Summary
A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader
and personal computer is described in detail. The use of such widely available instrumentation,
the convenience and rapid throughput suggest the assay could be developed as a reference
method with which to measure the potency of tissue plasminogen activator (t-PA) in
conjunction with the WHO reference preparation. The method has been used to investigate
molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does
not appear toinfluence the mechanism of plasminogen activation and clot lysis by plasmin.
High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in
a change in the lysis profile of turbidity versus time. This is probably the result
of plasminogen binding to fibrin and consequent restriction of the access of plasmin
to its sites of cleavage in the fibrin. A simple model is proposed, and equations
have been derived, for the kinetics of lysis which adequately describe the mechanism
and which are confirmed by experimental data. This model results in estimates of the
Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately
150 nM and0.1 s-1, respectively, in excellent agreement with published values The
assay should therefore prove useful in quantitative evaluationsof the molecular phenomena
occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen
by t-PA was confirmed. However, evidence was obtained that the lys-form binds more
tightly to fibrin by the same factor. This observation suggested that the appropriate
substrate in the kinetic model is fibrin-bound plasminogen. When the data were re-analysed
using published values ofthe affinities of the two forms, t-PA was found to activate
both forms indistinguishably on the fibrin surface, consistent with suggestions that
the conformation of glu-plasminogen changes to one more like that of lys-plasminogen
upon binding to fibrin.
