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 wrapped in tin foil and stored in an airtight plastic bag at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Seed the endothelial cells at a density of 1 x 106 cells per well in 2 mL of growth medium containing 10% fetal bovine serum (FBS). Incubate the cells for 24 h at 37 &#176;C (5% CO2). 
	NOTE: Here, blood outgrowth endothelial cells (BOECs) between passages 5 and 15 were used. BOECs were isolated from healthy volunteers with a wildtype VWF sequence using a method referenced in Starke et al.22 in a manner similar to one previously described by JoVE23.</li>
	<li>Remove the medium after 24 h and wash the cells with HBSS.</li>
	<li>Add 1 mL of serum-free medium (minimal essential growth medium without FBS) containing phorbol 12-myristate 13-acetate (PMA; 100 nM), unfractionated histone (UH), and HK or HR (all at 25 &#181;g/mL) to the cells for 2 h at 37 &#176;C (5% CO2). 
	NOTE: This concentration of histone was based on previous in vitro cytotoxicity reports by Xu et al.24 and Abrams et al.25 that used 50 &#181;g/mL.</li>
	<li>Collect the medium after 2 h and store it at -20 &#176;C until analysis.</li>
</ol>
2. VWF Quantification by Enzyme-linked Immunosorbent Assay (ELISA)
<ol>
	<li>Coat a microplate with 100 &#181;L of coating buffer (10 mM dibasic sodium phosphate and 145 mM sodium chloride, pH 7.2) containing 10 &#181;g/mL polyclonal anti-VWF antibody overnight at 4 &#176;C.</li>
	<li>Wash the plate three times with 250 &#181;L of wash buffer (10 mM dibasic sodium phosphate, 500 mM sodium chloride, and 0.1% Tween 20, pH 7.2).</li>
	<li>Dilute the medium samples 1:2 and 1:4 with wash buffer and use normal reference plasma for a standard curve, beginning at a 1:10 dilution.</li>
	<li>Plate 100 &#181;L of diluted samples and standards overnight at 4 &#176;C.</li>
	<li>Wash the plate three times with 250 &#181;L of wash buffer.</li>
	<li>Add 100 &#181;L of diluted anti-VWF, HRP-conjugated detection antibody (1.1 &#181;g/&#181;L in wash buffer) to the plate and incubate for 1 h at room temperature.</li>
	<li>Wash the plate and incubate for 10 min with O-Phenylenediamine dihydrochloride (OPD) reagent, as per the manufacturer&#39;s instructions.</li>
	<li>Stop the reaction with 100 &#181;L of 1 M H2SO4 and read the plate at 492 nm.</li>
</ol>
3. Solid-phase Histone-VWF Binding Assay
<ol>
	<li>Coat a microplate with histones diluted in 0.5% sodium deoxycholate 50 mM carbonate buffer (pH 9.6) to 10 &#181;g/mL overnight at 4 &#176;C.</li>
	<li>Wash the plate three times with 250 &#181;L of PBS supplemented with 0.1% Tween 20, incubating for 10 min between washes.</li>
	<li>Block the plate with 200 &#181;L of PBS supplemented with 0.1% Tween 20 and 1% bovine serum albumin (BSA) for 1 h at room temperature.</li>
	<li>Repeat step 3.2.</li>
	<li>Serially dilute plasma-derived human VWF/factor VIII complex from 200 mU/mL to 25 mU/mL in saline buffer (10 mM dibasic sodium phosphate, 500 mM sodium chloride, and 0.1% Tween 20, pH 7.2). Add 100 &#181;L to the plate for 2 h at room temperature.</li>
	<li>Repeat step 3.2.</li>
	<li>Detect the binding of VWF to immobilized histone using 100 &#181;L of a solution containing PBS-0.1% Tween 20 and 1:1,000 rabbit polyclonal anti-VWF antibody conjugated to HRP for 1 h at room temperature.</li>
	<li>Repeat step 3.2.</li>
	<li>Incubate the plate for 30 min with 100 &#181;L of OPD reagent and stop the reaction with 100 &#181;L of 1 M H2SO4. Measure the optical density at 492 nm.</li>
</ol>
4. Seeding Endothelial Cells onto Flow Chambers
<ol>
	<li>Load 0.5 mL of collagen-coating buffer (described in step 2.1) into a Luer-lock 1-mL syringe.</li>
	<li>Twist the Luer-lock end of the syringe onto the reservoir of a flow chamber slide.</li>
	<li>Press the plunger slowly until the entire lane is coated and each reservoir is full. Repeat for each lane and incubate the chamber at 37 &#176;C for 24 h.</li>
	<li>Aspirate the collagen-coating buffer with a 200 &#956;L pipette. Load another syringe with 1 mL HBSS and press the plunger slowly to wash each lane of the flow chamber. Aspirate the wash with a 200 &#956;L pipette at the opposing end of the flow chamber. Repeat twice.</li>
