"Going with the flow" in modeling fibrinolysis

Abstract

The formation of thrombi is shaped by intravascular shear stress, influencing both fibrin architecture and the cellular composition which has downstream implications in terms of stability against mechanical and fibrinolytic forces. There have been many advancements in the development of models that incorporate flow rates akin to those found in vivo. Both thrombus formation and breakdown are simultaneous processes, the balance of which dictates the size, persistence and resolution of thrombi. Therefore, there is a requirement to have models which mimic the physiological shear experienced within the vasculature which in turn influences the fibrinolytic degradation of the thrombus. Here, we discuss various assays for fibrinolysis and importantly the development of novel models that incorporate physiological shear rates. These models are essential tools to untangle the molecular and cellular processes which govern fibrinolysis and can recreate the conditions within normal and diseased vessels to determine how these processes become perturbed in a pathophysiological setting. They also have utility to assess novel drug targets and antithrombotic drugs that influence thrombus stability.

Keywords: fibrin; fibrinolysis; flow; plasminogen; platelets; shear; thrombus.

Figure 1

Fibrinolytic assays and models. Turbidimetric clot lysis assays involve clotting plasma in 96 well plates with the addition of calcium and thrombin in the presence of plasminogen activators. Change in absorbance is then measured using a spectrophotometer, increasing as clots form and decreasing during fibrinolysis. The Euglobulin fraction containing plasminogen, plasminogen activators and fibrinogen can be isolated by precipitation on ice cold acetic acid and collected via centrifugation. The pellet is then washed and solubilized before clotting with thrombin and the fibrinolytic activity then measured by change in absorbance. The Halo assay requires whole blood which is clotted with tissue factor in a “Halo” around the outside of a well of a 96 well plate. Plasminogen activators are then added and the absorbance increases as the clot lyses and is released into the well. Viscoelastic measurements are made in whole blood with added plasminogen activator using an analyzer. Data is outputted as traces [TEG^® (Haemonetics Corporation) and ROTEM^® (Werfen)] or a dial output [Quantra^® (Hemosonics LLC)]. Chandler model thrombi are formed after recalcifying whole blood or plasma with added fluorescently labeled fibrinogen and enclosing in tubing under rotation. Thrombi are then removed and bathed in plasminogen activator and samples are taken at regular intervals and change in fluorescence release is measured as a marker of fibrin degradation. The Badimon chamber uses non-anticoagulated blood taken directly from the donor. Plasminogen activators and fluorescently labeled fibrinogen can be mixed with the blood ex vivo which then flows over thrombogenic tissue within the specialized perfusion chambers at predefined shear stresses. Markers of fibrinolysis can be measured from collected effluent. Additionally, thrombi formed on the thrombogenic surface can be removed and fibrinolysis monitored as with the Chandler model. Whole blood microfluidic models use syringe pumps to either pull or push blood over microfluidic biochips. These can be commercially sourced or prepared in house and can have different channel sizes and conformations. Thrombus formation is initiated on thrombogenic coatings within the biochips and viewed via fluorescence microscopy. Various markers and fluorescently labeled proteins can be incorporated pre or post thrombus formation. Fibrinolysis can be monitored as change in fluorescence signal over time and effluents can be collected to monitor markers of fibrinolysis. Figure created with BioRender.com.

Figure 2

Whole blood thrombi under flow. (A) Chandler model thrombi formed from citrated whole blood that was recalcified (10.9 mM CaCl₂). Thrombi were formed under rotation at a constant speed of 30 rpm for 90 min before being removed and washed in 0.9% saline. The platelet-rich white head is visible. (B) Recalcified whole blood thrombi were formed on a collagen (100 ng) and tissue factor (300 pM) surface in Cellix biochips. Thrombi formed under high shear (1,000 s⁻¹) are more platelet-rich with less fibrin than those formed at low shear (250 s⁻¹). DiOC6 (0.5 μg/ml) was included to label platelets (green) and fibrinogen-AF546 (75 μg/ml) added (red). Scale bars represent 10 μm.