Binding of tissue‐type plasminogen activator to human melanoma cells

Abstract

We have shown (Bizik et al., Cell Regul 1:895-905, 1990) that tPA can activate plasminogen on the surface of human melanoma cells in the presence of alpha 2-macroglobulin (alpha 2M) secretion. In the present study, we investigated the binding of tPA on the surface of Bowes melanoma cells, selected since they lacked production of PAI-1 and alpha 2M. Elution of tPA from the cell layers indicated that polylysine (5 micrograms/ml) and tranexamic acid (10 mM), an analog of lysine, were the most efficient agents for disrupting the interaction between tPA and cell surface component(s). Using a panel of monoclonal antibodies against individual domains of tPA revealed that an antibody directed to the kringle-2 domain of tPA interfered most significantly with cell-surface plasmin generation. As tPA is a glycoprotein, interactions between the tPA sugar moieties and cell surface were also tested by the use of a series of monosaccharides. N-acetyl-D-glucosamine (100 mM) was the most potent sugar to release tPA from melanoma cells, but the results indicated that the oligosaccharides of tPA play only a supportive role in the binding of tPA to the cell surface. Quantitative comparison of the cell surface localized tPA, which was eluted by tranexamic acid, with the total cellular tPA showed that cell surface bound tPA could represent up to 10%. We conclude that tPA interacts with the melanoma cell surface in a similar manner as has been described for binding of tPA to fibrin and to the putative endothelial cell surface receptor.

Human Co115 colon carcinoma cells potentiate the degradation of laminin mediated by tissue‐type plasminogen activator

“…We developed a 1251-labeled laminin degradation assay with glutaraldehyde-immobilized laminin which allows the quantitative measurement of the laminin degradation potential of tumor cells, particularly of the PA system. Our results show that co115 cells which produced only t-PA were able to degrade laminin with the same efficacy as C0112 cells which produce only u-PA. Laminin degradation by co115 cells is plasminogen dependent and is mediated by t-PA. Our results extend to a human colonic epithelial malignant cell type those reported by others which suggested that activation of plasminogen by t-PA is localized on the surface of human melanoma cells (Bizik et al, 1990(Bizik et al, ,1993Meissauer et al, 1992). However, whether t-PA is bound or not to a specific receptor on the surface of tumor cells is still unknown.…”

The Voltage-dependent Anion Channel (VDAC) Binds Tissue-type Plasminogen Activator and Promotes Activation of Plasminogen on the Cell Surface

“…It is known that t-PA-mediated plasmin generation coincides with rapid necrosis and formation of aggregates in injured neurons (40). During this stage, there is a significant reduction in cell size, which depends on plasmin-mediated proteolytic degradation (39). Our data suggest that during injury VDAC overexpressed at the cell surface may originate from the migration of mitochondria toward the surface, and we know from a previous study that mitochondrial VDAC binds Pg K5 (6).…”

“…2C), suggesting that VDAC only affects t-PA activity. We also assessed the effect of VDAC on t-PA amidolytic activity in the presence of tranexamic acid, which inhibits kringle-dependent binding of t-PA to fibrin or cell receptors (38,39). Increasing amounts of tranexamic acid (1-10 mM) did not change the stimulatory activity of VDAC (100 nM) on t-PA amidolytic activity (Fig.…”

Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases.

“…Thus, PAI-1 and pro-uPA can be present together in the sample without interference. tPA was assayed by a slightly modified procedure using 20 was allowed to proceed for 45 minutes at 37°C and the plasmin produced was assayed by its thioesterase activity.25 To estimate the amount of enzyme produced, high molecular weight two-chain uPA (80 000 IU/mg; American Diagnostica) and two-chain tPA (650 000 IU/mg; American Diagnostica) were used as standards.…”