Binding of tPA to VSMCs determined by functional assay. VSMCs grown to confluency in 24-well tissue culture plates were preincubated with varying concentrations of tPA. The cells were than washed and plasmin generation was determined after the addition of plasminogen (200 nmol/L) and H-D-Val-Leu-Lys-AMC (0.2 mmol/L). Data are shown for plasmin generation assayed in the absence (▪) and presence (•) of 6AHA (1 mmol/L) and represent the mean ± SD (n = 4). The solid lines in both the upper and lower panels show the best fit of binding models to the data. In the presence of 6AHA, the best fit is to a single-site model; in the absence of 6AHA, the best fit is to a two-site model. The latter is highlighted in the lower panel, in which the data are plotted semilogarithmically. The single-site (- - - - -) and two-site (─) fits for data in the absence of 6AHA are shown; in the upper part of the panel, the residuals, ie, the differences between the measured and theoretical values, for each model are shown. Analysis of parameter dependencies, sum of squared residuals, and extended runs of positive and negative residuals all indicate the goodness of fit of the two-site model. In the presence of 6AHA, both models give identical binding constants and the fitted curves are therefore superimposed. The binding constants derived from these data are shown in Table 2.