Name: investigating von willebrand factor pathophysiology using a flow chamber model of von willebrand factor-platelet string formation
Uploaded: 2017-08-14T14:00:00
Description: Watch this Scientific Journal Video about Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation at JoVE.com

Alison Michels is a recipient of a Frederick Banting and Charles Best Canada Graduate Scholarship from the Canadian Institutes of Health Research (CIHR). Laura L. Swystun is the recipient a CIHR fellowship. David Lillicrap is the recipient of a Canada Research Chair in Molecular Hemostasis. This study was funded in part by a CIHR operating grant (MOP-97849).

These studies were approved by the Research Ethics Board of Queen&#39;s University, Canada.
1. Endothelial Cell Stimulation
<ol>
	<li>Collagen-coat a 6-well tissue culture plate.
	<ol>
		<li>24 h in advance, coat a 6-well tissue culture plate at 37 &#176;C with 1 mL of collagen buffer (50 &#181;g/mL rat tail collagen type 1 with 0.02 M glacial acetic acid).</li>
		<li>Wash the wells twice with 2 mL of Hank&#39;s Balanced Salt Solution (HBSS). 
		NOTE: Plates can be wrap...

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...

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 (&#60;50%) result in the bleeding disorder known as von Willebrand disease (VWD)2 and high levels (&#62;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 &#945;-granules and WPBs, respectively. Upon hemostatic challenge, VWF can be released from endothelial WPBs to tether circulating platelets to activated endothelial cells5 or exposed collagen on the vessel wall6. Anchoring of VWF to endothelial cells has been shown to be mediated by P-selectin7 and integrin &#945;v&#946;38. The subsequent release of platelet &#945;-granule stores can further increase localized VWF concentrations to stabilize platelet-platelet interactions for platelet plug formation, the scaffold needed for the propagation of the coagulation cascade and fibrin deposition. The platelet-binding activity of VWF is regulated by its multimeric structure, with high-molecular weight multimers possessing greater hemostatic activity9,10. In circulation, VWF also acts as a carrier for the coagulation factor VIII.
Fluid shear stress is an essential regulator of VWF physiology. In the absence of shear stress, VWF exists in a globular form, concealing binding domains for platelet glycoprotein Ib adhesion11. When shear stress is present, the cleavage site for a metalloprotease, A disintegrin and metalloprotease with thrombospondin motif (ADAMTS13), is exposed. ADAMTS13 cleaves naked and platelet-decorated VWF strings to regulate multimer size, thereby reducing its hemostatic activity12.
VWF is an acute-phase protein, and numerous stimuli, including hypoxia13, infection14, and proinflammatory cytokines, have been shown to mediate VWF release from endothelial cells. Similar to other inflammatory agents, extracellular histones have also been shown to induce systemic VWF release in mice15,16 and the activation of platelets in vitro17,18,19. This was shown to be dependent upon histone subtype, as differences in lysine and arginine content may influence function15. Our study aims to establish a flow chamber model to investigate the influence of lysine-rich (HK) and arginine-rich (HR) histone subtypes and secretagogues on endothelial VWF release and real-time platelet capture, potential early events in inflammation-induced thrombosis.
This flow chamber methodology recapitulates in vivo interactions between subendothelial collagen, endothelial cells, VWF, and platelets in an in vitro system that is visual, reproducible, and quantifiable. It allows for the real-time assessment of all aspects of the pathway that regulates VWF-platelet interactions, including WPB secretion, platelet activation, and VWF proteolysis. Studies of VWF under controlled shear stress conditions have been used to evaluate VWD mutations that impair VWF release and platelet-binding function20, WPB physiology21, and VWF cleavage by ADAMTS135. We use this methodology to quantify VWF-platelet string formation as a consequence of an inflammatory stimulus: extracellular histones.

