Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Summary

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Abstract

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.

A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

Introduction

Platelets are anucleate cells that circulate in the blood stream. Their main function is to seal vascular breaches at sites of injury and to prevent blood loss. At these sites of injury, subendothelial collagen fibers become exposed and are subsequently covered by the multimeric protein, von Willebrand factor (VWF). VWF interacts with the platelets in circulation in a mechanism that depends upon the glycoprotein Ibα-IX-V complex on the cell surface¹, slowing down the speed of the platelets. This is particularly important at high shear rates. The platelets subsequently undergo morphological changes while receiving activating impulses from c....

Protocol

The local Medical Ethical Committee of the University Medical Center Utrecht approved the drawing of blood for ex vivo research purposes, including those of this study.

1. Solution Preparation

1. Prepare a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Tyrode's buffer by dissolving 10 mM HEPES, 0.5 mM Na₂HPO₄, 145 mM NaCl, 5 mM KCl, 1 mM MgSO₄, and 5.55 mM D-glucose in distilled water.
   1. Make two variants of the buffer: set the pH levels at 6.5 and at 7.4, respectively. Make fresh buffer for each day of experiments. Supplement with 10 ng/mL prostac....

Results

Figure 1 shows images of the flow chamber and experimental setup; the position and dimensions of the silicon sheet; and tubing connections. Figure 2 provides details on the dimensions of the flow chamber. Figure 3 and Movie 1 show a time-series of images of platelet adhesion and spreading on immobilized VWF. CD63 is a transmembrane protein that is inserted into the membrane of intrac.......

Discussion

Worldwide, thrombosis is a leading cause of death and morbidity, and platelets play a central role in its development. This work describes a method for the live-cell imaging of platelet degranulation under flow. It is generally assumed that, when platelets become activated, all granular contents are directly released into solution. The accompanying results suggest that this is not necessarily the case. During adhesion and degranulation, platelets retain a significant amount of polyphosphate (Figure 4). A.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	VWR	441476L
Na2HPO4	Sigma	S-0876
NaCl	Sigma	31434
KCl	Sigma	31248
MgSO4	Merck KGaA	1.05886
D-glucose	Merck KGaA	1.04074
Prostacyclin	Cayman Chemical	18220
Tri-sodium citrate	Merck KGaA	1.06448
Citric acid	Merck KGaA	1.00244
Cover glasses	Menzel-Gläser	BBAD02400500#A	24 x 50 mm, No. 1 = 0.13 - 0.16 mm thickness.
Chromosulfuric acid (2% CrO3)	Riedel de Haen	07404	CAS [65272-70-0].
Von Willebrand factor (VWF)	in-house purified
Fibrinogen	Enzyme Research Laboratories	FIB3L
4 well dish, non-treated	Thermo Scientific	267061
Human Serum Albumin Fraction V	Haem Technologies Inc.	823022
Blood collection tubes, 9 mL, 9NC Coagulation Sodium Citrate 3.2%	Greiner Bio-One	455322
Cell analyser	Abbott Diagnostics	CELL-DYN hematology analyzer
Paraformaldehyde	Sigma	30525-89-4
Syringe pump	Harvard Apparatus, Holliston, MA	Harvard apparatus 22
10 mL syringe with 14.5 mm diameter	BD biosciences	305959	Luer-Lok syringe
Anti-CD63-biotin	Abcam	AB134331
Anti-CD62P-biotin	R&D Systems	Dy137
4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI)	Polysciences Inc.	9224
Streptavidin, Alexa Fluor 488 conjugate	Thermo Scientific	S11223
Immersion oil	Zeiss	444963-0000-000
Detergent solution	Unilever, Biotex
Glycine	Sigma	56-40-6
Polyvinyl alcohol	Sigma	9002-89-5	Mowiol 40-88.
Tris hydrochloride	Sigma	1185-53-1
1,4-Diazabicyclo[2.2.2]octane (DABCO)	Sigma	280-57-9
Sheep Anti-hVWF pAb	Abcam	AB9378
Alexa fluor 488-NHS	Thermo Scientific	A20000
Glycerol	Sigma-Aldrich	15523-1L-R
Parafinn film	Bemis	PM-996	4 in. x 125 ft. Roll.
Silicone sheet non-reinforced	Nagor	NA 500-1	200 mm x 150 mm x 0.125 mm.
Customized cut silicone sheet with perfusion and vacuum channels	in-house made		Made of Silicone sheet non-reinforced (Nagor, NA 500-1)
1.5 mL tubes	Eppendorf AG	T9661-1000AE
Fluorescent microscope	Zeiss Observer Z1		Equiped with LED excitation lights.
Microscope software	Zeiss ZEN 2	blue edition
18 G needle (18 G x 1 1/2")	BD biosciences	305196
NaCl	Riedel de Haen	31248375
Tris	Roche	10708976
Plastic pasteur pipet	VWR	612-1681	7 mL non sterile, graduated up to 3 mL.
Silicone tubing	VWR	228-0656	Inner diameter. x Outer diameter x Wall thickness = 1.02 x 2.16 x 0.57 mm.
Microscope slides	Thermo Scientific	ABAA000001##12E	76 x 26 x 1 mm, ground edges 45 °, frosted end.