Interference of an activity assay of tissue-type plasminogen activator in human plasma by endogenous factors.

Abstract

The estimation of the activity of circulating tissue-type plasminogen activator (t-PA) using the Coaset t-PA or Spectrolyse/fibrin t-PA reagent kits involves incubation of plasma with excess plasminogen in the presence of human fibrin fragments, and detection of the plasmin generated by an end-point amidolytic assay. We examined the interference of endogenous inhibitors such as plasminogen activator inhibitor-1 (PAI-1) and alpha 2-antiplasmin, and that of an endogenous activator namely single-chain urokinase-type plasminogen activator (scu-PA) on the Coaset t-PA assay. An extra acidification of the samples already collected into an acidified anticoagulant raised the mean Coaset t-PA activity of 15 samples from 1.39 +/- 0.25 (mean +/- SEM) to 1.71 +/- 0.27 (P < 0.001). In twelve of these samples, the PAI-1 and alpha 2-antiplasmin activities were determined. The increase in the t-PA activity by acidification was found to correlate inversely with PAI-1 (r = -0.66; n = 12; P = 0.019) but not with alpha 2-antiplasmin, demonstrating that the extra acidification step released t-PA from the t-PA-PAI-1 complex, but had no influence on the endogenous alpha 2-antiplasmin in the assay system. Incubation with monoclonal antibody against alpha 2-antiplasmin increased the Coaset t-PA activity in a concentration-dependent manner, linearly up to 2 micrograms/ml (final) of the antibody, irrespective of the extra acidification, the maximal rise being 41.5% and 19.7% in an in-house and commercial plasma pools respectively. In contrast to alpha 2-antiplasmin antibody, monoclonal antibody against scu-PA decreased the Coaset t-PA activity in the in-house and commercial plasma pools again in a concentration-related manner demonstrating that scu-PA in addition to t-PA was being measured by this method. Incubation with an irrelevant antibody (anti-DNA monoclonal antibody) did not affect the Coaset t-PA values. The mean Coaset t-PA activity of 40 subjects decreased from 1.76 +/- 0.10 (mean +/- SEM) to 0.73 +/- 0.07 when incubated with 8 micrograms/ml (final) monoclonal antibody against scu-PA (P < 0.001). The 't-PA' activity in the absence of scu-PA antibody correlated with the u-PA antigen (r = 0.53; n = 40; P < 0.001), and the activity in the presence of scu-PA antibody correlated with the t-PA activity measured by the bio-functional immunosorbent assay (r = 0.86; n = 40; P < 0.001). Hence, unless the Coaset t-PA activity is measured with appropriate amounts of antibodies to alpha 2-antiplasmin and scu-PA, it is best to qualify the activity measured by this method as being that of total plasminogen activators.