Comparison of affinities of urokinase and tissue plasminogen activator for fibrin clots

Abstract

Affinities of low molecular weight two-chain urokinase (UK) and tissue plasminogen activator (t-PA) for fibrin clots were investigated by using clot lysis rates to estimate an affinity (K d) between activator and fibrin clots. Lysis rates were obtained using a simple spectrophotometric based clot lysis assay which is described here. Fibrin clots, containing residual plasminogen, were suspended in a 1 ml cuvette and the increase in absorbance at 280 nm due to release of soluble fibrin peptides measured over a 150 to 250 minute time period. Lysis rates were obtained from plots of time squared vs absorbance change. Plots of activator concentration vs reciprocal rates yielded regression coefficients of 0.999 and K d values of nM for the affinity of both activators for fibrin clots. Although both activators are known to differ in affinity for fibrin, they nonetheless had similar affinities and lysis rates for the insoluble fibrin clots. This assay also suggested possible synergism; rates over twice that expected by an additive effect were observed when the two activators were mixed at 0.3 to 7.6 nM each.