|Title:||Physiologically-Relevant in vitro Platform for Testing Thrombolytic Therapies|
Christodoulides, Alexei, Department of Emergency Medicine, Indiana University School of Medicine; Ziqian Zeng, Department of Emergency Medicine, Indiana University School of Medicine; Dr. Nathan J. Alves, Department of Emergency Medicine, Indiana University School of Medicine
Background/Significance/Rationale: A great need exists for the development of a more representative in-vitro model to efficiently screen novel thrombolytic therapies.
Methods: We herein report the design, validation and characterization of a highly reproducible, physiological scale, flowing clot lysis platform with real-time fibrinolysis monitoring to screen thrombolytic drugs utilizing a FITC-labeled clot analog.
Results/Findings: Using this Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF Assay), a tPa-dependent degree of thrombolysis was observed both via clot mass loss as well as fluorometrically monitored release of FITC-labeled fibrin degradation products. Percent clot mass loss ranged from 33.6% to 85.9% with fluorescence release rates of 0.53 to 1.17 RFU/min in 40 and 1,000 ng/mL tPa conditions, respectively. The platform can be easily adapted to produce pulsatile flows. Hemodynamics of human main pulmonary artery (MPA) is mimicked through matching dimensionless flow parameters calculated using clinical data. Increasing pressure amplitude (4 to 40 mmHg) result in an 20% increase of fibrinolysis at 1000 ng/mL tPA. Increasing shear flow rate (205 s-1 to 913 s-1) result in significantly increased fibrinolysis and mechanical digestion.
Conclusions/Discussion: The proposed in-vitro clot model offers real-time digestion monitoring and represents a versatile testing platform for thrombolytic drug screening.
Translational/Human Health Impact: Numerous present platforms for testing novel thrombolytics fail to capture true in-vivo like conditions and the reliance on animal models for translational work often proves cumbersome, expensive, and presents numerous challenges for fine-tuning parameters such as drug delivery or flow dynamics. Our platform aims to serve as a stepping-stone from which novel thrombolytic therapies can be developed and translated into humans in a much more cost-efficient and tuneable manner.