The production of plasminogen activator (PA) by the vascular endothelium has been implicated in the maintenance of vessel patency. Cultured bovine aortic endothelial (BAE) cells were employed to study and compare the cell associated and secreted forms of PA. Samples were fractionated by polyacrylamide gel electrophoresis in the presence of SDS. PA activity in the gel was localized by the fibrin-overlay technique. Cellular PAs were found to be membrane associated and to consist of a major form of Mr 48,000 (C48) and minor forms of 53,000 (C53), 74,000 (C74), and 100,000 (C100). Incubation of the cell extracts at 37°C resulted in the appearance of two additional forms of Mr 41,000 and 33,000 suggesting that these forms were degradation products. Serum-free conditioned medium (CM) contained secreted PAs of Mr 52,000 (S52), 55,000 (S55), 74,000 (S74) and 100,000 (S100). In addition, a broad zone of fibrinolytic activity was observed in the region between Mr 80,000 and 95,000. Cellular PAs have isoelectric points of pH 8.5-8.6 and 7.5 while secreted PAs demonstrate activity at pH 8.6, 8.5, 8.0, and 7.5. The forms showed differential sensitivities to DFP with S74 and C74 being inactivated by 1mM DFP within one Hr at 37°C while S52 and C48 were still partially active after treatment with 10mM for two Hrs. S100 was completely retractile to treatment with 40 mM DFP. The addition of fresh serum to confluent cultures resulted in the disappearance of C48 and C74, and of S52 and S74, but caused an increase in C100 and S100. These studies indicate that several forms of PA are produced by endothelial cells and suggest that the production of each may be independently regulated.
Cell Communication in a Coculture System Consisting of Outgrowth Endothelial Cells and Primary Osteoblasts