	<li>Pipette approximately 100 &#181;L of a 3 x 106 cells/mL endothelial cell (BOEC) solution in growth medium with 10% FBS into the lane of a flow chamber. 
	NOTE: The seeding density of endothelial cells exceeds the capacity of the chamber. This is to ensure maximal channel coverage; non-adherent cells are removed in the subsequent wash step.</li>
	<li>Incubate the chamber at 37 &#176;C for 24 h. Change the medium every 8-12 h using Luer-lock syringes (as in step 4.4). Ensure that there are no visible bubbles in the lanes.</li>
</ol>
5. Isolating Platelets from Human Whole Blood
<ol>
	<li>In a biological safety cabinet with a 30 G needle, add 300 &#181;L of acid-citrate dextrose buffer (85 mM trisodium citrate, 111 mM D-glucose, and 71 mM citric acid, pH 4.5) to an additive-free 3 mL blood collection vacutainer.</li>
	<li>Collect whole blood from healthy volunteers (free from anti-platelet and non-steroidal anti-inflammatory drugs for seven days) by venipuncture (three tubes of 3 mL each), following a protocol similar to that published by Bernardo et al26.</li>
	<li>Discard the first tube, as the initial tissue damage associated with the venipuncture can cause spontaneous platelet activation.</li>
	<li>Gently invert the vacutainers three times to mix the blood with anticoagulant.</li>
	<li>Centrifuge the vacutainers at 150 x g for 15 min at 24 &#176;C (room temperature).</li>
	<li>Slowly remove the platelet-rich plasma (supernatant) with a pipette in a biological safety cabinet and carefully transfer it to a 15-mL conical tube. Transfer the platelet-rich volume of each vacutainer to a single conical tube to avoid potential cross-activation. 
	NOTE: Platelet isolation should be performed in a biological safety cabinet to prevent bacterial contamination and potential artificial platelet and/or endothelial activation.</li>
	<li>Centrifuge the conical tubes at 900 x g for 10 min at 24 &#176;C (room temperature) to pellet the platelets.</li>
	<li>Pipette and discard the platelet-poor supernatant.</li>
	<li>Wash the pellet carefully with 1 mL of citrate-glucose-sodium (CGS) buffer (13 mM sodium citrate, 30 mM D-glucose, and 120 mM sodium chloride, pH 7.0). 
	NOTE: The platelets will appear in wisp-like formations when the CGS buffer is added and the pellet is gently resuspended. In samples contaminated by platelet activation, the platelets will not easily resuspend and will appear clumpy.</li>
	<li>Once fully resuspended, centrifuge the samples at 900 x g for 10 min at 24 &#176;C (room temperature).</li>
	<li>Remove the CGS wash buffer and resuspend the pellet in 3 mL of Tyrode&#39;s buffer (138 mM sodium chloride, 5.5 mM D-glucose, 12 mM sodium bicarbonate, 2.9 mM potassium chloride, and 0.36 mM dibasic sodium phosphate, pH 7.4). Ensure complete resuspension of the platelets and, if aggregations are observed, discard the sample.</li>
	<li>Add 1.5 &#181;L from a 20 mM DiOC6 stock solution (dissolve 57.25 mg in 5 mL of methanol) to each 3 mL conical tube of washed platelets for a final concentration of 1 &#181;M. 
	NOTE: DIOC6 is a green fluorescent mitochondrial dye used in this experiment to label platelets.</li>
	<li>Aliquot 1 mL of the washed platelet solution to three 1.5-mL microcentrifuge tubes. 
	NOTE: The platelet volume required for this experiment is calculated based on the size of the flow chamber, the desired shear stress, and the length of the experiment (step 6.5). This protocol can be adjusted as conditions change to ensure that there is enough volume for fluid flow.</li>
	<li>Use the washed platelets within 2 h of isolation.</li>
</ol>
6. Flow Apparatus Assembly
<ol>
	<li>Attach a 70 cm piece of sterile silicone tubing (interior diameter of 1.6 mm) to a male elbow Luer connecter at one end and a female Luer lock adaptor at the other.</li>
	<li>Connect the female Luer lock adaptor attached to the tubing to a 20 mL syringe.</li>
