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EoE Sub Articles

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Buffer Preparation for Human Blood Collection and Washed Platelet Isolation
<ol>
	<li>Prepare a 100 mL aqueous solution of acid-citrate-dextrose (ACD) by dissolving 1.4 g citric acid monohydrate (C6H8O7&#8226;H2O, 66.6 mM), 2.5 g trisodium citrate dihydrate (Na3C6H5O7&#8226;2 H2O, 85 mM) and 2 g of anhydrous D(+)-glucose. The pH of the solution is about 4.5.</li>
	<li>Prepare stock solutions for Tyrode&#39;s buffer as follows
	<ol>
		<li>Prepare stock solution 1 by dissolving 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 10 g sodium bicarbonate (NaHCO3), and 0.58 g sodium dihydrogen phosphate monohydrate (NaH2PO4*H2O) in 500 mL of distilled H2O. The respective final concentrations are 2.73 M, 53.6 mM, 238 mM, and 8.4 mM. Keep the solution at 4 &#176;C.</li>
		<li>Prepare stock solution 2 by dissolving 10.15 g magnesium dichloride hexahydrate (MgCl2*6 H2O) (100 mM) in 500 mL distilled water (H2O). Keep the solution at 4 &#176;C.</li>
		<li>Prepare stock solution 3 by dissolving 10.95 g (100 mM) calcium chloride hexahydrate CaCl2*6 H2O in 500 mL distilled H2O. Keep the solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare Tyrode&#39;s buffer by diluting 2.5 mL of stock solution 1 in a final volume of 50 mL with distilled H2O. This corresponds to final concentrations of 136.5 mM NaCl, 2.68 mM KCl, 11.9 mM NaHCO3, and 0.42 mM NaH2PO4*H2O. Adjust the pH to 7.35 and sterilize by filtering with 0.22-&#956;m filters.</li>
	<li>Prepare Tyrode&#39;s albumin 0.35% buffer (TA buffer) by diluting 5 mL of stock solution 1, 1 mL of stock solution 2, 2 mL of stock solution 3, 0.5 mL 1M HEPES, 1.8 mL of 200 g/L human serum albumin and 0.1 g of anhydrous D(+)-glucose in a final volume of 100 mL distilled H2O.
	<ol>
		<li>Adjust the pH to 7.35 with 1N hydrochloric acid (HCl) and set the osmolarity to 295 mOsm/L by adding distilled H2O (10% of total volume). The final concentrations in this solution are 124 mM NaCl, 2.44 mM KCl, 10.82 mM NaHCO3, 0.38 mM NaH2PO4*H2O, 0.91 mM MgCl2*6 H2O, 1.82 mM CaCl2*6 H2O. Keep TA buffer at 37 &#176;C during the whole experiment.</li>
	</ol>
	</li>
</ol>
2. Blood Collection
<ol>
	<li>Collect 45-50 mL of venous blood, from the antecubital vein using a 19 G needle and no or low tourniquet, into 50 mL tubes containing ACD anticoagulant (1 volume ACD for 6 volumes of blood). Discard the first 1-2 mL of blood to avoid the presence of thrombin and tissue factor.
	<ol>
		<li>After collection, mix the blood with the ACD by gently inverting the tube. Incubate the sample for 10 min at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Preparation of Human Washed Platelets
<ol>
	<li>Pre-heat the centrifuge to 37 &#176;C. All centrifugation steps below are performed at this temperature.</li>
	<li>Dispatch the collected blood into 15 mL tubes (5 mL per tube) and centrifuge at 250 x g for 13 min to obtain platelet-rich plasma, PRP.&#160; 
	NOTE: This centrifugation step results in the production of three layers in the sample: 1) The upper layer, composed of plasma, platelets, and a small fraction of white blood cells. 2) The intermediate layer, a portion rich in white blood cells. 3) The bottom layer, which is essentially composed of red blood cells.</li>
	<li>Collect the PRP by pipetting the upper layer carefully into a new 15 mL tube to maximally prevent contamination with red and white blood cells, and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge the PRP at 2,200 x g for 12 min (for 5 mL PRP). 
	NOTE: This centrifugation step should be performed with low brake or without brake.</li>
	<li>Remove the supernatant (platelet-poor plasma), and carefully resuspend the pellet with 10 mL of TA buffer containing 2 &#181;L/mL of heparin (5,000 U/mL) and 2.5 &#181;L/mL of 25 &#956;M prostacyclin (PGI2) using a plastic Pasteur pipet. Incubate for 10 min at 37 &#176;C.</li>
	<li>Add 2.5 &#181;L/mL of 25 &#956;M prostacyclin-I2 (PGI2)&#160;and centrifuge for 8 min at 1,900 x g (with low brake or without brake).</li>
	<li>Remove the supernatant and resuspend the pellet with 5 mL TA buffer containing 2.5 &#181;L/mL of 25 &#956;M PGI2&#160;using a plastic Pasteur pipet. Incubate for 10 min at 37 &#176;C.</li>
	<li>During the incubation period, pipet 150 &#181;L of the platelet suspension into a 1.5 mL tube and count platelets, using an automatized cell counter (that detects the size of blood cells by measuring the changes in direct-current resistance).</li>
	<li>After the 10 min incubation, add 2.5 &#181;L/mL of 25 &#956;M PGI2&#160;to the platelet suspension and immediately centrifuge at 1,900 x g for 8 min.</li>
	<li>Remove the supernatant and resuspend the pellet to a concentration of 250,000 platelets/&#181;L with an adequate volume of TA buffer (i.e.&#160;if the cell count is 500,000 per &#181;L, resuspend in 10 mL TA buffer) containing 32 &#181;L/mL of apyrase at 0.01 U/mL (final concentration 0.32 U/mL). 
	NOTE: A high concentration of apyrase is used to avoid the desensitization of P2X purinoceptor 1, P2X1 receptors induced by spontaneous secretion of adenosine triphosphate (ATP)&#160;in the absence of agonists. This is important because collagen-induced responses are induced by fast paracrine/autocrine activation of P2X1 by ATP released from activated platelets. If the platelet signaling pathway does not critically require the preservation of P2X1 function, use 0.02 U/mL apyrase. Several studies demonstrated that 0.02 U/mL apyrase avoids adenosine diphosphate ADP receptor P2Y purinoceptor 1, and P2Y1 desensitization with negligible P2X1 responses.</li>
