Conversion of Glu-Plasminogen to Lys-Plasminogen Is Necessary for Optimal Stimulation of Plasminogen Activation on the Endothelial Cell Surface
2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês
10.1074/jbc.m101387200
ISSN1083-351X
AutoresYun Yun Gong, Sun-OK Kim, Jordi Félez, Davida K. Grella, Francis Castellino, Lindsey A. Miles,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoWhen 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. 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. the native form of plasminogen with N-terminal Glu a recombinant human plasminogen with Asp646 mutated to Glu epsilon aminocaproic acid bovine serum albumin Hanks' balanced salt solution high molecular weight urokinase human umbilical vein endothelial cells [D646E]Glu-Pg cleaved by plasmin low molecular weight urokinase a proteolytic derivative of Glu-Pg with N-terminal Met68, Lys77, or Val78 plasmin polyacrylamide gel electrophoresis tissue plasminogen activator 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 (1Miles L.A. Plow E.F. J. Biol. Chem. 1985; 260: 4303-4311Abstract Full Text PDF PubMed Google Scholar, 2Stricker R.B. Wong D. Tak Shiu D. Reyes P.T. Shuman M.A. Blood. 1986; 68: 275-280Crossref PubMed Google Scholar, 3Hajjar K.A. Harpel P.C. Jaffe E.A. Nachman R.L. J. Biol. Chem. 1986; 261: 11656-11662Abstract Full Text PDF PubMed Google Scholar, 4Loscalzo J. Vaughan D.E. J. Clin. Invest. 1987; 79: 1749-1755Crossref PubMed Scopus (123) Google Scholar, 5Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar, 6Duval-Jobe C. Parmely M.J. J. Biol. Chem. 1994; 269: 21353-21357Abstract Full Text PDF PubMed Google Scholar, 7Felez J. Miles L.A. Fabregas P. Jardi M. Plow E.F. Lijnen R.J. Thromb. Haemost. 1996; 76: 577-584Crossref PubMed Scopus (85) Google Scholar, 8Lopez-Alemany R. Longstaff C. Fabregas P. Jardi M. Merton E. Felez J. Fibrinolysis. 1996; 10, Supp. 3: 5Google Scholar, 9Longstaff C. Merton R.E. Fabregas P. Felez J. Blood. 1999; 93: 3839-3846Crossref PubMed Google Scholar, 10Sinniger V. Merton R.E. Fabregas P. Felez J. Longstaff C. J. Biol. Chem. 1999; 274: 12414-12422Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). 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 (11Wiman B. Eur. J. Biochem. 1973; 39: 1-9Crossref PubMed Scopus (66) Google Scholar, 12Wiman B. Wallen P. Eur. J. Biochem. 1973; 36: 25-31Crossref PubMed Scopus (138) Google Scholar, 13Violand B.N. Castellino F.J. J. Biol. Chem. 1976; 251: 3906-3912Abstract Full Text PDF PubMed Google Scholar) and at additional minor sites (14Horrevoets A.J.G. Smilde A.E. Fredenburgh J.C. Pannekoek H. Nesheim M.E. J. Biol. Chem. 1995; 270: 15770-15776Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) 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 (15Hoylaerts M. Rijken D.C. Lijnen H.R. Collen D. J. Biol. Chem. 1982; 257: 2912-2919Abstract Full Text PDF PubMed Google Scholar, 16Markus G. Evers J.L. Hobika G.H. J. Biol. Chem. 1978; 253: 733-739Abstract Full Text PDF PubMed Google Scholar, 17Markus G. Priore R.L. Wissler F.C. J. Biol. Chem. 1979; 254: 1211-1216Abstract Full Text PDF PubMed Google Scholar).) 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 (1Miles L.A. Plow E.F. J. Biol. Chem. 1985; 260: 4303-4311Abstract Full Text PDF PubMed Google Scholar, 2Stricker R.B. Wong D. Tak Shiu D. Reyes P.T. Shuman M.A. Blood. 1986; 68: 275-280Crossref PubMed Google Scholar, 3Hajjar K.A. Harpel P.C. Jaffe E.A. Nachman R.L. J. Biol. Chem. 1986; 261: 11656-11662Abstract Full Text PDF PubMed Google Scholar, 4Loscalzo J. Vaughan D.E. J. Clin. Invest. 1987; 79: 1749-1755Crossref PubMed Scopus (123) Google Scholar, 5Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar, 6Duval-Jobe C. Parmely M.J. J. Biol. Chem. 1994; 269: 21353-21357Abstract Full Text PDF PubMed Google Scholar, 7Felez J. Miles L.A. Fabregas P. Jardi M. Plow E.F. Lijnen R.J. Thromb. Haemost. 1996; 76: 577-584Crossref PubMed Scopus (85) Google Scholar, 8Lopez-Alemany R. Longstaff C. Fabregas P. Jardi M. Merton E. Felez J. Fibrinolysis. 