Platelet accumulation in an endothelium-coated elastic vein valve model of deep vein thrombosis is mediated by GPIb α-VWF interaction

Abstract

Deep vein thrombosis is a life-threatening disease that takes millions of people's lives worldwide. Given both technical and ethical issues of using animals in research, it is necessary to develop an appropriate in vitro model that would recapitulate the conditions of venous thrombus development. We present here a novel microfluidics vein-on-a-chip with moving valve leaflets to mimic the hydrodynamics in a vein, and Human Umbilical Vein Endothelial Cell (HUVEC) monolayer. A pulsatile flow pattern, typical for veins, was used in the experiments. Unstimulated human platelets, reconstituted with the whole blood, accumulated at the luminal side of the leaflet tips proportionally to the leaflet flexibility. Platelet activation by thrombin induced robust platelet accrual at the leaflet tips. Inhibition of glycoprotein (GP) IIb-IIIa did not decrease but, paradoxically, slightly increased platelet accumulation. In contrast, blockade of the interaction between platelet GPIbα and A1 domain of von Willebrand factor completely abolished platelet deposition. Stimulation of the endothelium with histamine, a known secretagogue of Weibel-Palade bodies, promoted platelet accrual at the basal side of the leaflets, where human thrombi are usually observed. Thus, platelet deposition depends on the leaflet flexibility, and accumulation of activated platelets at the valve leaflets is mediated by GPIbα-VWF interaction.

Keywords: DVT; glycoprotein Ibα; hydrodynamics; mechanical properties; microfluidics; platelets; von Willebrand factor.

Figure 1

Chamber coated with endothelial monolayer. (A) General view of the device. (B) Subdivision of the valve into four areas (2 for each leaflet) for quantitation. HUVEC cells were seeded in the flow chamber (C) and incubated for 4 h without flow (D) followed by 20 h underflow (1 µl/min; E, bar 150 µm). Representative images of the entire chamber (F) or the valve area (G) stained for CD31 (Fi,Gi), nuclei (DAPI, Fii,Gii), VE-cadherin (Fiii,Giii) and merge (Fiv,Giv). Scale bar 200 and 150 µm of F and G, respectively.

Figure 2

Deposition of unchallenged platelets on symmetrical and non-symmetrical valves. Blood reconstituted with fluorescently labelled platelets was perfused through either symmetrical (A) or non-symmetrical (B) valves, and resting platelets were preincubated with 9 mM eptifibatide (C) through a symmetrical valve for 5 min. Quantitation of resting platelet accumulation on more flexible (TL area, circles; TS area, open triangles) and less flexible/immobile (TL area, squares; TS area, Xs) leaflets of the symmetrical (D) or non-symmetrical (E) valve. (F,G) Quantitation of eptifibatide-treated platelet accumulation at the TL (F) and TS (G) regions. Scale bar 100 µm. Error bars represent SEM, n = 3.

Figure 3

Deposition of platelets is increased by thrombin. Platelets were preincubated with 0.1 U/ml thrombin (A,C), and with 0.1 U/ml thrombin + 9 µM eptifibatide (B,D), Blood reconstituted with fluorescently labelled platelets was perfused through a symmetrical valve for 5 min. (C,D), left row represents TL region, and the right row represents TS region; (more flexible, circles; less flexible, squares). Scale bar 100 µm. Error bars represent SEM, n = 3.

Figure 4

Deposition of platelets is mediated by the VWF-GPIbα axis. Platelets were preincubated with OS-1 peptide (M3456 CTERMALHNLC, Alta Bioscience, 12 µM) (A), or with 0.1 U/ml thrombin + OS-1 peptide (B) for 5 min, fluorescently labelled, reconstituted with whole blood, and perfused through a symmetrical valve for 5 min. (C,D), Quantitation of platelet accumulation on more flexible (TL area, circles; TS area, open triangles) and less flexible (TL area, squares; TS area, Xs) leaflets. Scale bar 100 µm. Error bars represent SEM, n = 3.

Figure 5

Histamine increases platelet deposition in the valve pockets (TS area). (A) HUVECs inside the chamber were incubated with histamine (10 µM, 15 min), and whole blood reconstituted with resting platelets was perfused through a symmetrical valve for 5 min. (B,C) quantitation of platelet accumulation on more flexible (circles) and less flexible (squares) leaflets, TL and TS region, respectively. Scale bar 100 µm. Error bars represent SEM, n = 3.