Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Summary

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

Abstract

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

Introduction

Thrombosis is a leading cause of mortality worldwide¹ and can develop in response todysregulated platelet activation and thrombin generation in both veinsand arteries. Plasma levels of VWF are a key regulator of blood coagulation, whereby low levels (<50%) result in the bleeding disorder known as von Willebrand disease (VWD)² and high levels (>150%) are associated with an increased risk of venous³ and arterial⁴ thrombosis.

VWF is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells and stored in platelet α-gr....

Protocol

These studies were approved by the Research Ethics Board of Queen's University, Canada.

1. Endothelial Cell Stimulation

1. Collagen-coat a 6-well tissue culture plate.
   1. 24 h in advance, coat a 6-well tissue culture plate at 37 °C with 1 mL of collagen buffer (50 µg/mL rat tail collagen type 1 with 0.02 M glacial acetic acid).
   2. Wash the wells twice with 2 mL of Hank's Balanced Salt Solution (HBSS).
      NOTE: Plates can be wrapped in tin foil and stored in an airtight plastic bag at 4 °C.
2. Seed the endothelial cells at a density of 1 x 10⁶

Results

To directly assess the effect of histones on VWF release from endothelial cells, we exposed confluent BOECs to serum-free medium containing PMA (positive control), UH, HR, and HK for 2 h. We showed that HK induced a 2-fold increase in VWF protein (VWF:Ag) in the medium of treated endothelial cells (Figure 1). Interestingly, when BOECs were stimulated with UH and HR, there was less VWF:Ag detected in the medium than in the untreated condition. We hypothesized .......

Discussion

While the physiological relevance of VWF-platelet strings remains controversial due to their rapid dissolution in the presence of the VWF-cleaving protease ADAMTS13, they serve as a quantifiable in vitro model of platelet recruitment by VWF to a site at which a thrombus might form in the presence of localized increases in histone levels⁵. Moreover, in pathologies lacking ADAMTS13 activity-such as thrombotic thrombocytopenic purpura (TTP)-or in inflammatory microenvironments-where ADAMTS13.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calf-thymus unfractionated histones (UH)	Worthington Biochemical	HLY	Reconstituted in serum-reduced media (5 mg/mL)
Calf-thymus lysine-rich histones (HK)	Sigma-Aldrich	H5505	Reconstituted in serum-reduced media (5 mg/mL)
Calf-thymus arginine-rich histones (HR)	Sigma-Aldrich	H4830	Reconstituted in serum-reduced media (5 mg/mL)
Phorbol 12-myristate 13-acetate (PMA)	Sigma-Aldrich	P8139	Reconstituted in DMSO (20 mM)
Histamine	Sigma-Aldrich	H7125-1G	Reconstituted in water (50 mg/mL)
3,3' Dihexyloxacarbocyanine Iodide (DiOC6)	Invitrogen	D273	Reconstituted in methanol (20 mM)
Rabbit Anti-VWF Coating Antibody	DAKO	A0082	For VWF ELISA
Rabbit Anti-VWF Detection Antibody, HRP conjugated	DAKO	P0026	For VWF ELISA and histone-VWF binding assay
Nunc MaxiSorp flat-bottom 96-well microplates	eBioscience	44-2404-21	For histone-VWF binding assay
Immulon 4 HBX Flat Bottom Microtiter 96-Well Plates	Thermo Scientific	3855	For VWF ELISA
Humate-P	CSL Behring	N/A	Plasma-derived human von Willebrand factor/factor VIII complex
Normal Reference Plasma	Precision BioLogic	CCNRP-05	For VWF ELISA standard curve
O-Phenylenediamine dihydrochloride (OPD) reagent	Sigma-Aldrich	P8287	Equivalent product available through ThermoFisher Scientific (Catalogue Number: 34006)
EGM-2 BulletKit	Lonza	CC-3162	For culturing and initial seeding of BOEC
Hank's Balanced Salt Solution (HBSS)	ThermoFisher Scientific	14025092
Rat-tail Collagen Type 1	Corning	354236
Gibco Opti-MEM I Reduced Serum Media	ThermoFisher Scientific	31985070	For endothelial cell stimulations
METAMORPH Microscopy Automation and Image Analysis Software	Molecular Devices	N/A
BD Vacutainer Blood Collection Tubes, No Additive	BD Biosciences	366703
µ-Slide III 0.1 (flow chambers)	Ibidi		This product has been discontinued. We suggest using µ-Slide VI 0.1 (#80661) or 0.4 (# 80601) and recalculating flow rate and platelet volume needed to maintain a shear stress of 4.45 dyn/cm2
Silicone Tubing 1.6 mm ID: 5 m, sterilized	Ibidi	10842
Luer Lock Connector Female: natural Polypropylene, sterilized	Ibidi	10825
Elbow Luer Connector Male: white Polypropylene, sterilized	Ibidi	10802
Blunted 18G Needle	BD Biosciences	305180
20 mL syringes	BD Biosciences	302830
Syringe Pump	New Era Pump Systems Inc. NE-1600 Multi-PhaserTM	N/A
Quorum WaveFX- 4X1 spinning disk microscope	Quorum Technologies	N/A
Image Processing Software	ImageJ	N/A