integrating fluorescently tagged fibrinogen into human blood clot for enhanced clot visualization

Combinando o Chandler Loop e o RT-FluFF para monitoramento de lise de coágulos ex vivo

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, acute treatment generally relies on the use of pharmacologic thrombolysis via plasminogen activators with the potential for mechanical thrombectomy under certain clinical circumstances2.
The development of novel pharmacologic treatment strategies fundamentally relies on both in-vivo animal models and in-vitro digestion models for preclinical testing3,4. In-vivo models naturally benefit from their ability to capture the complex interaction of various physiologic parameters on treatment efficacy that include clearance of pharmaceutical agents as well as cellular interactions with drugs. However, this same complexity often makes such models quite costly and introduces additional issues when attempting to isolate underlying pharmaco-dynamics/kinetics in animals that significantly differ from human physiology. The development of in-vitro models has helped by facilitating a distilled testing setting in which drug development and screening can be performed but often lacks the fidelity necessary to recapitulate the disease state being studied. 
Commonly found in-vitro protocols for testing novel thrombolytics rely on the utilization of clots formed and lysed under static conditions whereby the residual clot mass serves as the primary endpoint5,6. Unfortunately, such techniques fail to account for the mechanical aspects of clot lysis such as turbulent flow and trans-thrombus pressure drops that can significantly alter the pharmacodynamics of test drugs. Additionally, clots formed under static conditions contain microarchitecture that differs from physiologic clots. The presence of shear during clot formation has reproducibly been shown to impact the resulting clot characteristics such as platelet activation and fibrin-crosslinking. Clots being produced under shear flow exhibit complex heterogeneity from tip-to-tail that is absent in statically formed clots7,8. Such departures from physiologic clot architecture may impact important drug development characterization that includes drug penetration within a thrombus and subsequent lysis efficiency9. 
To address some of these limitations associated with the use of static clotting/clot-lysis models, the adoption of the Chandler loop for both clot formation and clot lysis in the presence of shear has seen a resurgence10. Although such systems allow for a better representation of flow dynamics and generate clots with more physiologically relevant architecture compared to relatively static assays, their simplified flow conditions still represent a deviation from physiologic conditions. Lastly, microfluidic approaches have also been undertaken due to their ease of imaging and uniform flow patterns; however, they remain a significant removal from the physiologic conditions expected within the larger vessels primarily affected in most clinically relevant thrombo-embolic disorders11,12. 
With the above discussion in mind, we developed a high-fidelity, in-vitro thrombolysis model for preclinical thrombolytic drug screening. The model aims at addressing some of the current pitfalls detailed above in the realm of novel thrombolytic therapy screening and was validated for reproducibility and sensitivity at varying concentrations of tissue plasminogen activator (tPA). The system described herein offers physiological shear flows utilizing a peristaltic pump, a pressure dampener, a heated reservoir, two pressure sensors, an in-line fluorometer, and a fluorescently labeled Chandler loop shear-formed clot analog to facilitate real-time tracking of fibrinolysis13. Taken together, the overall system is called the Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF Assay)14 and this manuscript will discuss the intricacies of successfully setting up and running assays in this high-fidelity in-vitro thrombolysis model.

Autor em destaque: Avançando no teste trombolítico integrando a dinâmica de fluxo em modelos in vitro

Rastreamento da fibrinólise de coágulos sanguíneos totais formados por alça de Chandler sob fluxo de cisalhamento em um modelo de trombólise in vitro

Integração de Fibrinogênio Marcado com Fluorescência no Coágulo de Sangue Humano para Visualização Aprimorada do Coágulo

Para começar, adicione fibrinogênio marcado com fluorescência a três mililitros de sangue total humano citrato para atingir uma concentração final de 60 microgramas por mililitro. Configure o Chandler Loop com um tambor de 120 milímetros de diâmetro em banho-maria de 37 graus Celsius. Corte um tubo PCR padrão de 200 microlitros e conecte as extremidades do tubo.Para iniciar a coagulação do sangue, adicione 176 microlitros de cloreto de cálcio 200 milimolares nos três mililitros de sangue total. Depois de misturar adequadamente com inversão suave, usando uma seringa de três mililitros, carregue o sangue no tubo. Coloque imediatamente a tubulação no tambor Chandler Loop, conectando as extremidades.Em condições de pouca luz, gire o tambor parcialmente submerso no banho-maria a 40 RPM. Após 40 a 60 minutos, remova os coágulos do tubo. As características do sangue total marcadas com fibrinogênio Gross Chandler Loop FITC mostraram dispersão uniforme da fluorescência sob luz ultravioleta.Além disso, variar o grau de fibrinogênio FITC no sangue total não altera o peso dos coágulos.

integrating-fluorescently-tagged-fibrinogen-into-human-blood-clot-for

Research

JoVE Journal

Bioengineering

Integrating Fluorescently Tagged Fibrinogen into Human Blood Clot for Enhanced Clot Visualization

98 visualizações. Para começar, adicione fibrinogênio marcado com fluorescência a três mililitros de sangue total humano citrato para atingir uma concentração final de 60 microgramas por mililitro. Configure o Chandler Loop com um tambor de 120 milímetros de diâmetro em banho-maria de 37 graus Celsius. Corte um tubo PCR padrão de 200 microlitros e conecte as extremidades do tubo.Para iniciar a coagulação do sangue, adicione 176 microlitros de cloreto de cálcio 200 milimolares nos três milil...

Vídeo: Integração de Fibrinogênio Marcado com Fluorescência no Coágulo de Sangue Humano para Visualização Aprimorada do Coágulo

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

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.

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.