surface Pg activation have not been distinguished in previous studies.

With both native Glu-Pg and [D646E]Glu-Pg, we detected <5% conversion to either Lys-Pg or [D646E]Lys-Pg following binding to HUVEC in the absence of a Pg activator. This finding allowed us to carry out our study to address the role of the Glu-Pg to Lys-Pg conversion in stimulation of Pg activation on the HUVEC surface. In contrast, in an earlier study ~50-60% of Glu-Pg was converted to Lys-Pg on the HUVEC surface, in the absence of the addition of exogenous Pm (18). (These studies also suggested that Pm was not the source of proteolytic activity for conversion of Glu-Pg to Lys-Pg (5, 18)). This difference from the previously published data may be ascribed to differences in the culture conditions of the HUVEC in different laboratories or to differences in the added ligand. Thus, the ability to convert Glu-Pg to Lys-Pg, in the absence of exogenous Pm or Pg activator is not a consistent property of the HUVEC surface.

We found that when Glu-Pg was bound to the cells and activated with either t-PA or hmw u-PA (conditions where Lys-Pg and Lys-Pm formation occurred), the rate of Pm formation was similar to that of cell-bound Lys-Pg. This resulted in stimulation of Glu-Pg activation 4-13-fold compared with the reaction in solution. This first observation would be compatible with either of the two activation mechanisms proposed above. To distinguish between these mechanisms we studied cleavage by t-PA of a mutant Glu-Pg, [D646E]Glu-Pg, that does not produce Pm activity following cleavage by Pg activators, so that a [D646E]Lys-Pg is not produced. Cleavage of [D646E]Glu-Pg on the HUVEC surface was not markedly enhanced compared with the reaction in solution. Furthermore, the rates of cleavage of cell-bound and soluble Lys-Pg were also similar, suggesting that the interaction of Lys-Pg with the cells also did not markedly stimulate its activation. Thus, in the absence of the reaction step in which Glu-Pg is converted to Lys-Pg, Pm formation was not markedly enhanced on the cell surface, i.e. the direct cleavage of [D646E]Glu-Pg was not markedly stimulated upon binding to the cell surface, and the cleavage of Lys-Pg was not markedly stimulated on the cell surface compared with the reaction in solution. In contrast, we observed a marked stimulation of activation of cell-bound Glu-Pg, a reaction in which the Glu-Pg to Lys-Pg conversion did occur.

In addition to the transition to the Lys-Pg form, the activation of both Glu-Pg and of Lys-Pg differs from that of [D646E]Glu-Pg because active Pm is formed in the former cases, but not in the latter case. Therefore, we considered whether other activities of Pm might account for the greater activation of Glu-Pg and Lys-Pg compared with the cleavage of [D646E]Glu-Pg on the cell surface. Firstly, treatment of U937 cells with Pm has been reported to increase the binding of Pg to this cell type (26). In the present study we found that the molecules of 125I-Glu-Pg bound to the cells did not increase in the presence when compared with the absence of t-PA. Furthermore, in the presence of t-PA, the molecules of ligand bound to the cell were not greater when cell-bound 125I-Glu-Pg was activated by t-PA compared with treatment of cell-bound 125I-[D646E]Glu-Pg with t-PA. These data suggest that the enhanced formation of Pm versus Pg on the cell surface could not be accounted for by enhanced binding of Glu-Pg versus [D646E]Glu-Pg to the cells because of the generation of new binding sites by Pm generated in the reaction. The differences between our data and the previous report (26) may be ascribed to differences in responsiveness of HUVEC to Pm and/or to lower levels of Pm generated in our experiments.

Pm also cleaves single-chain t-PA to two-chain t-PA and hmw u-PA to lmw u-PA. Although, the catalytic efficiencies of both forms are similar (24, 25) we excluded other potential effects of cleavage of the Pg activator by Pm by examining cleavage of cell-bound Pg forms by lmw u-PA (a Pg activator that is not cleaved by Pm). Glu-Pg activation was also markedly stimulated on the HUVEC surface by lmw u-PA under conditions where cleavage of [D646E]Glu-Pg was not detected. Because similar differences in activation were seen using both lmw and hmw u-PA, these results suggest that cleavage of the Pg activator by Pm did not account for the observed differences in Pg activation on the HUVEC surface with the different Pg forms.

The number of molecules of Lys-Pg bound to the cells compared with both Glu-Pg and [D646E]Glu-Pg, was higher as reflected by the data in Table I and consistent with previous reports of a higher affinity of Lys-Pg versus Glu-Pg for cells (27, 28). However, this increased binding did not appear to increase stimulation of Pg activation on the cell surface because the rates of activation of Glu-Pg and Lys-Pg (when bound to the cell surface) were similar. Although Lys-Pg and Lys-Pm were formed on the cell surface when cell-bound Glu-Pg was activated with t-PA, the number of molecules of ligand bound/cell was not increased. This suggests that the affinity of Glu-Pg for the cells remained the same even in the presence of t-PA when Lys-Pg was formed on the cell surface, most likely because >94% of the ligand in solution remained as Glu-Pg (data not shown).

Cleavage of the variant Pg, [D646E]Glu-Pg, was not markedly stimulated when bound to the HUVEC, although [D646E]Glu-Pg was still susceptible to cleavage by both t-PA and by u-PA when bound to the cell surface. Thus, cleavage of [D646E]Glu-Pg by Pg activators can occur (as it does in the solution phase) but direct cleavage of [D646E]Glu-Pg does not appear to be stimulated upon binding of the ligand to the cell. Analogously, a small amount of direct activation of native Glu-Pg on the cell surface, should provide a source of Pm for conversion of cell-bound Glu-Pg to Lys-Pg, leading to amplification of Pg activation on these cells.

The rate of cleavage of cell-bound Lys-Pg by t-PA was similar to the rate of cleavage of cell-bound Glu-Pg. However, binding to the cells only minimally increased Lys-Pg activation by the Pg activators compared with the reaction in the solution phase. The small amount of stimulation of activation of [D646E]Glu-Pg and Lys-Pg when bound to the cell surface could be because of the effect of concentration of reactants as previously suggested (10), particularly because the HUVEC express binding sites for both t-PA (29-36) and for u-PA (23, 32, 37). Alternatively, or in addition, some common conformational change induced in both Glu-Pg and Lys-Pg upon binding to cells may contribute to the enhancement in activation. Furthermore, although these experiments were performed under conditions where only a small proportion of t-PA receptors would be saturated and at an excess of hmw u-PA that would allow cleavage by soluble as well as by cell-bound u-PA, we cannot exclude that the effect of concentration of Pg on the cell surface, as well as the presence of receptors for the Pg activators is also absolutely required for stimulation of activation on the cell surface.

Our results also suggest an additional new profibrinolytic function of localization of plasmin(ogen) on the cell surface: Conversion of Glu-Pg to Lys-Pg by Pm was enhanced when the ligand was cell-associated compared with being in the solution phase. This may be because of colocalization and concentration of Pm and Pg on the cell surface, enhanced enzymatic activity of cell-bound Pm as described for U937 cells (26) and/or a more accessible conformation of cell-associated Glu-Pg compared with the solution phase.

Taken together, our results suggest that the conversion of Glu-Pg to Lys-Pg by Pm is necessary for maximal enhancement in Glu-Pg activation on cell surfaces relative to the reaction in solution and that the conversion of Glu-Pg to Lys-Pg is enhanced when Glu-Pg is bound to cells. Thus, the enhancement of formation of the more readily activated Lys-Pg allows cells to promote Pg activation on their surfaces, a key step in both thrombolysis and in physiologic and pathophysiologic processes involving cell migration.

    ACKNOWLEDGEMENT

We thank Holly Hapworth for excellent editorial assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL38272, HL45934, and HL31950 (to L. A. M.) and HL13423 (to F. J. C.), American Heart Association Grant-in-aid 9650636N (to L. A. M.), and Marato-TV3/Cardiovascular and F.R. Areces (to J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present Address: Han Nong Central Research Center, Dongbu Advanced Research Inst., 103-2 Moonji-dong, Daeduck Science Town, Taejon 305-380 Korea.

|| To whom correspondence should be addressed: The Scripps Research Inst., CVN-26 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7105; Fax: 858-784-7374; E-mail: lmiles@scripps.edu.

Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M101387200

    ABBREVIATIONS

The abbreviations used are: Glu-Pg, the native form of plasminogen with N-terminal Glu; [D646E]Glu-Pg, a recombinant human plasminogen with Asp646 mutated to Glu; EACA, epsilon aminocaproic acid; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution; hmw u-PA, high molecular weight urokinase; HUVEC, human umbilical vein endothelial cells; [D646E]Lys-Pg, [D646E]Glu-Pg cleaved by plasmin; lmw u-PA, low molecular weight urokinase; Lys-Pg, a proteolytic derivative of Glu-Pg with N-terminal Met68, Lys77, or Val78; Pm, plasmin; PAGE, polyacrylamide gel electrophoresis; t-PA, tissue plasminogen activator.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Miles, L. A., and Plow, E. F. (1985) J. Biol. Chem. 260, 4303-4311[Abstract]
2. Stricker, R. B., Wong, D., Tak Shiu, D., Reyes, P. T., and Shuman, M. A. (1986) Blood 68, 275-280[Abstract]
3. Hajjar, K. A., Harpel, P. C., Jaffe, E. A., and Nachman, R. L. (1986) J. Biol. Chem. 261, 11656-11662[Abstract/Free Full Text]
4. Loscalzo, J., and Vaughan, D. E. (1987) J. Clin. Invest. 79, 1749-1755[Medline] [Order article via Infotrieve]
5. Ellis, V., Behrendt, N., and Dano, K. (1991) J. Biol. Chem. 266, 12752-12758[Abstract/Free Full Text]
6. Duval-Jobe, C., and Parmely, M. J. (1994) J. Biol. Chem. 269, 21353-21357[Abstract/Free Full Text]
7. Felez, J., Miles, L. A., Fabregas, P., Jardi, M., Plow, E. F., and Lijnen, R. J. (1996) Thromb. Haemost. 76, 577-584[Medline] [Order article via Infotrieve]
8. Lopez-Alemany, R., Longstaff, C., Fabregas, P., Jardi, M., Merton, E., and Felez, J. (1996) Fibrinolysis 10, Supp. 3, 5
9. Longstaff, C., Merton, R. E., Fabregas, P., and Felez, J. (1999) Blood 93, 3839-3846[Abstract/Free Full Text]
10. Sinniger, V., Merton, R. E., Fabregas, P., Felez, J., and Longstaff, C. (1999) J. Biol. Chem. 274, 12414-12422[Abstract/Free Full Text]
11. Wiman, B. (1973) Eur. J. Biochem. 39, 1-9[Medline] [Order article via Infotrieve]
12. Wiman, B., and Wallen, P. (1973) Eur. J. Biochem. 36, 25-31[Medline] [Order article via Infotrieve]
13. Violand, B. N., and Castellino, F. J. (1976) J. Biol. Chem. 251, 3906-3912[Abstract]
14. Horrevoets, A. J. G., Smilde, A. E., Fredenburgh, J. C., Pannekoek, H., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 15770-15776[Abstract/Free Full Text]
15. Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919[Abstract/Free Full Text]
16. Markus, G., Evers, J. L., and Hobika, G. H. (1978) J. Biol. Chem. 253, 733-739[Medline] [Order article via Infotrieve]
17. Markus, G., Priore, R. L., and Wissler, F. C. (1979) J. Biol. Chem. 254, 1211-1216[Medline] [Order article via Infotrieve]
18. Hajjar, K. A., and Nachman, R. L. (1988) J. Clin. Invest. 82, 1769-1778[Medline] [Order article via Infotrieve]
19. Grella, D. K., and Castellino, F. J. (1997) Blood 89, 1585-1589[Abstract/Free Full Text]
20. Deutsch, D. G., and Mertz, E. T. (1970) Science 170, 1995-1996
21. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve]
22. Menhart, N., Hoover, G. J., McCance, S. G., and Castellino, F. J. (1995) Biochemistry 34, 1482-1488[Medline] [Order article via Infotrieve]
23. Miles, L. A., Levin, E. G., Plescia, J., Collen, D., and Plow, E. F. (1988) Blood 72, 628-635[Abstract]
24. Rijken, D. C., Hoylaerts, M., and Collen, D. (1982) J. Biol. Chem. 257, 2920-2925[Free Full Text]
25. Saksela, O., and Rifkin, D. B. (1988) Annu. Rev. Cell Biol. 4, 93-126[CrossRef]
26. Gonzalez-Gronow, M., Stack, S., and Pizzo, S. V. (1991) Arch. Biochem. Biophys. 286, 625-628[Medline] [Order article via Infotrieve]
27. Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719[Medline] [Order article via Infotrieve]
28. Miles, L. A., Dahlberg, C. M., and Plow, E. F. (1988) J. Biol. Chem. 263, 11928-11934[Abstract/Free Full Text]
29. Hajjar, K. A., Harpel, P. C., and Nachman, R. L. (1986) Circulation 74 Suppl. II, 233
30. Beebe, D. P. (1987) Thromb. Res. 46, 241-254[Medline] [Order article via Infotrieve]
31. Beebe, D., and Wood, L. (1989) Blood 74, 1116
32. Hajjar, K. A., and Hamel, N. M. (1990) J. Biol. Chem. 265, 2908-2916[Abstract/Free Full Text]
33. Ramakrishnan, V., Sinicropi, D. V., Dere, R., Darbonne, W. C., Bechtol, K. B., and Baker, J. B. (1990) J. Biol. Chem. 265, 2755-2761[Abstract/Free Full Text]
34. Russell, M. E., Quertermous, T., Declerck, P. J., Collen, D., Haber, E., and Homcy, C. J. (1990) J. Biol. Chem. 265, 2569-2575[Abstract/Free Full Text]
35. Felez, J., Chanquia, C. J., Levin, E. G., Miles, L. A., and Plow, E. F. (1991) Blood 78, 2318-2327[Abstract]
36. Fukao, H., Hagiya, Y., Nonaka, T., Okada, K., and Matsuo, O. (1992) Biochem. Biophys. Res. Commun. 187, 956-962[Medline] [Order article via Infotrieve]
37. Barnathan, E. S., Kuo, A., Rosenfeld, L., Karikó, K., Leski, M., Robbiati, F., Nolli, M. L., Henkin, J., and Cines, D. B. (1990) J. Biol. Chem. 265, 2865-2872[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.