The cofactor activity of injured cells leads to their proteolytic degradation. (A) tPA (50 nM), plasminogen (50 nM), or tPA (0.5 nM) + plasminogen (50 nM) were added to uninjured (37°C incubated) or injured (56°C incubated) THP-1 cells for 15 minutes. Cells were then washed and cellular proteins harvested and subjected to immunoblot analysis for tPA, plasmin(ogen), and β-tubulin. Although viable THP-1 cells are known to possess cell-surface plasminogen-activating cofactors,³⁸  injuring these cells substantially increases both tPA-binding and the rate of plasminogen generation. This result corroborates the notion that dead cells are a potent plasminogen-activating cofactor. The increased binding of plasmin to nonviable cells is presumably due to a positive feedback loop where plasmin-mediated proteolysis of nonviable cells reveals more lysine residues (a phenomenon seen also on fibrin). In addition, plasmin activity leads to a loss of β-tubulin signal only in nonviable cells, presumably because the intact plasma membrane of viable cells prevents plasmin-mediated degradation of intracellular tubulin. (B) THP-1 cells treated at 56°C were incubated with 100 nM tPA, 100 nM plasminogen, or 1 nM tPA + 100 nM plasminogen for 15 minutes. Cells were then washed and incubated at room temperature. Cells were then assessed by flow cytometry 24 hours later, with forward scatter taken as a measure of cell size. Histograms show that only tPA + plasminogen–treated cells become significantly smaller, presumably as a result of proteolytic degradation (supplemental Figure 5).