Neutralization of Human Extrinsic (Tissue-Type) Plasminogen Activator in Human Plasma: No Evidence for a Specific Inhibitor

Abstract

Human extrinsic plasminogen activator (EPA), highly purified from a melanoma cell culture fluid is inactivated in human plasma with a half-life (t ½) of 90–105 min. Gel filtration on Ultrogel AcA 34 of mixtures of ¹²⁵I-labeled EPA and human plasma, incubated at 37°C, revealed the progressive formation of two radioactive components, one with an apparent M_r of 150,000 and one eluting at the void volume. The component with an M_r of 150,000 was identified as consisting at least in part of EPA-α₂-antiplasmin complex since: 1) it reacted with antibodies against α₂-antiplasmin, but not with antibodies against the other known plasma protease inhibitors, and 2) formation of this component was strongly reduced in plasma specifically depleted in α₂-antiplasmin or when the active site of EPA was blocked. The component eluting at the void volume was identified as consisting at least in part of EPA-α₂-macroglobulin complex since: 1) it only reacted with antibodies against these two proteins and 2) was not formed in plasma depleted in α₂-macroglobulin or when the active site of EPA was blocked. In purified systems α₂-antiplasmin inhibited one-chain EPA with a rate constant of 60 M^-1s^-1 and two-chain EPA with a rate constant of 130 M^-1s^-1, which corresponds to a t ½ in plasma of 180 min or 90 min, respectively. α₂-Macroglobulin inhibited one-chain EPA with a rate constant of 15 M^-1s^-1 and two-chain EPA with a rate constant of 30 M^-1s^-1, which corresponds to a t ½ plasma of 4 or 2 hrs. All these findings taken together indicate that EPA is slowly neutralized in human plasma primarily by α₂-antiplasmin and to a lesser extent by α₂-macroglobulin. There appears to be no specific inhibitor in human plasma, which would inactivate EPA either rapidly or to a significant extent.