A precise and rapid microtitre plate clot lysis assay: methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis.

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 to influence 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 and 0.1 s-1, respectively, in excellent agreement with published values. The assay should therefore prove useful in quantitative evaluations of 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The determination of recombinant human tissue-type plasminogen activator activity by turbidimetry using a microcentrifugal analyzer.

A method is described for determining the activity of recombinant human tissue-type plasminogen activator (rt-PA) by turbidimetry using a microcentrifugal analyzer (MCA). A mixture of thrombin and rt-PA is centrifuged into a mixture of fibrinogen and plasminogen to initiate clot formation and subsequent clot dissolution. The resultant profile of absorbance versus time is analyzed to determine the assay endpoint. Different rt-PA assay concentration ranges were studied in conjunction with profile endpoints for assay optimization. Spiked placebo recovery studies were used to evaluate assay accuracy and precision, which were determined to be 99.5 and 5% (relative standard deviation or RSD), respectively. Assay sensitivity was 0.5 ng/ml. Typical analysis time, including calculations, for a standard curve plus 14 samples was less than 30 min. The application of turbidimetry with the MCA for determining rt-PA activity provides rapid sample analysis and high throughput while maintaining accuracy and precision.