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

Abstract

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.

Plasminogen activator inhibitor (PAI‐1) in plasma and platelets

“…Equally it can be applied to high PAI-1 concentrations, the recovery of purified PAI-1 added to PAI-1-depleted plasma being complete over the entire range tested (1.5-500 ng/ml). This assay has a lower detection limit than the published radioimmunoassays for PAI-1 (Kruithof et al, 1987: Urden et al, 1987 and compares well with the recently published ELISA of Declerk et a1 (1 988). The mean PAI-1 antigen concentration in citrated plasma from 20 normal males aged 20-23 was 21.0f 7.2 ng/ml, in good agreement with the data of Declerk et a1 (1988).…”

“…It is notable that the range of PAL1 antigen in normal plasma was much less broad than that of PA1 activity, which varied from zero to 4.8 units/ml. Other workers have also observed a similarly wide range of activity in normal subjects (Chmielewska et a!, 1983: Kruithof et al, 1987; in the latter study a wide range of PAI-1 antigen was also noted.…”

“…The PAI-I is in the euglobulin was shown by zymography to consist both of free and complexed material, while all the PAL1 in the supernatant was free. Thus in studies where both ELT and PAL1 are being measured good correlations between these measurements age and plasma PAI-1 has been reported {Hashimoto et Kruithof et al, 1987). The time of day at which these samples were taken (2 pm.)…”

Differential growth state‐dependent regulation of plasminogen activator inhibitor type‐1 expression in senescent IMR‐90 human diploid fibroblasts

“…Only specific isolates (e.g., clones 13-5A and 13-2A) which exhibit significantly increased SAP fraction PAI-1 protein accumulations have actually reached the point of replicative senescence. At the cellular level, such increases in PAI-1 expression are reminiscent of the augmented serum PAI-1 activity typically seen in elderly individuals Kruithof et al, 1987;Aillaud et al, 1986). The in vivo significance of PAI-1 overexpression is not known, but an altered fibrinolytic balance potentially underlies a variety of age-associated pathologies associated with inbalances in ECM deposition and function.…”

Plasminogen Activator Inhibitor‐1: Physiologic Role, Regulation, and the Influence of Common Pharmacologic Agents

“…10 PAI-1 is produced by the vascular endothelium but is also present in vascular smooth muscle, platelets, and the liver. 11,12 When present in active form (i.e., the form synthesized in vascular endothelial cells and platelets), PAI-1 rapidly binds to endogenous thrombolytic molecules in a 1:1 fashion, thereby forming a stable complex that is eliminated hepatically. 13,14…”