Plasminogen carbohydrate side chains in receptor binding and enzyme activation: A study of C6 glioma cells and primary cultures of rat hepatocytes

Abstract

The human [Glu1]-plasminogen carbohydrate isozymes, plasminogen type I (Pg 1) and plasminogen type II (Pg 2), were separated by chromatography and studied in cell binding experiments at 4 degrees C with primary cultures of rat hepatocytes and rat C6 glioma cells. In both cell systems, Pg 1 and Pg 2 bound to an equivalent number of receptors, apparently representing the same population of surface molecules. The affinity for Pg 2 was slightly higher. With hepatocytes, the KD for Pg 1 was 3.2 +/- 0.2 microM, and the KD for Pg 2 was 1.9 +/- 0.1 microM, as determined from Scatchard transformations of the binding isotherms. The Bmax was approximately the same for both isozymes. With C6 cells, the KD for Pg 1 was 2.2 +/- 0.1 microM vs. 1.5 +/- 0.2 microM for Pg 2. Again, the Bmax was similar with both isozymes. 125I-Pg 1 and 125I-Pg 2 were displaced from specific binding sites by either nonradiolabeled isozyme. The KI for Pg 2 was slightly lower than the KI for Pg 1 with hepatocytes (0.9 vs. 1.3 microM) and with C6 cells (0.6 vs. 1.1 microM). No displacement was detected with miniplasminogen at concentrations up to 5.0 microM. Activation of Pg 1 and Pg 2 by recombinant two-chain tissue-plasminogen activator (rt-PA) was enhanced by hepatocyte cultures. The enhancing effect was greater with Pg 2. Hepatocyte cultures did not affect the activation of miniplasminogen by rt-PA or the activation of plasminogen by streptokinase. Unlike the hepatocytes, C6 cells did not enhance the activation of plasminogen by rt-PA or streptokinase; however, plasmin generated in the presence of C6 cells reacted less readily with alpha 2-antiplasmin.

Fibrinogenolytic and fibrinolytic activity of cell-associated plasmin.

“…24 In the present investigation, we confirmed these results. C6 cells were incubated with 0.1 juM plasmin, washed, and then incubated with S-2251 and a 2 AP simultaneously for 30 minutes.…”

“…19 Each preparation included a mixture of carbohydrate variants I and II (60-70% type II). 24 Plasminogen (100-200 fig) was activated with 50 units of u-PA for 15 minutes at 37°C. Streptokinase was purified from Kabikinase (KabiVitrum) by the method of Castellino et al 25 Fibrinogen was depleted of plasminogen by chromatography on lysine-Sepharose.…”

“…- 24 In previous studies of plasminogen binding affinity, capacity, dissociation rate constant, and reactivity of bound plasmin with proteinase inhibitors, C6 cells have behaved equivalently to these other cell types. The greatly decreased activity of plasmin toward fibrinogen when the former is bound to C6 cells or HUVECs represents another equivalence that is demonstrated here.…”

Cell-surface Cytokeratin 8 Is the Major Plasminogen Receptor on Breast Cancer Cells and Is Required for the Accelerated Activation of Cell-associated Plasminogen by Tissue-type Plasminogen Activator

“…Unlabeled plasminogen and ⑀ACA are equally effective at displacing 125 I-plasminogen from specific cellular binding sites (12,13). At the conclusion of an incubation, the cultures were washed three times.…”

“…Plasminogen is activated more readily by u-PA and t-PA when both enzyme and substrate are bound to the surfaces of certain cell types (6,(12)(13)(14). Plasmin that is cell-associated reacts very slowly with ␣ 2 -antiplasmin and ␣ 2 -macroglobulin, which are rapid inhibitors of solution-phase plasmin (15,16).…”