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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

Abstract

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.

The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Introduction

Diseases fundamentally stemming from thrombo-embolic etiologies present a major source of morbidity and mortality in present-day society. Manifestations of thrombo-embolic pathogenesis include, but are not limited to, myocardial infarctions, ischemic strokes, deep venous thromboses, and pulmonary emboli1. A tremendous amount of ongoing research, spanning multiple disciplines, revolves around the development of safe and effective methods for dealing with pathogenic thrombosis. Variations in arterial and venous manifestations of thrombosis and varying anatomic locations have resulted in the development of different treatment approaches. However, ....

Protocol

All methods mentioned below are in accordance with institutional review board (IRB) protocols and the institutional human research ethics committee. All healthy volunteers provided written and informed consent prior to blood donation. Of note, all materials referenced within the protocol can be found in the Table of Materials. While human WB and plasma are discussed throughout this protocol, the use of research animal blood and factor-depleted blood products can be purchased and substituted.

Representative Results

Chandler loop clot formation
In forming clots, we generally aimed for quadruplicates to ensure that if any clot outliers (based on gross morphology and mass) existed, we still had the ability to run triplicate thrombolysis assays. Assuming optimal loading conditions, clots should all be quite uniform in length (~3.3 cm), weight (~100 mg), and appearance as is represented in Figure 3. When employing FITC-Fg, we also aimed to examine clots under UV light to ensure relati.......

Discussion

Clot formation and labeling
The Chandler loop has been demonstrated to provide an easy and effective means of reproducibly generating clots that mimic in-vivo thrombi16. Fine-tuning parameters such as tubing size, rotational speeds, drum diameter, and clotting time allow for the rapid generation of clots under differing shear conditions that can capture architectural features appreciated in a range of thrombi mimicking both arterial and venous sources. The additiona.......

Acknowledgements

Research reported in this publication was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R01HL167877. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Materials

NameCompanyCatalog NumberComments
30 G Disposable Hypodermic NeedlesExel International 26439Other Consumables
6 mm HSS Lathe Bar Stock Tool 150 mm LonguxcellB07SXGSQ82Chandler loop, 
96-Well Clear Flat Bottom UV-Transparent MicroplateCorning3635Other Consumables, Non-treated acrylic copolymer, non-sterile
Air-Tite Luer-lock Unsterile 60 mL SyringesAir-TiteMLB3RT-FluFF Apparatus , dampeners
Arium Mini Plus Ultrapure Water SystemSartoriusNADI water source
Calcium ChlorideMillipore SigmaC5670Other Consumables
Disposable BP TransducersAD InstrumentsMLT0670RT-FluFF Apparatus
Drager Siemans HemoMed PodDrager5588822RT-FluFF Apparatus
Drager Siemans Patient MonitorDragerSC 7000RT-FluFF Apparatus
Drum (cylinder, diameter 120 mm, width 85 mm)Chandler loop,
Face ShieldMoxeSHIELDS10Chandler loop, 
Fibrinogen From Human Plasma, Alexa Fluor 488 ConjugateThermo ScientificF13191Other Consumables
Fitting, Polycarbonate, Four-Way Stopcock, Male Luer Lock, Non-SterileMasterflex30600-04RT-FluFF Apparatus
Fluorescein (FITC)Thermo Scientific119245000Other Consumables
General-Purpose Water BathThermo Scientific2839Chandler loop, 
Hotplate 4 × 4Fisher Scientific1152016HRT-FluFF Apparatus
Human Source Plasma Fresh-FrozenZen-BioSER-SPLOther Consumables, CPDA-1 anticoagulant
Human Whole Blood Zen-BioSER-WB-SDS Other Consumables, CPDA-1 anticoagulant
L/S Easy-Load II Pump Head for High-Performance Precision Tubing, PPS Housing, SS RotorMasterflex77200-62RT-FluFF Apparatus, Pump Head
L/S Variable-Speed Digital Drive Pump with Remote I/O, 6 to 600 rpm; 90 to 260 VACMasterflex7528-10RT-FluFF Apparatus, Pump
Motor Speed ControllerCoCocinaZK-MGChandler loop, 
Nalgene Tubing T-Type ConnectorsThermo Scientific6151-0312RT-FluFF Apparatus
Peristaltic pump tubing Masterflex06424-15 Other Consumables
Phosphate buffered salineMillipore SigmaP3813Other Consumables, Powder, pH 7.4, for preparing 1 L solutions
SpectraMax M5 multi-detection microplate reader system (or other fluorescence detection)Molecular DevicesM5RT-FluFF Apparatus
Switching Power SupplySoulBayUC03UChandler loop, 
Thermo Scientific National Target All-Plastic Disposable Syringes 10 mLThermo ScientificS751010Other Consumables
Tissue plasminogen activator, humanMillipore SigmaT0831Other Consumables
Tubing ID 1/4'', OD 3/8''Fisher ScientificAGL00017Other Consumables, cut into 1.5cm sections use to connect tubing to T-type connectors
Tubing ID 5/32", OD 7/32"TygonND-100-65, ADF 00009 Other Consumables
V3 365 nm Mini - Black Light UV FlashlightuvBeastuvB-V3-365-MINIChandler loop, used to check completed clots
ZGA37RG ZYTD520 DC Motor, 12 V, 100 rpmPangyooZGA37RGChandler loop, 

References

  1. Ali, M. R., et al. Aspect of thrombolytic therapy: a review. Scientific World Journal. 2014, 586510 (2014).
  2. Bhogal, P., Andersson, T., Maus, V., Mpotsaris, A., Yeo, L. Mechanical thrombectomy-A brief review of a re....

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