A plasmin generation method for the determination of tissue plasminogen activator (t-PA) activity in blood

A plasmin generation method to determine tissue plasminogen activator (t-PA) activity in plasma is described. A protein solution of homogenized fibrin was used as a stimulator in the presence of plasminogen and the plasmin generated was measured by the release of para-Nitroanilide (p-NA) from the chromogenic substrate S-2251. Plasmin generation by 5 iu/mL t-PA in the presence of 1 CU/mL of plasminogen and 850 micrograms/mL of fibrin solution reaches a peak at about 5 h incubation whilst in plasma, plasmin generation peaks after about 16 h incubation. The highest t-PA activity in plasma was determined using an assay involving 18 h incubation. In the 21 subjects studied by this method the t-PA activity at rest ranged from 0.34 to 0.92 iu/mL with a mean of 0.57 +/- 0.15 iu/mL of plasma whilst in the post-occlusion state the activity ranged from 1.12 to 18.0 iu/mL, with a mean of 5.25 +/- 4.49 iu/mL of plasma. We also found that subjects who developed petechiae during occlusion had significantly higher t-PA activity both at pre- and post-occlusion when compared with those who did not develop petechiae. The t-PA activity of acid-treated plasma stored at -70 degrees C showed no significant changes in activity after 12 weeks of storage when compared with the t-PA activity of the same plasma tested prior to storage.