Name: investigating von willebrand factor pathophysiology using a flow chamber model of von willebrand factor-platelet string formation
Uploaded: 2017-08-14T14:00:00.000+00:00
Duration: 8 min 30 s
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 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 activity is inhibited-VWF-platelet strings can be observed in vivo32 and may contribute to platelet microaggregation and leukocyte extravasation, linking the hemostatic and proinflammatory functions of VWF33. Finally, it has been hypothesized that VWF-platelet string formation might be an initiating event in venous and/or arterial thrombosis, forming the nidus around which a pathological thrombus is built.
VWF-platelet string formation in the flow chamber system can be used to study WPB exocytosis, ADAMTS13 cleavage, and the platelet binding capacity of VWF. There are several critical steps to successfully executing these experiments. However, depending on the application of the model, modifications can be made to the protocol.
Endothelial cells from specific vascular beds are heterogeneous in their response to mechanical and biochemical signals34. It may therefore be useful to select a cell type suitable for the application of this methodology. BOECs were used in the current study because of their proliferative potential; maintenance of phenotype across several (10+) passages; and robust VWF expression, facilitating the quantification of VWF exocytosis. Other investigators have used human umbilical vein endothelial cells (HUVECs)5,8,28 and non-endothelial cells (human embryonic kidney 293 cells) transfected with VWF cDNAs expressing VWD mutations35. It has been demonstrated that VWF expression varies significantly between tissue and vessel physiology36. Cultured endothelial cells arising from various micro- and macrovasculature have observable differences in VWF protein basal secretion37, and there is evidence that calcium channel differences between endothelial cell subtypes influence WPB exocytosis in response to secretagogues38.
Histones were used in this study as a novel secretagogue. They were compared to the positive control, histamine, a frequently described physiological stimulant of VWF release. A VWF:Ag ELISA was used to verify VWF release from endothelial cells under static conditions. There are two intracellular mechanisms that trigger the release of VWF: the elevation of intracellular calcium39 or pf cyclic AMP levels40. Differential activation of these pathways can be induced by secretagogues such as thrombin/histamine or epinephrine/vasopressin (or, relevant to VWD, desmopressin), respectively, and it may be interesting to compare these two pathways using this flow chamber model of VWF-platelet string formation. In the context of thrombo-inflammatory disease, further elucidation of inflammatory biomarkers, such as cytokines41 or damage/pathogen-associated molecular patterns (DAMPS/PAMPS), and their roles in VWF-platelet string formation is encouraged.
Fluid shear stress is important when globular VWF unfolds to expose the A1 domain binding site for glycoprotein Ib on platelets11. It has been demonstrated that a minimum fluid shear stress of approximately 0.73 dyn/cm2 is required for platelet binding to VWF strings42. Furthermore, these strings form on endothelial cells at relatively low shear stresses (2.5 dyn/cm2) but can be dislodged at shear stresses of 20 dyn/cm2 or above8. In the current investigation, we selected a venous shear stress (4.45 dyn/cm2) that allowed for interactions between the endothelium, VWF, and platelets while conserving the perfusion buffer for a 10 min experiment.
Using this flow chamber system to evaluate ADAMTS13 cleavage of VWF-platelet strings requires an increase in the fluidic shear stresses over those used in this experiment. Maximal ADAMTS13 cleavage of VWF-platelet strings occurs between 10 and 30 dyn/cm2 12, a range that encompasses high venous to moderate arterial shear stress43. Modifying the shear rate in this system may be useful for comparing VWF-platelet string formation and dissolution, contributing factors in the development of venous, arterial, or microvascular thrombosis.
Additional modifications of this technique can be made to the perfusion material and the flow chamber. Secretagogues can be administered under flow conditions to resemble in vivo environments. Priming platelets with agonists to evaluate their reactivity or including leukocyte populations are extensions of this technique. Furthermore, including therapeutic agents targeting VWF multimer length, platelet binding capacity, or platelet reactivity may elucidate important mechanisms that regulate the initiation of thrombus development. Other investigators have used either in-house44 or purchased glass coverslip flow chambers45, as opposed to the polymer-based slides used in this study, and seem to obtain comparable results.
A critical consideration in the VWF-platelet string protocol is the preparation of washed platelets. It is not recommended to use whole blood in this protocol due to the presence of ADAMTS13. Agents preventing platelet activation, such as prostacyclin or apyrase, should be avoided, as this may later adversely influence platelet adhesion to VWF. Ensuring that the buffers are brought to room temperature prior to platelet isolation and using careful pipette techniques (i.e., avoiding bubbles and harsh shear stresses) are key to maintaining the platelets in their inactive state. Furthermore, labeling platelets with a mitochondrial stain, such as DIOC6 or rhodamine 6G, is preferred over interfering with extracellular domains that may be important for VWF binding. However, as DIOC6 is a membrane-permeable dye, it may also be taken up in small quantities by the endothelial cells over the course of the experiment. In the absence of a fluorescent microscope, other studies have observed VWF-platelet strings using bright-field microscopy46.
In conclusion, this flow chamber methodology of VWF-platelet string formation can provide mechanistic, real-time data to support in vivo observations in the pathogenesis of thrombosis. As exposure to shear strongly influences the pathophysiological role that VWF plays in thrombo-inflammatory disease, the flow chamber model is a vital investigative tool for any lab interested in understanding endothelial cell-platelet interactions in this context.

