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

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. 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. 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. were to distinguish between two potential mechanisms by which Pm formation is enhanced on the cell surface compared with the 1) direct activation of Glu-Pg forms by Pg activators is promoted on the cell surface compared with the reaction in or 2) plasminolytic cleavage is necessary for stimulation of Glu-Pg activation on the cell surface. Furthermore, we whether conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was enhanced on the HUVEC surface. Glu-Pg was human into and was to on Scopus Google Scholar) in with and by molecular on Pg was at using an of Lys-Pg was the for and was by Lys-Pg with high molecular weight for at Pm was [D646E]Glu-Pg was by of 1987; PubMed Scopus Google Scholar) as N. Castellino F.J. 1995; PubMed Scopus Google Scholar) and in cells, by on as Scopus Google Scholar). of this mutant been (19Grella D.K. Castellino F.J. Blood. 1997; 89: 1585-1589Crossref PubMed Google Scholar). Pg forms were using a as (1Miles L.A. Plow E.F. J. Biol. Chem. 1985; 260: 4303-4311Abstract Full Text PDF PubMed Google Scholar). t-PA was molecular weight u-PA was HUVEC were and cells of and were used in these HUVEC were to in bovine human of Pg forms to HUVEC was as our L.A. J. Collen D. Plow E.F. Blood. 1988; PubMed Google Scholar). to in were with Pg forms were with the cells in a of at a of were by the the and the with was with of of the cell-associated was by this as by bound to the cells to with were to that distinguish Pm the Pg and were to were on an To the activation by was employed because kinetic assays could not be on [D646E]Glu-Pg because its Pm form is found that in the absence of added of the added remained in Glu-Pg and Pg activation was not on these cells of the by t-PA was compared on HUVEC with the reaction in the solution phase, in the absence of cells. was and to which distinguish native Glu-Pg Lys-Pg and distinguish the of and of the was by the Pm formation was by the of the of the and by the of the of the Glu-Pg and Lys-Pg and the and of Pm does not in to the and was not used in the of cell-associated activation was enhanced at compared with the reaction in the solution phase and was observed at but the extent of enhancement was because the Pm formation that could be was form of the Pm to the consistent with production of Pm activity Glu-Pg and Lys-Pg were on the cell surface following t-PA Lys-Pg formation was not when the reaction was in solution. Lys-Pg for of the in Pg at and of the in Pg at similar of Glu-Pg and Lys-Pg was observed also at and at of cleavage of Pg forms by t-PA on HUVEC and in solution. in were Pm formation was by the of the of the and by the of the of the Glu-Pg and Lys-Pg and the and Pm in the was are as t-PA t-PA Pg t-PA the as in previous 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 it was not possible to whether Glu-Pg or Lys-Pg was the for To address this cleavage of by t-PA was of by t-PA was the reaction in the absence of cells at and form of the Pm was form, and conversion to was observed on the cell surface, consistent with the absence of Pm the extent of Pm formation was bound to the cells was less that observed when was bound to the cells and extent of cleavage of by t-PA was similar to that and However, at the activation of Lys-Pg was the reaction in solution. cells afforded this minimal enhancement of Lys-Pg activation by compared with the solution phase, because Lys-Pg was activated more in solution was of the Lys-Pg in solution was cleaved to Pm of the Glu-Pg in solution was cleaved to To that the in Pm with the cells was not because of a or of the cells for Pg, we compared the of forms bound to the cells in either the presence or absence of t-PA of bound to the cells not in the presence compared with the absence of Furthermore, in the presence of the of bound to the cell were not when was activated by t-PA compared with of with that the enhanced formation of Pm Pg on the cell surface could not be for by enhanced of Pm to the of t-PA on plasminogen to HUVEC in of were with of either or in either the presence or absence of t-PA for at were by the the with and the as are of two in a new HUVEC in of were with of either or in either the presence or absence of t-PA for at were by the the with and the as are of two also cleavage of the Pg forms by Pg of Glu-Pg to Pm by u-PA was stimulated at when Glu-Pg was bound to the HUVEC surface compared with the reaction in solution and form of the Pm was In Lys-Pg for of the Pg on the cell surface. In contrast, cleavage of cell-associated was markedly less cleavage of cell-associated and cleavage of cell-associated was not enhanced compared with the reaction in solution formation of [D646E]Lys-Pg was not on the cell surface. cleavage of Lys-Pg on the cell surface was similar to that of Glu-Pg, but cleavage of Lys-Pg in solution was markedly that of of the Lys-Pg in solution was cleaved to Pm of the Glu-Pg in solution was cleaved to Hence, cleavage of Lys-Pg was enhanced that was less readily cleaved either cell-associated or which is consistent with less direct cleavage of Glu-Pg forms compared with Lys-Pg. However, the case of was also that of the because Pm was not following cleavage by the Pg activators. Pm t-PA to t-PA D.C. M. Collen D. J. Biol. Chem. 1982; 257: Full Text PDF PubMed Google the catalytic of both forms are D.C. M. Collen D. J. Biol. Chem. 1982; 257: Full Text PDF PubMed Google Scholar). Pm also u-PA to u-PA, which also catalytic activity Biol. 1988; PubMed Scopus Google Scholar). To potential of cleavage of the Pg activator by Pm as an for our we compared cleavage and by u-PA is not cleaved by cleavage of these Pg forms by u-PA was compared when the were either bound to the HUVEC or in solution and extent of Pm formation when cell-associated Glu-Pg was activated with u-PA was similar to that when cell-associated Glu-Pg was activated with u-PA and When cell-associated Glu-Pg was activated with u-PA, the form of the Pm was and Lys-Pg for of the Pg on the cell surface. these activation of Glu-Pg in solution was not of bound to HUVEC was not these results that the in extent of cleavage of compared could not be to an of Pm in the on the Pg activator. whether localization of Glu-Pg on the cell surface could enhance its conversion to Lys-Pg by exogenous Pm, compared with the solution was used in this to of the added to the was with either or HUVEC in the presence of of Pm for at conversion of the and solution phase to [D646E]Lys-Pg was by enhancement in conversion to the was observed when the was bound to the compared with the solution phase of the was in the of the in the solution phase was in the In this we the first that activation of Glu-Pg is markedly enhanced when its conversion to Lys-Pg on the cell surface is This 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, conversion of Glu-Pg to Lys-Pg is necessary, that formation of Pm is enhanced because the more readily activated Lys-Pg becomes the for Pg activators. two potential mechanisms for this key in cell surface Pg activation not been in previous both native Glu-Pg and [D646E]Glu-Pg, we conversion to either Lys-Pg or [D646E]Lys-Pg following to HUVEC in the absence of a Pg activator. This us to our 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 of Glu-Pg was converted to Lys-Pg on the HUVEC surface, in the absence of the of exogenous Pm (18Hajjar K.A. Nachman R.L. J. Clin. Invest. 1988; 82: 1769-1778Crossref PubMed Scopus (73) Google Scholar). also that Pm was not the of proteolytic activity for conversion of Glu-Pg to Lys-Pg (5Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google K.A. Nachman R.L. J. Clin. Invest. 1988; 82: 1769-1778Crossref PubMed Scopus (73) Google This the be to in the of the HUVEC in or to in the added Thus, the to Glu-Pg to Lys-Pg, in the absence of exogenous Pm or Pg activator is not a consistent of the HUVEC surface. found that when Glu-Pg was bound to the cells and activated with either t-PA or u-PA Lys-Pg and formation the of Pm formation was similar to that of Lys-Pg. This in stimulation of Glu-Pg activation compared with the reaction in solution. This first be with either of the two activation mechanisms To distinguish between these mechanisms we cleavage by t-PA of a mutant Glu-Pg, [D646E]Glu-Pg, that does not Pm activity following cleavage by Pg that a [D646E]Lys-Pg is not of [D646E]Glu-Pg on the HUVEC surface was not markedly enhanced compared with the reaction in solution. Furthermore, the of cleavage of and Lys-Pg were also suggesting that the interaction of Lys-Pg with the cells also not markedly its activation. Thus, in the absence of the reaction in which Glu-Pg is converted to Lys-Pg, Pm formation was not markedly enhanced on the cell surface, the direct cleavage of [D646E]Glu-Pg was not markedly stimulated upon 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 stimulation of activation of Glu-Pg, a reaction in which the Glu-Pg to Lys-Pg conversion In to the to the Lys-Pg form, the activation of both Glu-Pg and of Lys-Pg that of [D646E]Glu-Pg because active Pm is in the but not in the we whether of Pm for the activation of Glu-Pg and Lys-Pg compared with the cleavage of [D646E]Glu-Pg on the cell surface. of U937 cells with Pm has been to the of Pg to this cell M. Biochem. 