Sensitive and specific detection of PAI-2 in body fluids was achieved by the use of a new zymographic system, where samples were separated by polyacrylamide gel electrophoresis (PAGE) without SDS. PAI-2 in pregnancy plasma separated on PAGE into two distinct bands. Fast PAI-2, the major form, was detectable from 6 weeks of pregnancy to 8 days post-delivery. The weaker, slow form was detectable from 9 weeks of pregnancy until 6 days after delivery. The fast and slow forms of PAI-2 correspond respectively to the 75 kDa and 130 kDa PAI-2 forms detected by SDS-PAGE, previously reported. Fluid aspirated from both malignant and benign ovarian cysts contained PAI-2, primarily in its fast form; the slow form was additionally detected in 2 of 8 samples studied. The fast form of PAI-2 was detectable in the plasma of 1 subject with a malignant cyst. Normal Graafian follicle fluid contained no PAI-2. PAI-2, formerly regarded as a pregnancy-associated protein, is detected also in non-pregnant women with pathological ovarian cysts.

During normal pregnancy maternal haemostasis alters to protect against bleeding with a rise in plasma clotting factor levels and increased inhibition of fibrinolysis. The latter is due in part to increased levels of the 48 kDa plasminogen activator inhibitor (PAI-1) present in the plasma of non-pregnant individuals. A second plasminogen activator inhibitor (PAI-2) occurs in placenta and in a cultured histiocytic lymphoma cell line. We report here the identification by SDS-PAGE and zymography of PAI-2 in plasma during normal pregnancy. PAI-2 was present in two molecular forms of about 75 and 130 kDa, which were detectable at 12 weeks gestation and which persisted in the maternal circulation for up to 7 days after delivery. These forms of PAI-2 appear to be distinct from purified PAI-2, which has a molecular mass of 47-60 kDa and which is not normally detectable in this zymographic system, since it is sensitive to denaturants. The novel forms of PAI-2 may represent complexes or aggregates that retain activity after SDS-PAGE.

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.