Plasminogen activator inhibitor 1: development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation

To study the effect of plasminogen activator inhibitors (PAI) on fibrinolysis it is essential to be able to specifically measure these proteins in plasma. To this end PAI-1 was purified from cortisol-stimulated HT 1080 fibrosarcoma cells and antisera raised in rabbits. The immunologic relationship of the purified inhibitor to PAI-1 in plasma and platelet extracts was established by immunoblotting and regular and reverse fibrin zymography. Furthermore, the purified product could be immunoprecipitated with antibodies to human or bovine endothelial cell-derived PAI-1. A radioimmunoassay was developed that measures both free and tissue-type plasminogen activator (t-PA)-bound PAI-1 in plasma and has an effective range of 8 to 250 ng/mL. PAI-1 antigen levels showed a twofold increase after 20 minutes of venous occlusion, partially due to hemoconcentration. Approximately one quarter of PAI-1 before and after venous occlusion is derived from platelets. After correction for hemoconcentration and the contribution of platelets to plasma PAI-1 levels, a still significant increase in PAI-1 levels was noted during venous occlusion, which suggests that the local vascular bed releases PAI-1. Concomitant with PAI-1, t-PA antigen levels increased eightfold and fibrinolytic activity 18-fold after 20 minutes of venous occlusion. PAI-1 and t-PA levels tend to augment with age: in a group of older healthy volunteers (mean age, 53 years) PAI-1 levels were twice and t-PA levels 1.7 times higher than those in a group with a mean age of 29 years. Determination of PAI-1 antigen levels before and after platelet aggregation demonstrated that 85% of PAI-1 in platelet-rich plasma is associated with platelets. The average amount of PAI-1 per platelet was 0.3 fg/platelet, ie, 4,000 molecules per platelet.