1991; PubMed Scopus Google Scholar). In the we found that the of bound to the cells not in the presence when compared with the absence of Furthermore, in the presence of the of bound to the cell were not when was activated by t-PA compared with of with that the enhanced formation of Pg on the cell surface could not be for by enhanced of Glu-Pg [D646E]Glu-Pg to the cells because of the generation of new sites by Pm in the between our and the previous M. Biochem. 1991; PubMed Scopus Google Scholar) be to in of HUVEC to Pm to of Pm in our Pm also t-PA to t-PA and u-PA to the catalytic of both forms are similar D.C. M. Collen D. J. Biol. Chem. 1982; 257: Full Text PDF PubMed Google Scholar, Biol. 1988; PubMed Scopus Google Scholar) we potential of cleavage of the Pg activator by Pm by cleavage of Pg forms by u-PA Pg activator that is not cleaved by Glu-Pg activation was also markedly stimulated on the HUVEC surface by u-PA cleavage of [D646E]Glu-Pg was not similar in activation were using both and u-PA, these results that cleavage of the Pg activator by Pm not for the observed in Pg activation on the HUVEC surface with the Pg of of Lys-Pg bound to the cells compared with both Glu-Pg and [D646E]Glu-Pg, was as by the in and consistent with previous of a of Lys-Pg Glu-Pg for cells K.A. Harpel P.C. Nachman R.L. J. Clin. Invest. 1987; PubMed Scopus Google Scholar, L.A. Plow E.F. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). However, this increased not to stimulation of Pg activation on the cell surface because the of activation of Glu-Pg and Lys-Pg bound to the cell were Although Lys-Pg and were on the cell surface when Glu-Pg was activated with the of of was not This that the of Glu-Pg for the cells remained the in the presence of t-PA when Lys-Pg was on the cell surface, because of the in solution remained as Glu-Pg not of the variant Pg, [D646E]Glu-Pg, was not markedly stimulated when bound to the [D646E]Glu-Pg was 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 it does in the solution but direct cleavage of [D646E]Glu-Pg does not to be stimulated upon of the to the a of direct activation of native Glu-Pg on the cell surface, a of Pm for conversion of Glu-Pg to Lys-Pg, leading to of Pg activation on these cells. of cleavage of Lys-Pg by t-PA was similar to the of cleavage of However, to the cells increased Lys-Pg activation by the Pg activators compared with the reaction in the solution of stimulation of activation of [D646E]Glu-Pg and Lys-Pg when bound to the cell surface could be because of the of of as 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 because the HUVEC sites for both t-PA K.A. Harpel P.C. Nachman R.L. 1986; Scholar, Thromb. 1987; Full Text PDF PubMed Scopus (40) Google Scholar, D. Blood. Google Scholar, K.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, V. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.E. Collen D. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Miles L.A. Plow E.F. Blood. 1991; PubMed Google Scholar, H. K. Biochem. PubMed Scopus Google Scholar) and for u-PA L.A. J. Collen D. Plow E.F. Blood. 1988; PubMed Google Scholar, K.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. M. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). or in conformational induced in both Glu-Pg and Lys-Pg upon to cells to the enhancement in activation. Furthermore, these were a of t-PA be and at an of u-PA that cleavage by as as by u-PA, we that the of of Pg on the cell surface, as as the presence of for the Pg activators is also required for stimulation of activation on the cell surface. results also an additional new of localization of on the cell of Glu-Pg to Lys-Pg by Pm was enhanced when the was cell-associated compared with being in the solution This be because of and of Pm and Pg on the cell surface, enhanced activity of Pm as for U937 cells M. Biochem. 1991; PubMed Scopus Google Scholar) a more of cell-associated Glu-Pg compared with the solution our results that the conversion of Glu-Pg to Lys-Pg by Pm is necessary for 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 cells to Pg activation on surfaces, a key in both and in and processes cell for