1996; 10, Supp. 3: 5Google Scholar, 9Longstaff C. Merton R.E. Fabregas P. Felez J. Blood. 1999; 93: 3839-3846Crossref PubMed Google Scholar, 10Sinniger V. Merton R.E. Fabregas P. Felez J. Longstaff C. J. Biol. Chem. 1999; 274: 12414-12422Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) 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 (5Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar) and HUVEC (18Hajjar K.A. Nachman R.L. J. Clin. Invest. 1988; 82: 1769-1778Crossref PubMed Scopus (73) Google Scholar) 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 (19Grella D.K. Castellino F.J. Blood. 1997; 89: 1585-1589Crossref PubMed Google Scholar). 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. 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 (20Deutsch D.G. Mertz E.T. Science. 1970; 170: 1995-1996Crossref Scopus (1670) Google Scholar) 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 (21Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar) as previously described (22Menhart N. Hoover G.J. McCance S.G. Castellino F.J. Biochemistry. 1995; 34: 1482-1488Crossref PubMed Scopus (37) Google Scholar) and expressed in baculovirus-infected lepidopteran cells, followed by purification on lysine-Sepharose as described (20Deutsch D.G. Mertz E.T. Science. 1970; 170: 1995-1996Crossref Scopus (1670) Google Scholar). The characteristics of this mutant have been described (19Grella D.K. Castellino F.J. Blood. 1997; 89: 1585-1589Crossref PubMed Google Scholar). Pg forms were radiolabeled using a modified chloramine T procedure as described (1Miles L.A. Plow E.F. J. Biol. Chem. 1985; 260: 4303-4311Abstract Full Text PDF PubMed Google Scholar). t-PA was from Genentech (South San Francisco, CA). Low molecular weight (lmw) u-PA was from Calbiochem (San Diego, CA). 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 (23Miles L.A. Levin E.G. Plescia J. Collen D. Plow E.F. Blood. 1988; 72: 628-635Crossref PubMed Google Scholar). Briefly, HUVEC, grown to confluence in 24-well culture dishes (6–8 × 104cells/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 mmEDTA, 10 mm benzamidine, 2 mmphenylmethylsulfonyl 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. To monitor the activation of125I-Glu-Pg, 125I-[D 646E]Glu-Pg and125I-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 and125I-[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 of125I-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.Figure 2Comparison 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: ▴, 125I-Glu-Pg + t-PA; ▵, 125I-Glu-Pg + t-PA + HUVEC; ●,125I-[D646E]Glu-Pg + t-PA; ○,125I-[D646E]Glu-Pg + t-PA + HUVEC; ▪,125I-Lys-Pg + t-PA; ■, 125I-Lys- Pg + t-PA + HUVEC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) From the foregoing experiments, as in previous studies with Pg activation by u-PA on U937 cells (5Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar), 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-associated125I-[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 the125I-[D646E]Glu-Pm form, and no conversion of125I-[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 when125I-[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-associated125I-Lys-Pg by t-PA was similar to that of125I-Glu-Pg (Fig. 1, panels A and Cand 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 Pmversus Pg, we compared the number of molecules of125I-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-bound125I-[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.Table IEffect of t-PA on plasminogen binding to HUVECAdded radiolabeled ligandPlasminogen boundBuffert-PAmolecules/cell × 105Glu-plasminogen1.01 ± 0.031.04 ± 0.16[D646E]Glu-Pg1.02 ± 0.101.20 ± 0.06Lys-plasminogen1.93 ± 0.702.09 ± 0.60Confluent 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. Open table in a new tab 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. 