Name: turbidimetry on human washed platelets: the effect of the pannexin1-inhibitor brilliant blue fcf on collagen-induced aggregation
Uploaded: 2017-04-06T13:00:00
Description: Watch this Scientific Journal Video about Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation at JoVE.com

This work was supported by grants from the Swiss National Science Foundation (310030_162579/1 to Brenda Renata Kwak and 320030_144150 to Pierre Fontana) as well as by a grant from the Swiss Heart Foundation.

Five unrelated healthy volunteers were recruited for blood sampling for platelet isolation and aggregation tests. Written informed consent was obtained and the protocol was approved by the Central Ethics Committee of the Geneva University Hospitals. All volunteers certified to be healthy and to have not taken any platelet-interfering drugs during at least the 10 days preceding the experiments.
1. Buffer Preparation for Human Blood Collection and Washed Platelet Isolation
<ol>
	<li>Prepare a ...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+volume+changes+associated+with+light+transmittance+changes+in+the+platelet+aggregometer:+dependence+upon+aggregating+agent+and+effectiveness+of+stimulus.">Particle volume changes associated with light transmittance changes in the platelet aggregometer: dependence upon aggregating agent and effectiveness of stimulus.</a> Thromb Res. 41 (5), 615-626 (1986).</li><li>Malinski, J. A., Nelsestuen, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+turbidity+to+the+stages+of+platelet+aggregation.">Relationship of turbidity to the stages of platelet aggregation.</a> Biochim Biophys Acta. 882 (2), 177-182 (1986).</li><li>Riess, H., Braun, G., Brehm, G., Hiller, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+evaluation+of+platelet+aggregation+in+whole+human+blood.">Critical evaluation of platelet aggregation in whole human blood.</a> Am J Clin Pathol. 85 (1), 50-56 (1986).</li><li>Hayward, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+methods+for+determining+reference+intervals+for+light+transmission+platelet+aggregation+tests+on+samples+with+normal+or+reduced+platelet+counts.">An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts.</a> Thromb Haemost. 100 (1), 134-145 (2008).</li><li>Frelinger, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+function+tests,+independent+of+platelet+count,+are+associated+with+bleeding+severity+in.">Platelet function tests, independent of platelet count, are associated with bleeding severity in.</a> ITP. Blood. 126 (7), 873-879 (2015).</li><li>Fusegawa, Y., Goto, S., Handa, S., Kawada, T., Ando, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+spontaneous+aggregation+in+platelet-rich+plasma+is+increased+in+habitual+smokers.">Platelet spontaneous aggregation in platelet-rich plasma is increased in habitual smokers.</a> Thromb Res. 93 (6), 271-278 (1999).</li><li>Davis, R. B., Boyd, D. G., McKinney, M. E., Jones, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+and+exercise+conditioning+on+blood+platelet+function.">Effects of exercise and exercise conditioning on blood platelet function.</a> Med Sci Sports Exerc. 22 (1), 49-53 (1990).</li><li>Yee, D. L., Sun, C. W., Bergeron, A. L., Dong, J. F., Bray, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregometry+detects+platelet+hyperreactivity+in+healthy+individuals.">Aggregometry detects platelet hyperreactivity in healthy individuals.</a> Blood. 106 (8), 2723-2729 (2005).</li><li>Cattaneo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+Standardization+of+Light+Transmission+Aggregometry:+A+Consensus+of+the+Working+Party+from+the+Platelet+Physiology+Subcommittee+of+SSC/ISTH.">Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.</a> J Thromb Haemost. , (2013).</li><li>Patel, D., Zhang, X., Veenstra, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+hemichannel+and+pannexin+channel+electrophysiology:+how+do+they+differ?.">Connexin hemichannel and pannexin channel electrophysiology: how do they differ?.</a> FEBS Lett. 588 (8), 1372-1378 (2014).</li><li>European Food Safety Authority. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+Opinion+on+the+re-evaluation+of+Brilliant+Blue+FCF+(E+133)+as+a+food+additive.">Scientific Opinion on the re-evaluation of Brilliant Blue FCF (E 133) as a food additive.</a> EFSA Journal. 8 (11), 1853-1889 (2010).</li><li>Lages, B., Scrutton, M. 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There is great interest in finding new drugs capable of modulating platelet function in order to prevent thrombosis without enhancing the risk of bleeding. For this purpose, in vitro laboratory tests which can reliably and reproducibly monitor aggregation responses in human platelets are absolutely necessary. Turbidimetric aggregometry is an easy technique to perform. However, some precautions need to be kept in mind. The measurements need to be performed under continuous stirring as the aggregation process is l...

Platelets are crucial components of blood and their main function-together with coagulation factors-is to stop bleeding after blood vessel injury. Platelets are small (2-3 &#181;m) anuclear fragments derived from megakaryocytes of the bone marrow1. Platelets circulate in non-activated state, during which they appear as lens-shaped structures. Upon interruption of the endothelium, platelets gather to the site of blood vessel injury to plug the hole, a process called primary hemostasis. Initially, platelets attach to sub-endothelial molecules, such as collagen and von Willebrand factor, that are exposed as a result of the injury-adhesion step2. Then, they change shape and secrete chemical messengers-activation step. Finally, they connect to each other by bridging receptors-aggregation step. Primary hemostasis is followed by a secondary process involving activation of the coagulation cascade with fibrin deposition, which stabilizes the initial thrombus2.
Acute ischemic events such as myocardial infarction3 often result from thrombi that form because of physical disruption (rupture) of an atherosclerotic plaque. Current anti-platelet drugs are the cornerstone of the treatment of this widespread disease but their clinical benefit is limited by an increased risk for bleeding. The most prescribed drugs in cardiovascular patients, aspirin and anti-P2Y12 compounds, target the thromboxane A2 and the ADP pathways4, respectively, which are the major pathways leading to platelet activation. However, innovative research towards new targets that would optimally balance antithrombotic effects and haemorragic risk is still necessary.
From the 1960s5 to today, turbidimetric aggregometry has played a crucial role in research, enhancing our knowledge of platelet reactivity and in the monitoring of the potency of anti-thrombotic reagents in humans. Turbidimetry was initially applied to PRP extracted from blood samples. Indeed, blood collection performed in tubes containing citrate allows fast and large production of PRP without having any effect on platelet integrity and function. However, the short-term stability (about 3 h) of PRP and the remaining plasmatic enzymes, such as thrombin, and the low calcium concentration associated with potentially artefactual aggregation profiles are of major inconvenience for the use of PRP. An important step forward has been the development of a method for platelet isolation with additional centrifugation and washing steps6. In short, PRP is isolated from whole blood collected on acid-citrate-dextrose (ACD) and platelets are isolated after serial centrifugation steps before being resuspended in an iso-osmotic phosphate buffer (Tyrode&#39;s buffer) containing glucose, human serum albumin and divalent cations (Ca2+ and Mg2+). To avoid changes in platelet reactivity, the pH of Tyrode&#39;s buffer is carefully kept at 7.35-7.4. Moreover, undesired activation of platelets is prevented by adding prostacyclin (PGI2) before some centrifugation steps. Finally, addition of apyrase prevents washed platelets from becoming resistant against the action of ATP/ADP. The resulting platelet suspension lacks coagulant factors and the stability of platelets is increased by at least two-fold as compared to PRP solutions. In addition, the fact that platelets are inactive but intact warrants the reproducibility of turbidimetric measurements and provides the ability to study the action of agonists or antagonists of platelet aggregation in an optimal way.
Using this method, we have shown in a recent study that inhibiting the formation of Panx1 channels by a genetic approach (knock-out mice) or decreasing Panx1 channel activity by pharmacological approaches reduced collagen-induced platelet aggregation7. Panx1 forms ATP-release channels, which are ubiquitously expressed in many cell types including human platelets7,8. In fact, we demonstrated by turbidimetry on human washed platelets that a 7 min preincubation with a panel of more-or-less specific chemical blockers (probenecid, mefloquine and 10Panx1 peptides) prior to the addition of various agonists, inhibited specifically collagen-induced platelet aggregation while platelet responses to AA and ADP were not affected. We demonstrated that ATP release through Panx1 channels specifically interferes in the GPVI signaling pathway leading to collagen-induced aggregation. Interestingly, multiple FDA-approved compounds with applications in other diseases (probenecid, mefloquine) affect the activity of Panx1 channels in platelets. On one hand, this opens new therapeutic perspectives to selectively modify platelet reactivity. On the other hand, one should consider potential secondary effects of these compounds. In this context, the safe food dye Brilliant Blue FCF used in multiple candies and energy drinks has been described as a selective inhibitor of Panx19. We describe here a protocol for the isolation of human washed platelets and turbidimetric measurements of platelet aggregation adapted to investigate the effect of the Brilliant Blue FCF dye as an antagonist of platelet aggregation.

<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&#160;</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&#160;</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>

turbidimetry on human washed platelets: the effect of the pannexin1-inhibitor brilliant blue fcf on collagen-induced aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

The aggregometer software automatically produces the aggregation curves and gives the values for maximal aggregation in percentage. The values can be copied to a data analysis software in order to perform statistical analysis and visualize maximal aggregation values in form of bar charts. Optionally, each individual point of the aggregation curves can be exported successively into a spreadsheet software and then to statistical software (e.g. GraphPad) in order to visualize the cu...

Watch this Scientific Journal Video about Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation at JoVE.com

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

The overall goal of this procedure is to describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. This method can help answer key questions in the field of human platelet biology such as the effects of pannexin-1 inhibition on agonist-induced platelet aggregation. The main advantage of this technique is that it is rapid and easy to perform while it allows reliable and reproducible monitoring of aggregation responses.Light transmission aggregometry is not only used for research purposes but it is also used for the investigation of platelet function in patients with bleeding disorders. To begin, dissolve 1.4 grams of citric acid monohydrate, 2.5 grams of trisodium citrate dihydrate, and two grams of anhydrous D-glucose in distilled water to prepare 100 milliliters of an ACD solution with a pH of approximately 4.5. Then prepare Tyrode's buffer by diluting 2.5 milliliters of stock solution one in a final volume of 15 milliliters of distilled water.Adjust the pH to 7.35 by adding one normal hydrogen chloride and filter sterilize the solution using a 0.22 micron filter. Finally, add five milliliters of stock solution one, one milliliter of stock solution two, two milliliters of stock solution three, 0.5 milliliters of one molar heaps, 1.8 milliliters of 200 grams per liter human serum albumin, and 0.1 gram of anhydrous d-glucose to a final volume of 100 milliliters distilled water to prepare the TA buffer. Using one normal hydrogen chloride, adjust the pH to 7.35 and set the osmolarity to 295 milliosmoles per liter with distilled water.Fill 50 milliliter tubes with one volume of ACD anticoagulant per anticipated six volumes of blood to be collected. Introduce a 19 gauge needle into an antecubital vein of a healthy donor. Discard the initial one to two milliliters of the withdrawn blood as it may contain thrombin and tissue factor and collect the following 45 to 50 milliliters.Gently invert the tube to mix the blood with the ACD solution. Then incubate the sample in an oven at 37 degrees Celsius for 10 minutes. To prepare platelet-rich plasma, transfer five milliliter aliquots of the collected blood into 15 milliliter tubes and centrifuge the samples at 250 times g in 37 degrees Celsius for 13 minutes.When centrifuged, aspirate the top layer of the samples, avoiding any red and white blood cell contamination. Then transfer the obtained platelet-rich plasma into a new 15 milliliter tube and incubate it at 37 degrees Celsius for 10 minutes. Centrifuge platelet-rich plasma at 2, 200 times g for 12 minutes.Next, remove the supernatant and using a plastic Pasteur pipette, carefully resuspend the pellet in 10 milliliters of the TA buffer containing two microliters per milliliter of heparin and 2.5 microliters per milliliter of 25 micromolar prostacyclin in Tris-HCL buffer. Incubate the sample for 10 minutes at 37 degrees Celsius. Then add 2.5 microliters per milliliter of 25 micromolar prostacyclin and centrifuge the sample at 1, 900 times g for eight minutes.After spinning, remove the supernatant and with a plastic Pasteur pipette, resuspend the pellet in five milliliters of the TA buffer containing 2.5 microliters per milliliter of 25 micromolar prostacyclin. Incubate the platelets at 37 degrees Celsius for 10 minutes one more time. While the sample is being incubated, transfer 150 microliters of the platelet suspension into a 1.5 milliliter tube and count the platelets using an automated cell counter.After incubation, add 2.5 microliters per milliliter of 25 micromolar prostacyclin to the platelet suspension and immediately centrifuge the sample at 1, 900 times g for eight minutes. Remove the supernatant and resuspend the pellet to achieve a density of 250, 000 platelets per microliter using an adequate volume of the TA buffer containing apyrase at 0.32 units per milliliter. Before the aggregametric measurements, incubate the cell suspension at 37 degrees Celsius for at least 30 minutes.To proceed with aggregometry, dissolve fibrinogen in Tyrode's buffer to obtain a 56 milligram per milliliter solution. Pipette 260 microliters of the platelet suspension into glass cuvettes containing 10 microliters of the fibrinogen solution and a magnetic stirring rod. Then incubate the suspensions for two to three minutes at 37 degrees Celsius in an incubation well within the aggregometer.Next, add 10 microliters of a 2.8 millimolar brilliant blue FCF stock solution prepared in distilled water and pre-incubate the suspension at 37 degrees Celsius for seven minutes. To calibrate the aggregometer, prepare a cuvette containing a solution of 10 microliters fibrinogen, 10 microliters brilliant blue FCF solution, and the TA buffer without platelets that corresponds to 100%aggregation. Place the cuvette in an aggregometer channel with automatic stirring turned on.Indicate the experimental channel in the software controlling the apparatus and measure the optical density of the solution. Then under constant stirring conditions, calibrate the aggregometer using a platelet sample with no agonist added corresponding to 0%aggregation. Place the cuvette in the aggregometer and indicate the experimental channel in the software controlling the apparatus.Wait for about 20 to 30 seconds to ensure no aggregation occurs. Then add 20 microliters of the desired agonist into the cuvette. Immediately start recording aggregation data.Proceed with a readout for six minutes and save the data when completed. Presented here are the aggregation curves obtained for collagen-stimulated platelets that were incubated with either water or different concentrations of brilliant blue FCF. The low brilliant blue FCF concentration did not influence the platelet response to collagen while brilliant blue FCF at one millimolar completely abolished the collagen-induced platelet aggregation.Finally, the platelet inhibiting activity of brilliant blue FCF was shown to be specific towards platelets activated by collagen stimulation whereas it did not influence the aggregation of platelets activated with arachidonic acid as demonstrated by five independent measures taken in healthy volunteers. Once mastered, this technique can be done in four to six hours, depending on the number of tests, if it is performed properly. After watching this video, you should have a good understanding of how to perform turbidimetry-based aggregation tests on washed platelets isolated from human blood.Don't forget that working with human blood can be hazardous and particular precautions such as wearing protective gloves should always be taken while performing this procedure.

Research

JoVE Journal

Medicine

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