A NOVEL MICROTITER PLATE ASSAY FOR FUNCTIONAL PLASMINOGEN-ACTIVA-TOR INHIBITOR (PAI) LEVELS IN WHOLE PLASMA

Currently, the most widely used functional assay for PAI levels in plasma is that developed by Chmielewska et al (Thromb. Res. 31:427-436, 1983). A major drawback of this assay is the considerable manipulation of plasma samples required by the necessity of acidification and reneutralization in order to determine remaining activator levels based on plasminogen to plasmin conversion. We have devised an assay based on microtiter plates coated with 20 U/ml urokinase. In this assay, aliquots of citrate- or EDTA-anticoagulated plasma are incubated in the coated wells for 30 min at room temperature to allow PAI in the plasma to complex with the solid state urokinase. The plasma is then removed, the wells extensively washed with Tris-saline buffer plus 1 mg/ml bovine serum albumin, and the remaining urokinase activity determined by adding plasminogen and S-2251 chroma-genic substrate for plasmin. Inhibitor levels can be conveniently expressed as percent inhibition relative to the urokinase activity in wells not exposed to plasma. Absolute units of inhibitor have also been derived by titration of plasma samples with known units of urokinase (calibrated by S-2444 chromagenic substrate for plasminogen activators). Between the range of 90% to 10% inhibition, a linear slope of 0.69±0.6 units ml-1/10% inhibition is obtained.In agreement with previous reports, we find a wide variation in control subjects and some tendency towards higher PAI levels in older age groups. Age group 20-39, n=29, 36.6 ± 14.9%. Age group 40 and over, n=16, 48.9 ±12.9%.Reproducible values (SEM ± 4.6%,n=5)are obtained for control* individuals sampled and assayed at various times over a period of several months. We have also assayed plasma samples from patients undergoing a variety of surgical procedures, and have followed rapid post-surgical increases in PAI levels with a gradual return to pre-operative levels. Evidence is presented that the post-surgical increase is predominantly from the platelet releasable pool.