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 plasminog en (Pg) activators is markedly enhanced compared with the reaction in solut ion. It is not known whether the direct activation of Glu-Pg by Pg activato rs is promoted on the cell surface or whether plasminolytic conversion of G lu-Pg to the more readily activated Lys-Pg is necessary for enhanced Pm for mation on the cell surface. To distinguish between these potential mechanis ms, 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 G lu-Pg, Lys-Pg, and a mutant Glu-Pg, [D646E]Glu-Pg, by either tissue Pg acti vator (t-PA) or urokinase (u-PA) were compared when these Pg forms were eit her bound to human umbilical vein endothelial cells (HUVEC) or in solution. ([D646E]Glu-Pg can be cleaved at the Arg(561)-Val(562) bond by Pg activato rs but does not possess Pm activity subsequent to this cleavage because of the mutation of Asp(646) Of the serine protease catalytic triad,) Glu-Pg ac tivation 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 w ith [D646E]Glu-Pg (similar to2-fold), Although the extent of activation of Lys-Pg on cells was similar to that of Glu-Pg, the cells afforded minimal e nhancement 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-P g 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. Furthe rmore, we found that conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogen ous Pm was markedly enhanced (similar to 20-fold) on the HUVEC surface, sug gesting that the stimulation of the conversion of Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.