	<li>Repeat steps 6.1 and 6.2 twice to generate three identical syringe-tubing connections.</li>
	<li>Use a syringe pump to generate fluid flow. Input the following settings into the syringe pump: interior diameter of the syringe and flow rate. 
	NOTE: A 20-mL syringe has an interior diameter of 19.55 mm. The flow rate is calculated based on the desired shear rate/shear stress and the dimensions of the flow chamber. For the flow chamber used in the current study, to achieve 1 dyn/cm2 (observed in low venous shear), a flow rate of 9.37 &#181;L/min is required. Because of evidence linking histones to venous thrombosis, a moderate venous shear of 4.45 dyn/cm2 is selected. This is also based on the assumption that the buffer has a similar viscosity to water. The flow rate is then equal to 41.7 &#181;L/min. Equations used to calculate the flow rate for various flow chamber dimensions can be found at:
	<ol>
		<li>Set the pump to withdraw fluid using the button option on the syringe pump.</li>
	</ol>
	</li>
	<li>Load the three syringes attached to tubing on the syringe pump and tightly secure brackets around the syringes and plungers.</li>
	<li>Attach a male elbow Luer to both ends of a second 20 cm piece of sterile silicone tubing.</li>
	<li>Connect one elbow Luer to a blunted 18-gauge needle.</li>
	<li>Repeat twice, creating three identical intake systems for the three lanes of the flow chamber. 
	NOTE: The silicon tubing and Luers can be washed with 10% bleach followed by distilled water before reuse. It is not recommended to reuse the flow chambers.</li>
</ol>
7. Microscope Settings
<ol>
	<li>Use a spinning disk microscope equipped with a fully automated stage. Perform illumination with a laser launch that provides 405, 458, 491, 543, and 633 nm laser lines. Capture all images with a 20X objective. Adjust all parameters with the analysis software (http://www.well.ox.ac.uk/_asset/file/metamorph-basic-commands.pdf).</li>
	<li>Select the channel settings (emission filter: 520/35), with excitation at 491 nm, for acquisition. Set the electron multiplying charge-coupled device to an electron-multiplying gain of 150, with an exposure time of 200 ms, to obtain the full dynamic data range. 
	NOTE: Platelets are labelled with a green fluorescent dye, DiOC6, with wavelength maxima (excitation, 482 nm; emission, 504 nm).</li>
	<li>Adjust the 491 nm laser power to 20%.</li>
	<li>Mark the centre of all three lanes and select 1 &#956;m for z range and 0.5 &#956;m for step size. Select the number of time points (i.e., duration) and the time between images (i.e., interval) to capture an image every 10 s; select &#34;60 time points&#34; per slide. Set the microscope to capture images from the three lanes and save them to a designated experiment folder. 
	NOTE: Because the flow chamber has three lanes, the microscope will need to toggle between three positions to capture the images.</li>
</ol>
8. VWF-platelet String Formation
<ol>
	<li>Remove the culture medium from the flow chamber (from step 4) using the same technique as in step 4.4.</li>
	<li>Add 100 &#181;L of either histamine (25 &#181;M), PMA (100 nM), UH, HK, or HR (all at 25 &#181;g/mL) diluted in serum-free medium (as in step 1.4) to stimulate the BOEC monolayer for 15 min at room temperature and ambient O2/CO2. 
	NOTE: Although not necessary for VWF release, performing this stimulation at 37 &#176;C will improve VWF exocytosis27.</li>
	<li>Ensure that one of the three separate lanes of the flow chamber remain untreated as an experimental control.</li>
	<li>Remove the medium using a 1 mL syringe and replace it with 100 - 200 &#181;L of PBS to wash the secretagogues off of the endothelial monolayer.</li>
	<li>Mount the flow chamber onto a slide stand using vascular clips to secure the chamber in place.</li>
	<li>Attach the available male Luer connector on the end of the 70 cm tubing (connected to the syringe) to the reservoir of the flow chamber. Repeat for the two other flow chamber lanes.</li>
	<li>Start the syringe pump to remove the PBS wash. Pipette additional PBS into the opposing flow chamber reservoir to ensure that the lanes do not run dry.</li>
