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Bound plasminogen is rate-limiting for cell-surface-mediated activation of plasminogen by urokinase.

ABSTRACT: The ability of U937 monocyte-like cells and KATO III cells (a human gastric carcinoma line) to potentiate activation of plasminogen by single-chain urokinase-type plasminogen activator (scu-PA), as mediated by the cell receptor for urokinase (u-PAR), was compared. It was observed that, although the concentration of u-PAR on these cell lines differed considerably (U937 cells: 5000 receptors/cell, Kd 0.35 nM; KATO III cells: 400 receptors/cell, Kd 0.85 nM), the rate of activation of plasminogen by scu-PA in the presence of the same density of each cell line was equivalent. From data generated in the presence of increasing concentrations of scu-PA, the kcat, for plasminogen activation in the presence of each cell line was calculated and found to differ by 26-fold (0.36 s-1 on U937 cells; 9.25 s-1 on KATO III cells). However, the Km for plasminogen with respect to the rate of formation of plasmin was lower than the Kd for binding (0.2 microM compared with 0.5 microM on U937 cells; 0.34 microM compared with 1.6 microM on KATO III cells). A rapid transformation from Glu-plasminogen (native plasminogen with N-terminal Glu) to Lys-plasminogen (plasmin-degraded plasminogen with primarily N-terminal Lys-77) occurred on the surface of U937 cells (unlike KATO III cells), but this transition did not coincide with faster rates of plasminogen activation. From this evidence it is concluded that the accessibility of bound plasminogen acts to limit the rate of activation by cell-bound urokinase. The significance of this proposal is that the proteolytic potential of the cell-mediated activation of plasminogen would be controlled by the accessibility of plasminogen for activation rather than by the concentration of u-PAR (the latter may act to localize proteolysis to appropriate domains on the surface of the cell).

SUBMITTER: Namiranian S

PROVIDER: S-EPMC1135727 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Project description:Data from clinical studies, cell culture, and animal models implicate the urokinase (uPA)/Plasminogen (Plg) system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/Plg stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression to clarify mechanisms of uPA/Plg-accelerated atherosclerosis and aneurysm formation. Microarray studies were performed to identify potential mediators of uPA-accelerated atherosclerosis. These studies identified S100A8 and S100A9 mRNA as the most highly upregulated transcripts in uPA-overexpressing macrophages; upregulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also upregulated in aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is upregulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Bioinformatics analysis of the microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm—in a second animal model—that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease. Six independent biological replicate RNA samples were prepared from thioglycollate-elicited peritoneal macrophages from mice of two different genotypes: SR-uPA+/0 transgenic (overexpress uPA in macrophages) and nontransgenic, respectively), for a total of 12 independent RNA samples. Both transgenic and nontransgenic mice were Apoe-/- and were fed Western diet from 5-15 weeks before peritoneal macrophages were elicited. In addition, from the six samples of each genotype, a pooled sample was prepared by combining the six genotype-specific samples. Each of the two pooled samples was assayed on the BeadChip in two technical replicates, for a total of 16 hybridizations performed using two Illumina Mouse Ref-8 v1.1 chips.

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Bound plasminogen is rate-limiting for cell-surface-mediated activation of plasminogen by urokinase.

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