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

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Abstract
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 850g/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 034 to 092 iu/mL, with a mean of 057 015 iu/mL of plasma whilst in the post-occlusion state the activity ranged from 112 to 180 iu/mL, with a mean of 525 449 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°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.

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