TY - JOUR
T1 - Conversion of Glu-Plasminogen to Lys-Plasminogen Is Necessary for Optimal Stimulation of Plasminogen Activation on the Endothelial Cell Surface
AU - Gong, Yun
AU - Kim, Sun Ok
AU - Felez, Jordi
AU - Grella, Davida K.
AU - Castellino, Francis J.
AU - Miles, Lindsey A.
PY - 2001/6/1
Y1 - 2001/6/1
N2 - 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-Val 562 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.
AB - 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-Val 562 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.
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U2 - 10.1074/jbc.M101387200
DO - 10.1074/jbc.M101387200
M3 - Article
C2 - 11264290
AN - SCOPUS:0035374672
SN - 0021-9258
VL - 276
SP - 19078
EP - 19083
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 22
ER -