	<li>Observe the flow system for equivalent flow across all three lanes and then pause the syringe pump.</li>
	<li>Select an area to capture the images in about the middle of each lane, in roughly the same area between experiments. 
	NOTE: The person operating the microscope can be blinded to the experimental conditions. This step ensures that the microscope is focused prior to platelet flow (refer to step 7).</li>
	<li>Attach a female luer coupler<a> </a>to a 5 mL syringe and then to the free male luer connector (step 6.8) and submerge the blunted needle in the 1.5 mL microcentrifuge tube containing the washed platelet solution (step 5.13). Slowly draw the platelet solution through the tubing to ensure no air bubbles within the system.</li>
	<li>Remove the 5 mL syringe and attach the male Luer to the open reservoir of the flow chamber. Repeat for the three lanes.</li>
	<li>Restart the syringe pump and capture images during platelet flow using the microscope and protocol settings from step 7 for 10 min in the dark.</li>
	<li>After 10 min, disconnect the Luer from the intake tubing on the flow chamber reservoir and fill the reservoir with PBS to maintain flow over the endothelial monolayer.</li>
</ol>
9. VWF-platelet String Quantification
NOTE: The continuous flow of PBS is required to maintain the VWF-platelet string elongation for image analysis. Cessation of flow will result in the VWF becoming globular and will impair VWF-platelet string quantification. It is essential to top up each reservoir with PBS during image capture. HBSS can replace PBS if cell shrinking or detachment is observed during this step.
<ol>
	<li>Beginning with the first lane, capture 10 images per slide without overlap using a 20X objective lens around middle of the slide. Repeat this for each lane. 
	NOTE: The areas surrounding the reservoirs should be avoided due to turbulent shear stress.</li>
	<li>Using image processing software, adjust the brightness by clicking &#34;Image,&#34; &#34;Adjust,&#34; and &#34;Brightness/Contrast.&#34; Drag the scroll bar until the adhered platelets can be viewed well enough for quantification.</li>
	<li>Record a VWF-platelet string when three or more aligned platelets are observed in the image. Count all the VWF-platelet strings in an image. Repeat for all 10 images and average for the experiment. 
	NOTE: The person performing the quantification can be blinded to the experimental conditions. It is not necessary to co-stain for VWF, as this has been confirmed by independent studies8,28,29.</li>
	<li>Repeat the experiment a minimum of three times to evaluate each secretagogue.</li>
</ol>

The authors declare no competing financial interests.

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 ...

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11.1K Views.  Queen's University. 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.

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

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 complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

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 (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

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 controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

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 system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

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 hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

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 from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

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 drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

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 (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

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

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

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 conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.