	<li>Incubate the cell suspension for at least 30 min at 37 &#176;C before performing the aggregometric measurements. The preparation is stable for 5 to 8 hours.</li>
</ol>
4. Aggregometry
<ol>
	<li>Prepare fibrinogen (56 mg/mL) in Tyrode&#39;s buffer.</li>
	<li>Pipet 260 &#181;L of platelet suspension into glass cuvettes (Figure 1A; left cuvette) containing 10 &#181;L of fibrinogen (56 mg/mL) and a magnetic stirring rod, then incubate the suspension for 2 - 3 min at 37 &#176;C in incubation wells present in the aggregometer (Figure 1B&#160;and&#160;1C).</li>
	<li>Pre-incubate with the Panx1 inhibitor Brilliant Blue FCF by adding 10 &#181;L of a 2.8 mM or 28 mM stock solution (final concentration 100 &#956;M and 1 mM, respectively) for 7 min at 37 &#176;C.</li>
	<li>Calibrate the aggregometer to an assumptive 100% aggregation value by measuring the optical density (OD) of a cuvette containing 10 &#181;L fibrinogen (56 mg/mL), 10 &#181;L Brilliant Blue FCF (2.8 mM or 28 mM), and TA buffer without platelets.
	<ol>
		<li>Place the cuvette in an aggregation well under automatic stirring and press the corresponding button on the keyboard of the computer linked to the aggregometer (i.e.&#160;press F1 if aggregation well 1 is used). 
		NOTE: This experiment described below includes Brilliant Blue FCF. The compound used for calibration has to be adjusted to the experimental condition.</li>
	</ol>
	</li>
	<li>Calibrate the aggregometer to an assumptive 0% aggregation value by using the same platelet sample that will be used for the experiment under automatic stirring.
	<ol>
		<li>Place the cuvette in the aggregation well and press the corresponding button on the keyboard of the computer linked to the aggregometer. Wait for about 20-30 s before proceeding. This delay serves to assure that no aggregation happens before adding the agonists.&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: As any difference in platelet number may have an effect on the measured OD, the 0% calibration step needs to be repeated for each individual measurement.</li>
	</ol>
	</li>
	<li>Add 20 &#181;L of desired agonist, such as 15 &#181;g/mL collagen (1 &#181;g/mL final) or 1.125 mM arachidonic acid (75 &#181;M final), into the cuvette. Immediately start the recording under continuous automatic stirring by pressing the corresponding button on the keyboard of the computer linked to the aggregometer. 
	NOTE: The addition of the agonist induces platelet activation. Platelet aggregates can clearly be distinguished in the glass cuvette at the end of the experiment (Figure 1A; right cuvette).</li>
	<li>The recording automatically stops after 6 min. At this point, save the data by clicking on the save icon on the computer. 
	NOTE: The calculation of the rate of aggregation is performed by the computer, which expresses the end results of the aggregation process as a percentage.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Citric acid monohydrate (C6H8O7*H2O)</td>
			<td>Roth</td>
			<td>5949-29-1</td>
			<td>danger of eye damage/irritation</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate (Na3C6H5O7*2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td>-</td>
		</tr>
		<tr>
			<td>D(+)-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium dihydrogenophosphate monohydrate (NaH2PO4*H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2*6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium chloride hexahydrate (CaCl2*6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>442909</td>
			<td>danger of eye damage/irritation</td>
		</tr>
		<tr>
			<td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes)</td>
			<td>ThermoFisher Scientific</td>
			<td>15630</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Human serum albumin</td>
			<td>CSL Behring</td>
			<td>00257/374</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Corrosive and irritative for the respiratory system. Can cause severe skin and eye damages.</td>
		</tr>
		<tr>
			<td>Eppendorf 5810 R</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Drosspharm AG/SA</td>
			<td>20810</td>
			<td></td>
		</tr>
		<tr>
			<td>Prostacyclin I2 (PGI2)</td>
			<td>Cayman</td>
			<td>18220</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Apyrase from potatoes</td>
			<td>Sigma-Aldrich</td>
			<td>A6535</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fibrinogen (Haemocomplettan)</td>
			<td>CSL Behring</td>
			<td>HS 73466011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Thrombo-aggragometer SD-Medical</td>
			<td>SD-Innovation</td>
			<td>TA8V</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Brilliant blue FCF (Erioglaucine disodium salt)</td>
			<td>Sigma-Aldrich</td>
			<td>80717</td>
			<td>Harmful to aquatic life with long lasting effects (Avoid release to the environnement)</td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Horm, Nycomed</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>Arachidonic acid</td>
			<td>Bio/Data corporation</td>
			<td>C/N 101297</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cell counter Sysmex KX-21N</td>
			<td>Sysmex Digitana</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glass cuvettes</td>
			<td>SD-Innovation</td>
			<td>THCV1000</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnetic stirrers</td>
			<td>SD-Innovation</td>
			<td>THA100</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