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. The foregoing data showed that cell-associated125I-[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 (24Rijken D.C. Hoylaerts M. Collen D. J. Biol. Chem. 1982; 257: 2920-2925Abstract Full Text PDF PubMed Google Scholar), although the catalytic efficiency of both forms are virtually identical (24Rijken D.C. Hoylaerts M. Collen D. J. Biol. Chem. 1982; 257: 2920-2925Abstract Full Text PDF PubMed Google Scholar). Pm also cleaves hmw u-PA to lmw u-PA, which also retains catalytic activity (25Saksela O. Rifkin D.B. Annu. Rev. Cell Biol. 1988; 4: 93-126Crossref PubMed Scopus (717) Google Scholar). To exclude other potential effects of cleavage of the Pg activator by Pm as an explanation for our results, we compared cleavage of125I-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 with125I-Glu-Pg could not be ascribed to an effect of Pm (produced in the latter reaction) on the Pg activator. 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. 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 (1Miles L.A. Plow E.F. J. Biol. Chem. 1985; 260: 4303-4311Abstract Full Text PDF PubMed Google Scholar, 2Stricker R.B. Wong D. Tak Shiu D. Reyes P.T. Shuman M.A. Blood. 1986; 68: 275-280Crossref PubMed Google Scholar, 3Hajjar K.A. Harpel P.C. Jaffe E.A. Nachman R.L. J. Biol. Chem. 1986; 261: 11656-11662Abstract Full Text PDF PubMed Google Scholar, 4Loscalzo J. Vaughan D.E. J. Clin. Invest. 1987; 79: 1749-1755Crossref PubMed Scopus (123) Google Scholar, 5Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar, 6Duval-Jobe C. Parmely M.J. J. Biol. Chem. 1994; 269: 21353-21357Abstract Full Text PDF PubMed Google Scholar, 7Felez J. Miles L.A. Fabregas P. Jardi M. Plow E.F. Lijnen R.J. Thromb. Haemost. 1996; 76: 577-584Crossref PubMed Scopus (85) Google Scholar, 8Lopez-Alemany R. Longstaff C. Fabregas P. Jardi M. Merton E. Felez J. Fibrinolysis. 1996; 10, Supp. 3: 5Google Scholar, 9Longstaff C. Merton R.E. Fabregas P. Felez J. Blood. 1999; 93: 3839-3846Crossref PubMed Google Scholar, 10Sinniger V. Merton R.E. Fabregas P. Felez J. Longstaff C. J. Biol. Chem. 1999; 274: 12414-12422Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). 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 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 (10Sinniger V. Merton R.E. Fabregas P. Felez J. Longstaff C. J. Biol. Chem. 1999; 274: 12414-12422Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), particularly because the HUVEC express binding sites for both t-PA (29Hajjar K.A. Harpel P.C. Nachman R.L. Circulation. 1986; 74 Suppl. II: 233Google Scholar, 30Beebe D.P. Thromb. Res. 1987; 46: 241-254Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 31Beebe D. Wood L. Blood. 1989; 74: 1116Crossref Google Scholar, 32Hajjar K.A. Hamel N.M. J. Biol. Chem. 1990; 265: 2908-2916Abstract Full Text PDF PubMed Google Scholar, 33Ramakrishnan V. Sinicropi D.V. Dere R. Darbonne W.C. Bechtol K.B. Baker J.B. J. Biol. Chem. 1990; 265: 2755-2761Abstract Full Text PDF PubMed Google Scholar, 34Russell M.E. Quertermous T. Declerck P.J. Collen D. Haber E. Homcy C.J. J. Biol. Chem. 1990; 265: 2569-2575Abstract Full Text PDF PubMed Google Scholar, 35Felez J. Chanquia C.J. Levin E.G. Miles L.A. Plow E.F. Blood. 1991; 78: 2318-2327Crossref PubMed Google Scholar, 36Fukao H. Hagiya Y. Nonaka T. Okada K. Matsuo O. Biochem. Biophys. Res. Commun. 1992; 187: 956-962Crossref PubMed Scopus (20) Google Scholar) and for u-PA (23Miles L.A. Levin E.G. Plescia J. Collen D. Plow E.F. Blood. 1988; 72: 628-635Crossref PubMed Google Scholar, 32Hajjar K.A. Hamel N.M. J. Biol. Chem. 1990; 265: 2908-2916Abstract Full Text PDF PubMed Google Scholar, 37Barnathan E.S. Kuo A. Rosenfeld L. Karikó K. Leski M. Robbiati F. Nolli M.L. Henkin J. Cines D.B. J. Biol. Chem. 1990; 265: 2865-2872Abstract Full Text PDF PubMed Google Scholar). 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 (26Gonzalez-Gronow M. Stack S. Pizzo S.V. Arch. Biochem. Biophys. 1991; 286: 625-628Crossref PubMed Scopus (44) Google Scholar) 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. We thank Holly Hapworth for excellent editorial assistance.
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