OC 17.5 - A Multichannel Microfluidic chemiluminescent Plasmin Generation Assay to asses fibrinolytic capacity in plasma

Background: Plasmin is the central protease of fibrinolysis, responsible for cleaving fibrin. Plasmin generation (PG) assays can be useful to diagnose abnormalities in fibrinolysis and monitor the effect of treatment with anti-fibrinolytic agents. By using a chemiluminescent-based substrate, the assay can be incorporated in a miniaturized microfluidic platform.

Aims: To develop a chemiluminescent-based Plasmin Generation Assay (PGAlum) on a microfluidic cartridge.

Methods: The assay uses a novel plasmin-specific substrate consisting of a tripeptide conjugated to an aminoluciferin moiety. Upon activation of plasmin, this substrate is cleaved, releasing the aminoluciferin, which is subsequently converted to a light quant by luciferase. PG was induced in tPA spiked recalcified plasma with tissue-factor initiated fibrin formation. Initial tests were performed in a 96-well plate format to determine the specificity and sensitivity to thrombomodulin activated TAFI, tranexamic acid (TXA), and α2-antiplasmin. Finally, the assay reagents were printed on a multichannel microfluidic cartridge. After the addition of plasma, the generated photons were detected with Single Photon Avalanche Diode sensors.

Results: The PGAlum was completely tPA dependent in normal pooled plasma activated with tissue-factor. The addition of 0.5 nM thrombomodulin inhibited plasmin generation by activated TAFI, as was confirmed by carboxypeptidase inhibitor. An inhibitor effect was also observed by the addition of 10 µM TXA. Furthermore, 2.7 µM α2-antiplasmin showed complete inhibition of the PG curve, while in α2-antiplasmin deficient plasma PG was accelerated. Results obtained with a microfluidic cartridge using only 110 nL demonstrated plasmin generation curves compared to the experiments performed in 60 µL in 96-well plates.

Conclusion(s): A chemiluminescent-based PGA sensitive for antifibrinolytics was developed which also can be performed in a microfluidic cartridge in a total volume of 110 nL. Together with other specific or global assays, this PGAlum can be implemented on the multichannel cartridge to provide a more complete overview of the hemostatic status.