Current status of activity assays for tissue plasminogen activator.

Tissue plasminogen activator (TPA) is a serine protease involved in the fibrinolytic system that dissolves blood clots. The enzyme catalyzes the conversion of a zymogen, plasminogen, to the enzymatically active form, plasmin, by limited proteolysis. In the course of searching for specific activity assays that might be useful in monitoring the purification of TPA, we have developed several coupled photometric assays. In addition, radioactive, agarose-plate, and other activity assays have also been considered and investigated for this purpose (Table 1). We have previously reported the one-step photometric procedure consisting of a thioester, thiobenzyl benzyloxycarbonyl-lysine (Z-Lys-S-Bzl) and fibrinogen-coated plates. It is simpler and more sensitive than the old two-step method without using immobilized fibrinogen. The new assay has been used successfully for protein purification and can be easily adapted to automated processes. Recently, other chromogenic substrates, D-Val-L-Leu-L-Lys-p-nitroanilide (Val-Leu-Lys-pNA) and D-Ile-L-Pro-L-Arg-p-nitroanilide (Ile-Pro-Arg-pNA) were also used in the one-step assay. It is found that TPA activity is greatly enhanced by immobilized fibrinogen and free fibrinogen when either the thioester. Val-Leu-Lys-pNA, or Ile-Pro-Arg-pNA were used in the colorimetric assay (Fig. 1, A-D). Enzyme kinetics studies indicate that the Km for plasminogen assayed on the thioester and fibrinogen-coated plates is 1.5 micrograms per ml, which is substantially lower than that observed in untreated plates (4.8 micrograms per ml). This is not due to the effect of fibrinogen on the second step of the coupled photometric assay because there is no change in the plasmin activity under these conditions (Fig. 1E). Similar results in TPA activation have also been observed, when fibrin-coated plates were used. Free fibrinogen, which is an activator of TPA, has been included in the standard assay mixture. We are able to detect less than 1 ng of TPA activity within a one-hour incubation time at 20 degrees C (Fig. 2A). In the thioester assay, however, high concentrations of reducing agents and nonspecific proteins cause significant background due to the interaction of DTNB with these reagents. 125I-labeled fibrin-coated plates had been extensively used in the past for urokinase and TPA assays. Although the sensitivity of the radioactive procedure is equivalent to that of the thioester photometric method, it appears that the kinetics of the enzyme are not easy to follow nor is the reproducibility great.(ABSTRACT TRUNCATED AT 400 WORDS)