Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

Summary

Multi-color flow cytometry for platelets can identify new platelet subtypes within classical platelet subpopulations, such as resting, aggregatory, procoagulant, and apoptotic platelets. This allows comparison of different subpopulation patterns induced by various platelet agonists. Here, a procedure for establishing a multi-color flow cytometry panel is described in detail.

Abstract

Platelets, or thrombocytes, are small anucleated blood cells that play a crucial role in hemostasis and thrombosis. Defects in platelet functions can cause bleeding or thrombotic events in patients with cardiovascular diseases. Therefore, it is important to characterize platelets phenotypically to be able to assign platelet subpopulations to platelet function. Stimulation of human platelets with platelet agonists and activators induces morphological and physiological changes in platelets, accompanied by changes in the surface receptor population. This leads to functionally diverse platelet subpopulations. Classically defined platelet subpopulations are resting, aggregatory, procoagulant, and apoptotic platelets. To characterize the effect of agonists on platelet subtypes, we established an assay using multi-color flow cytometry consisting of 10 different antibodies and dyes (anti-CD62P, anti-CD63, anti-CD61, anti-CD41, anti-CD42b, anti-Integrin_αIIbβ3 (clone: PAC-1), anti-CXCR4, anti-ACKR3, Annexin V, Zombie NIR). Isolated human platelets were incubated with platelet agonists and stained with specific fluorophore-conjugated antibodies and dyes. Afterwards, they were measured by flow cytometry. This allows us to define agonist-specific subtypes within the classic four subpopulations. In conclusion, the combination of activation markers (anti-CD62P, anti-CD63, anti-Integrin_αIIbβ3 (clone: PAC-1)), inflammatory markers (anti-CXCR4, anti-ACKR3) and apoptotic markers (Annexin V, Zombie NIR) that compose the 10-color flow cytometry panel described in this manuscript opens up the possibility to define further platelet subtypes that could be linked to specific platelet function. This method can be applied in basic research on platelet function and physiology, as well as in defining new platelet subtypes in disease models and patient studies.

Introduction

Thrombocytes, also called platelets, are small, nucleus-free blood cells essential for hemostasis and the development of thrombosis^1,2. They can be activated by various platelet agonists. Classical, physiological platelet agonists are adenosine diphosphate (ADP), which binds to the P2Y12 and P2Y1 receptors, thrombin that activates platelets through the PAR1 and PAR4 receptors, and collagen that interacts with glycoprotein VI. Collagen can be substituted in in vitro experiments by the collagen-related peptide (CRP-XL)^3,4. Recently, hemin has be....

Protocol

Blood collection and handling for research purposes were approved by the Ethics Committee at the Medical Faculty of the Eberhard Karls University and the University Hospital of Tübingen (ethics vote 238/2018B02), and the study methodologies conformed to the standards set by the Declaration of Helsinki. Healthy donors gave written informed consent for blood collection. The baseline characteristics for healthy donors are listed in Table 1. Figure 1 gives an overview of the flow cytometry protocol.

1. Isolation of human platelets from whole blood

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Results

There are two options for analyzing the raw data collected by a multi-color flow cytometry panel. One option is to analyze it manually with the analysis software (Figure 3 and Figure 4). Therefore, the raw data needs to be dragged and dropped into the software window. First, the cells need to be found by adjusting the SSC/FSC scale in the scatterplot display mode to show the platelet population as a distinct point cloud. Then, the positive cells for each marker .......

Discussion

Multi-color flow cytometry allows to define distinct platelet clusters/subpopulations associated with discrete functions^12,17,18. Decisive for the results of the subpopulations and the receptor surface expression is the quality of the isolated human platelets. Therefore, one key step in the presented protocol is the preparation of the platelets. They need to be gently isolated to avoid activation prior to sample treatment with d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x Binding Buffer	Invitrogen, Carlsbad, California, USA	88-8103-74	Buffer
Adenosine diphosphate (ADP)	Probe & go Labordiagnostica GmbH Lemgo, Germany	70212	platelet agonist
Annexin V PE-Cyanine7	Invitrogen, Carlsbad, California, USA	88-8103-74	dye
anti-CD41 AF 700	BioLegend, San Diego, CA, USA	303728	antibody
anti-CD41 Alexa Fluor 700	BioLegend, San Diego, CA, USA	303727	antibody
anti-CD42b PerCP/Cyanine5.5	BioLegend, San Diego, CA, USA	303918	antibody
anti-CD61 BV605	BD Bioscience Franklin Lakes, NJ, USA	744382	antibody
anti-CD62P PE	Beckman Coulter, Brea, California, USA	IM1759U	antibody
anti-CD63 PE/Cy5	Abcam, Cambrigde, UK	ab 234251	antibody
anti-CXCR4 BV650	BD Bioscience Franklin Lakes, NJ, USA	740599	antibody
anti-CXCR7 BV421	BD Bioscience Franklin Lakes, NJ, USA	566234	antibody
anti-PAC-1 FITC	BD Bioscience Franklin Lakes, NJ, USA	340507	antibody
Bovine serum albumin (BSA)	Applichem, Darmstadt, Germany	A1391	-
Citrit acid	Carl Roth, Karlsruhe, Germany	6490.1	-
CRP-XL	CambCol Laboratories, Ely, UK	CRP-XL	platelet agonist
D-(+)-Glucose	Sigma Aldrich Co., St. Louis, Missouri, USA	G7528	-
Dubecco’s Phosphate Buffered Saline (PBS)	Sigma Aldrich Co, St. Louis, Missouri, USA	D8537	-
Hemin	Sigma Aldrich Co., St. Louis, Missouri, USA	H9039-10G	platelet agonist
HEPES	Carl Roth, Karlsruhe, Germany	9105.3	-
Potassium chloride	Carl Roth, Karlsruhe, Germany	6781.3	-
Sodium chloride	Carl Roth, Karlsruhe, Germany	3957.1	-
Sodium hydrogencarbonate	Sigma Aldrich Co, St. Louis, Missouri, USA	401676	-
Thrombin	Roche	10602400001	platelet agonist
tri-Sodium citrate-dihydrat	Applichem, Darmstadt, Germany	1416551211	-
Zombie NIR	BioLegend, San Diego, CA, USA	423105	amine-reactive dye