Summary
Two monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the
quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) antigen and activity
in plasma were constructed and validated. The intra-assay, interassay, and interdilution
coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA
and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA,
of either latent or active recombinant porcine PAI-1 (10 and 50 ng/ml) added to plasma
were 86 ± 9% and 92 ± 22%, respectively, for the latent form and 89 ± 9% and 87 ±
7% for the active form (mean ± SD, n = 3 to 4). In the immunofunctional assay, recoveries
for the same concentrations of active PAI-1 were 108 ± 16% and 92 ± 21%, respectively.
In male porcine plasma the level of PAI-1 antigen was 31 ± 11 ng/ ml and the activity,
34 ± 16 ng/ml (mean ± SD, n = 10). In female plasma PAI-1 antigen levels were 20 ±
5.2 ng/ml and the PAI-1 activity 42 ± 17 ng/ml (n = 13). A linear correlation was
found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r =
0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1
antigen or activity levels. Incubation of plasma samples at 37° C for 16 h resulted
in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37°
C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of
the female samples in the PAI-1 antigen assay increased two-fold.
In lysed platelet-rich plasma males had 990 ± 470 ng/ml antigen and 160 ± 80 ng/ml
activity and females, 920 ± 500 ng/ml antigen and 150 ± 98 ng/ml activity corresponding
to 2.1 ± 0.77 fg PAI-1 antigen per platelet. Only 16% of PAI-1 released from platelets
was found to be active. Linear correlations between PAI-1 antigen and activity were
found for both males (r = 0.61) and females (r = 0.67).
The assays are both sensitive and specific and may, therefore, aid the elucidation
of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis
and other thrombotic disorders.
Key words
Fibrinolysis - pig - PAI-1 - atherosclerosis - ELISA