a turbidimetric analysis to monitor collagen-induced aggregation of pretreated human platelets

Source: Molica, F. et al., <a href="https://app.jove.com/v/55525">Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates turbidimetric analysis to assess collagen-induced platelet aggregation in pre-treated, washed human platelets. Control wells show collagen-induced aggregation, whereas a high concentration of inhibitors blocks Pannexin-1 channels, thereby inhibiting collagen-induced platelet aggregation,&#160; evident from unchanged turbidity.

<img alt="Figure 1" src="/files/ftp_upload/21995/21995_Figure1.jpg" /> 
Figure 1: Aggregometry.&#160;(A) Representative image of glass cuvettes (containing a stirring magnet) used for aggregometry. The cuvette on the left shows resting platelets while the cuvette on the right illustrates platelet aggregates after collagen-induced activation. (B) Representative image of an 8-well aggregometer for turbidimetric measurements. The wells ...

A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Begin with glass cuvettes containing pre-treated, human platelet suspension containing non-activated platelets and fibrinogens &#8212; a plasma glycoprotein.
The cuvettes include a stirring device for uniform mixing.
Place the control cuvette in an aggregometer that measures the aggregation by assessing turbidity.
Introduce collagen &#8212; an agonist for platelet aggregation, interacting with platelet&#39;s surface receptors and activating downstream signaling cascades.
This results in ATP release via the Pannexin 1 channel &#8212; a platelet surface channel, initiating platelet activation and shape change, leading to a depression in the aggregation curve.
Fibrinogen further crosslinks activated platelets, promoting collagen-induced platelet aggregation, altering turbidity, and causing a gradual rise in the aggregation curve.
Treat the test cuvette with a high concentration of an inhibitor of the Pannexin 1 channel that blocks this channel.
Add collagen to the cuvette. Inhibitor interactions prevent ATP release and hinder collagen-induced platelet aggregation, evident from unchanged turbidity and a flat aggregation curve.

a-turbidimetric-analysis-to-monitor-collagen-induced-aggregation

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

81 Views. Źródło: Molica, F. et al., Zmętnienie na ludzkich płukanych płytkach krwi: Wpływ inhibitora pannexin1 Brilliant Blue FCF na agregację indukowaną kolagenem.J. Vis. Exp.(2017) Ten film pokazuje analizę mętnościową w celu oceny indukowanej kolagenem agregacji płytek krwi w wstępnie potraktowanych, umytych ludzkich płytkach krwi. Studzienki kontrolne wykazują agregację indukowaną kolagenem, podczas gdy wysokie stężenie inhibitorów blokuje kanały Pannexin-1, hamując w ten spo...

Video: Analiza turbidymetryczna w celu monitorowania indukowanej kolagenem agregacji wstępnie leczonych ludzkich płytek krwi - Concept

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.

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 ...

JoVE Journal

Medicine

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 ...

Biology

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets

Transkrypcja

A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets