Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis

The subcellular localization of plasminogen activator (PA) in human neutrophils was studied. The cells were disrupted by nitrogen cavitation and fractionated on Percoll density gradients into three major components containing the plasma membranes, the specific granules, and the azurophilic granules. The biochemical markers we used to identify these organelles were alkaline phosphatase, vitamin B12- binding protein, and beta-glucuronidase, respectively. Using the radioactive fibrin plate method, PA activity and plasminogen- independent fibrinolytic activity were measured. In resting neutrophils, PA was associated mainly with the membranes of the specific granules. In five individual experiments the activity of this fraction varied from 79 to 100% of the total; the remaining activity was found to be associated with the plasma membrane, and no activity was present in the azurophilic granules. In neutrophils that were activated by exposure to PMA (20 ng/ml for 15 min at 37 degrees C), the total recoverable PA activity remained unchanged; however, the main peak of activity (85% of total) shifted from the specific granules to the plasma membranes. The magnitude of the reduction of the enzyme in the specific granules paralleled that of vitamin B12-binding protein. PMA-activated, intact neutrophils had approximately 12-fold more surface-bound PA activity than resting cells. Recovery of PA activity from neutrophils was critically dependent on pretreatment of the intact cells with DFP before cavitation; 100-fold more PA activity was detected in DFP-pretreated cells. At the same time, this pretreatment reduced the plasminogen-independent fibrinolytic activity by approximately sevenfold. We determined that PA present in the neutrophils is of the urokinase (UK) type and that the enzyme is produced and stored as a pro-UK, a form insensitive to DFP inhibition. The reduction in the level of proteases (measured as fibrinolytic activity) and the resistance of pro-UK to DFP are most likely the two major reasons for the greatly improved recovery of PA from the DFP- pretreated cells. These findings show that in resting neutrophils PA is stored in the specific granules, and that during activation, it translocates to the outer surface of the plasma membranes, thus equipping the cell with an ecto-proteolytic potential.