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&#039; 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&#039;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>&amp;micro;-Slide III 0.1 (flow chambers)</td><td>Ibidi</td><td></td><td>This product has been discontinued. We suggest using &amp;micro;-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 that because histones can bind directly to VWF30, they may differentially interfere with VWF:Ag detection in the culture medium. To evaluate histone binding to VWF, we used a solid-phase binding assay. We showed that UH and HR bound more strongly to VWF than HK (Figure 2), supporting the detection interference demonstrated in Figure 1.
Extracellular histones have been shown to activate platelets in vitro19, and our data suggest that histones mediate VWF release from endothelial cells. To characterize the effect of histones on VWF-platelet interactions, we used a flow chamber model of VWF-platelet string formation in which labelled platelets were perfused on stimulated endothelial cells. Representative images were captured post-flow, and the number of VWF-platelet strings were quantified.
We observed that, following the initiation of flow conditions, untreated cells had very few VWF-platelet strings (&#60;1 string per review field), while histamine and PMA-treated cells had an average of 2.10 and 3.10 strings, respectively. Significantly more VWF-platelet strings were formed when cells were treated with HK (3.52 strings, P = 0.019), HR (6.00 strings, P = 0.004), and UH (5.51 strings, P = 0.012). A graphical representation of the VWF-platelet string count is shown in Figure 3G. Real-time VWF-platelet string formation in response to UH compared to untreated cells is shown in Videos 1A and B.
These studies, along with investigations by other groups, show that histones28 and other proinflammatory agents such as histamine5 and cytokines31 stimulate VWF secretion from endothelial cells, mediating subsequent platelet capture in vitro. We demonstrated that the static stimulation of endothelial cells was not sufficient to elucidate VWF release due to the capability of histones to bind and sequester VWF. The design of a flow chamber model of VWF release and platelet adhesion was therefore required to more completely characterize and model the in vivo interactions between the endothelium, VWF, and platelets.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55917/55917fig1.jpg" /> 
Figure 1: Lysine-rich Histones Induces VWF Release from Cultured Endothelial Cells. BOECs were stimulated with unfractionated histone (UH), lysine-rich (HK), and arginine-rich (HR) histone fractions. HK stimulated VWF release into the culture medium. UH and HR treatment decreased the detection of VWF:Ag in the endothelial cell medium relative to the untreated (Untr) control. N &#8805;3 individual experiments were performed. The data are shown as the mean values &#177; SE. *P &#60;0.05, **P &#60;0.01, and ***P &#60;0.001 indicate the significance relative to the untreated condition. This figure has been modified from Michels et al15. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55917/55917fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55917/55917fig2.jpg" /> 
Figure 2: VWF-histone Binding Interferes with VWF Detection In Vitro. Plasma-derived VWF binds more strongly to HR and UH in a dose-dependent fashion compared to lysine-rich (HK) histones in a solid-phase binding assay. The data are shown as the mean values &#177; SE. N&#8805;3 individual experiments were performed. This figure has been modified from Michels et al15. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55917/55917fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55917/55917fig3.jpg" /> 
Figure 3: Histones Mediate VWF-platelet String Formation. Endothelial cells seeded on flow chamber slides were treated with unfractionated histone (UH), lysine-rich (HK), or arginine-rich (HR) histone fractions or histamine/PMA (positive controls) and were perfused at a shear stress of 4.45 dyn/cm2 for 10 min with washed, fluorescently labeled platelets. Representative images were obtained post-platelet flow from the untreated control (A) and cells treated with histamine (B), Phorbol 12-myristate 13-acetate (PMA) (C), HK (D), HR (E), and UH (F). PMA treatment resulted in an average of 3.1 strings per field of view, comparable to histamine. VWF-platelet strings were quantified from 10 consecutive review fields and averaged for n&#8805;3 individual experiments (G). The data are shown as the mean values &#177; SE. *P &#60;0.05 and **P &#60;0.01 indicate significance relative to the untreated condition. This figure has been modified from Michels et al15. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55917/55917fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Videos. VWF-platelet String Formation in Response to Unfractionated Histone (2) Compared to No Treatment (1): This video has been modified from Michels et al15. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55917/Supplemental_Videos.zip">Please click here to download these videos.</a>

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 ...

investigating-von-willebrand-factor-pathophysiology-using-flow

Universidad San Sebastian

Research

JoVE Journal

Medicine

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

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Biology

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Bioengineering

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Immunology and Infection

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

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).

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.

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Platform for Assessing Platelet Function in Trauma and Transfusion

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

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

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Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

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In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

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Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

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Microfluidics in Assessing Platelet Function