Inhibition Of Human Tissue Plasminogen Activator By Human Plasma : No Evidence For A Specific Antiactivator

Human tissue plasminogen activator (TPA) was highly purified (one-chain or two-chain form) from the culture fluid of a melanoma cell line and labeled with 125 Gel filtration of mixtures of human plasma with trace amounts of labeled and 10 to 1,000 units/ml of unlabeled TPA revealed formation of two radioactive complexes with apparent Mr of 150,000 and 800,000, which were devoid of lytic activity of fibrin plates. These radioactive complexes were precipitated (90 and 63 percent) by antisera against α2 antiplasmin (α2AP) and α2-macroglobulin (α2M) respectively, but not by antisera against other known plasma protease inhibitors. In plasma specifically depleted in α2AP formation of the complex with Mr ≃ 150,000 did not occur, whereas removal of α2M abolished formation of the complex with Mr ≃ 800,000. The initial rates of formation of these two complexes in plasma were very similar to those obtained with mixtures of TPA and 1 μM α2AP or 3.5 μM α2M respectively. Complex formation was completely abolished by blocking the active site serine of TPA.In purified systems TPA was inhibited by α2AP with a rate constant of 140 M-1s-1 and by α2M with a rate constant of 30 M-1s-1. These rate constants correlate well with the rate of formation in plasma of the Mr ≃ 150,000 complex and the Mr ≃ 800,000 complex respectively.All these data indicate that TPA is slowly inhibited in human plasma by α2AP (tl/2 ≃ 60') and by α2M (tl/2 ≃ 120'). We found no evidence for the existence of another significant inhibitor in plasma. Both the one-chain and two-chain forms of TPA behaved very similarly in all experiments.