Thrombus Profiling Assay: A Microfluidics-Based Platform for Comprehensively Characterizing Biomechanical Thrombogenesis

Summary

This work describes a microfluidic assay that uses shear stress to trigger thrombus formation in blood and characterizes the thrombus by multiple dimensions of readout. The assay can measure the prothrombotic tendency of blood samples, and is thus useful in disease diagnosis, drug discovery, as well as basic mechanistic studies related to thrombosis.

Abstract

Thrombosis is a pathological condition describing the abnormal accumulation of platelets and clotting factors in a blood vessel. While many works focused on platelet activation by soluble agonists as an underlying mechanism of thrombosis, it has often been overlooked that blood flow also facilitates thrombus formation. Especially, in the arteries, thrombosis is generally associated with arterial stenosis, which elevates the shear stress in the blood flow and facilitates the process of thrombogenesis, a phenomenon termed biomechanical thrombogenesis. For a long time, no bioassay was available to provide all-around and detailed insights into the process of biomechanical thrombogenesis. To address this, a thrombus profiling assay was developed by combining microfluidics with multi-color fluorescence imaging, which allows comprehensive characterization of biomechanical thrombogenesis with seven readouts covering the size and composition of the thrombus as well as platelet activation level. This thrombus profiling assay can be used to evaluate the prothrombotic tendency in humans and the efficacy of anti-thrombotic agents, and it is also useful for further understanding the mechanisms underlying arterial thrombosis.

Introduction

Thrombosis is a main cause of cardiovascular diseases, responsible for millions of deaths worldwide every year¹. Currently, no bioassay is available in standard clinical settings for evaluating risks of thrombosis. Among the commercialized laboratory and point-of-care hematological function assays, conventional coagulation assays and aggregometry have been proven unreliable in predicting thrombosis or major adverse cardiovascular events^2,3,4. Global thrombosis test⁵, PFA-100/200⁶, and global coagulation a....

Protocol

This protocol follows the guidelines of and has been approved by the Human Research Ethics Committee of The University of Texas Medical Branch. The experimental hardware setup consists of a microfluidic device, perfusion components (connectors, tubing, syringe, and syringe pump), and an inverted microscope with optical components enabling bright-field and multi-color fluorescence imaging. A multi-LED light house and a multi-pass filter cube are used in the microscope to split 4 fluorescent channels with minimal mutual bleed-through: excitation: 391/32, 479/33, 554/24, 638/31, and emission: 435/30, 519/25, 594/32, 695/58. Wear personal protective equipment, including g....

Results

The thrombus profiling assay perfuses blood through a stenotic channel to allow shear-driven thrombus formation, and performs real-time multi-color fluorescence imaging to collect multi-dimensional information on the formed thrombus. By completing experiments using both Set 1 and Set 2 sensors, one should be able to characterize the thrombus in aspects including size, enrichment of crosslinking proteins (von Willebrand factor, fibrinogen), as well as platelet activation level (reflected by P-selection expression, PS expo.......

Discussion

By combining the microfluidic stenosis assay with multi-color fluorescence imaging, the thrombus profiling assay provides a convenient and powerful approach to study biomechanical thrombogenesis and platelet mechanobiology. Meanwhile, the assay is useful in a wide range of applications. For instance, it can be used to screen anti-thrombotic agents that specifically inhibit biomechanical platelet aggregation, where the molecular target and mechanism of action can be deduced from the effect barcode²¹

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-mL Syringes	Henke Sass Wolf	5100-X00V0	Blood sample collection
15-mL falcon tubes	Genesee Scientific	28-101	Blood sample storage
1-mL gastight glass syringe	Hamilton	81331	Hardware assembly
2.2.9	MERU VasImmune		Thrombus staining
20 X1/2 Probe Needles	McMaster-Carr	M919	Making PDMS devices
20-mL Syringes	Henke Sass Wolf	5200-X00V0	Blood sample collection
70% ethanol	Sigma-Aldrich	EX0281-1	Disinfection of gastight syringes and cleaning of glass slides
AK4-Alexa Fluor 647	BioLegend	304918	Thrombus staining
Alexa Fluor 405 NHS Ester	Invitrogen	A30000	Fibrinogen labeling
Alexa fluor 555 antibody labeling kit	Invitrogen	A88065	MBC 370.2 and 2.2.9 labeling
Annexin V-Pacific Blue	Invitrogen	501121505	Thrombus staining
Cleaning Duster	Office Depot	911245	Making PDMS devices
Craft Hobby Knife Set with Wooden Box	Excel Blades	44282	Making PDMS devices
Deionized water	ThermoFisher Scientific	751-628	Washing of gastight syringes
Desiccator	Bel Art	F420270000	Degasing PDMS
Disposable Multipurpose Laboratory Spatula	LevGo	17211	Mixing the silicone elastomer base and curing agent
Dye and Biotin Removal Spin Column	Zeba	A44296S	Fibrinogen labeling
Fibrinogen	Innovative Research	IHUFBG25MG	Thrombus staining
Fusion 200-X syringe pump	Chemyx Inc.	0720X	Hardware assembly
Heparin	Sigma-Aldrich	H3149	Blood sample anticoagulation
High Frequency Generator	Electro-technic product Inc.	BD-20	Making PDMS devices
Human VWF monomer	Sino Biological Inc.	10973-H08C	Microfuidic device coating
Image J v1.53 software	Fiji, National Institute of Health		Data analysis
Inverted microscope	Leica	DM IL LED; filter cube: Leica DFT51010; lighthouse: LED5	Hardware assembly
Kimwipes	Kimberly- Clark Professional	34120	Cleaning of glass slides
Luer lock caps	International Medical Industries, Inc.	57100B	Blood sample collection
MBC 370.2	Kerafast	EBW104	Thrombus staining
Microscope cover glasses	Paul Marienfeld GmbH & Co. KG	ES0107222	Making PDMS devices
Mini-Razor Blade Scraper	Stanley	28-100	Making PDMS devices
NanoDrop 2000 UV-Vis Spectrophotometer	Thermo Scientific	ND-2000	Protein concentration and F/P ratio measurement
Oven	Lab-Line Instruments	3512	Making PDMS devices
PAC-1-Alexa Fluor 647	BioLegend	362806	Thrombus staining
Plastic cup	ThermoFisher Scientific	S04589	Mixing the silicone elastomer base and curing agent
SU-8 photoresist master  mold	UC San Diego Nano3 Nanofabrication Cleanroom Facility		Making PDMS devices
Sylgard 184 Silicone Elastomer Kit	Krayden Dow	DC4019862	PDMS
SZ22-FITC	Beckman Coulter	IM 1756U	Thrombus staining
Tubing	Cole -Palmer Instrument Co.	06422-01	Hardware assembly