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Current liquid-phase methods for thedetermination of tPA in plasma involve inactivation of both tPA and plasmin inhibitors by either plasma acidificationor euglobulin fractionation. In a firststep, our liquid-phase assay was performed in euglobulin fractions (EF) and the inactivation of PAI was monitored.In a second step, we studied the influenceof PAI onthe tPA assay in plasma.When EF were used (1:10 dilution, pH 5.9), the reference curve was done with tPA (0 to 1 IU/ml) diluted in buffer.The diluted samples (tPA or EF) were incubated with fibrinogen fragments as Stimulator (0.5 mg/ml), an excess of plasminogen (0.15 pM/ml) and chromogenic substrate CBS 30.41 (3.75 pM/ml).The tPA level of EF obtained after venous stasis was found tobe2.7 IU/ml, whatever the dilution was (1:4 to 1:64). For EF obtained at rest, a paradoxical increase in tPA activity was found from 1:4 to 1:16 until a plateau was reached (usually ar dilution 1:16 to 1:32).This is due to the partial dissociation of tPA/PAI complexes which increases with the dilution as demonstrated by adding tPA to highly diluted (1:64) EF : noinhibition occurred. To avoid the influence of PAI and to measure the total tPA activity, a sufficient dilution was needed.When the tPA assay was performed in plasma, ∝ 2 antiplasmin (∝ 2AP) was shown to be mainly responsiblefor a gradual loss of tPA and for inhibition of plasmin generation (using anti ∝2AP IgG). To maintain the concentration of ∝2AP constant in samples, they were 1:10 diluted in a tPA/PAI depleted plasma (plasmin inhibitors were brought in excess by the depleted plasma). The reference curve was done with tPA (0 to2 IU/ml) diluted in the depleted plasma instead of buffer. TPA activities were shown in plasma obtained after venous stasis, when low PAI values (0 to 2.5 IU/ml) were associated. No tPA activity was found in plasma obtained at rest, associated with normal PAI values. It can be concluded that a tPA activity is displayed only when PAI is saturated by released tPA.A free tPA activity can be detected in plasma, while in the EF, the tPA measured is dissociated from tPA/PAI complexes.

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