<table>
	<tbody>
		<tr>
			<td>Calf-thymus unfractionated histones (UH)</td>
			<td>Worthington Biochemical</td>
			<td>HLY</td>
			<td>Reconstituted in serum-reduced media (5 mg/mL)</td>
		</tr>
		<tr>
			<td>Calf-thymus lysine-rich histones (HK)</td>
			<td>Sigma-Aldrich</td>
			<td>H5505</td>
			<td>Reconstituted in serum-reduced media (5 mg/mL)</td>
		</tr>
		<tr>
			<td>Calf-thymus arginine-rich histones (HR)</td>
			<td>Sigma-Aldrich</td>
			<td>H4830</td>
			<td>Reconstituted in serum-reduced media (5 mg/mL)</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td>Reconstituted in DMSO (20 mM)</td>
		</tr>
		<tr>
			<td>Histamine</td>
			<td>Sigma-Aldrich</td>
			<td>H7125-1G</td>
			<td>Reconstituted in water (50 mg/mL)</td>
		</tr>
		<tr>
			<td>3,3&#39; Dihexyloxacarbocyanine Iodide (DiOC6)</td>
			<td>Invitrogen</td>
			<td>D273</td>
			<td>Reconstituted in methanol (20 mM)</td>
		</tr>
		<tr>
			<td>Rabbit Anti-VWF Coating Antibody</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td>For VWF ELISA</td>
		</tr>
		<tr>
			<td>Rabbit Anti-VWF Detection Antibody, HRP conjugated</td>
			<td>DAKO</td>
			<td>P0026</td>
			<td>For VWF ELISA and histone-VWF binding assay</td>
		</tr>
		<tr>
			<td>Nunc MaxiSorp flat-bottom 96-well microplates</td>
			<td>eBioscience</td>
			<td>44-2404-21</td>
			<td>For histone-VWF binding assay</td>
		</tr>
		<tr>
			<td>Immulon 4 HBX Flat Bottom Microtiter 96-Well Plates</td>
			<td>Thermo Scientific</td>
			<td>3855</td>
			<td>For VWF ELISA</td>
		</tr>
		<tr>
			<td>Humate-P</td>
			<td>CSL Behring</td>
			<td>N/A</td>
			<td>Plasma-derived human von Willebrand factor/factor VIII complex</td>
		</tr>
		<tr>
			<td>Normal Reference Plasma</td>
			<td>Precision BioLogic</td>
			<td>CCNRP-05</td>
			<td>For VWF ELISA standard curve</td>
		</tr>
		<tr>
			<td>O-Phenylenediamine dihydrochloride (OPD) reagent</td>
			<td>Sigma-Aldrich</td>
			<td>P8287</td>
			<td>Equivalent product available through ThermoFisher Scientific (Catalogue Number: 34006)</td>
		</tr>
		<tr>
			<td>EGM-2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>For culturing and initial seeding of BOEC</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat-tail Collagen Type 1</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Opti-MEM I Reduced Serum Media</td>
			<td>ThermoFisher Scientific</td>
			<td>31985070</td>
			<td>For endothelial cell stimulations</td>
		</tr>
		<tr>
			<td>METAMORPH Microscopy Automation and Image Analysis Software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tubes, No Additive</td>
			<td>BD Biosciences</td>
			<td>366703</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide III 0.1 (flow chambers)</td>
			<td>Ibidi</td>
			<td></td>
			<td>This product has been discontinued. We suggest using &#181;-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</td>
		</tr>
		<tr>
			<td>Silicone Tubing 1.6 mm ID: 5 m, sterilized</td>
			<td>Ibidi</td>
			<td>10842</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock Connector Female: natural Polypropylene, sterilized</td>
			<td>Ibidi</td>
			<td>10825</td>
			<td></td>
		</tr>
		<tr>
			<td>Elbow Luer Connector Male: white Polypropylene, sterilized</td>
			<td>Ibidi</td>
			<td>10802</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunted 18G Needle</td>
			<td>BD Biosciences</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL syringes</td>
			<td>BD Biosciences</td>
			<td>302830</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>New Era Pump Systems Inc. NE-1600 Multi-PhaserTM</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Quorum WaveFX- 4X1 spinning disk microscope</td>
			<td>Quorum Technologies</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Processing Software</td>
			<td>ImageJ</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating von willebrand factor pathophysiology using a flow chamber model of von willebrand factor-platelet string formation

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.

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 ...

Watch this Scientific Journal Video about Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation at JoVE.com

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

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.

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Research

JoVE Journal

Medicine

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Orthotopic Small Bowel Transplantation in Rats

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome (ARDS)

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome ARDS

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial ...