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

这项工作是由来自瑞士国家科学基金会资助（310030_162579 / 1布伦达雷娜塔郭某和320030_144150皮埃尔丰塔纳），以及由来自瑞士心脏基金会的资助。

五名例健康志愿者招募采血进行血小板分离和聚集测试。获得书面知情同意书和协议批准了日内瓦大学医院的中央伦理委员会。认证所有的志愿者是健康的，并在至少试验前10天都没有采取任何血小板干扰药物。 1.缓冲液配制人类血液收集和洗涤过的血小板分离<ol><li>通过溶解1.4克柠檬酸一水合物（C 6 H 8 O 7•H 2 O，66.6毫摩尔），将2.5g柠檬酸...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+human+platelet+activation+by+surface+pannexin-1+channels.">Amplification of human platelet activation by surface pannexin-1 channels.</a> J Thromb Haemost. 12 (6), 987-998 (2014).</li><li>Wang, J., Jackson, D. G., Dahl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+food+dye+FD&amp;C+Blue+No.+1+is+a+selective+inhibitor+of+the+ATP+release+channel+Panx1.">The food dye FD&amp;C Blue No. 1 is a selective inhibitor of the ATP release channel Panx1.</a> J Gen Physiol. 141 (5), 649-656 (2013).</li><li>Mahaut-Smith, M. P., Jones, S., Evans, R. 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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=Preparation+of+washed+platelet+suspensions+from+human+and+rodent+blood.">Preparation of washed platelet suspensions from human and rodent blood.</a> Methods Mol Biol. 272, 13-28 (2004).</li><li>Jarvis, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation:+turbidimetric+measurements.">Platelet aggregation: turbidimetric measurements.</a> Methods Mol Biol. 272, 65-76 (2004).</li><li>Thompson, N. T., Scrutton, M. C., Wallis, R. 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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有一个在寻找能够以防止血栓形成，而不增加出血的风险调控血小板功能的新药极大的兴趣。为了这个目的， 体外实验室试验能可靠和可重复地监测人体血小板凝集反应是绝对必要的。比浊集合度​​是执行一个简单的技术。然而，一些注意事项需要牢记。测量需要在连续搅拌下将要执行的聚集过程在很大程度上取决于搅拌。同样重要的是，保持在血小板的37℃的生理温度，以避免任何类?...

血小板是血液，其主要有凝血功能在一起的重要组成部分的因素，是阻止血管损伤后出血。血小板是从骨髓1的巨核细胞衍生的小（2-3微米）的无核片段。血小板循环在非激活状态，在此期间，它们显示为透镜形结构。在内皮的中断，血小板聚集到血管损伤部位，以堵塞漏洞，一个称为原发性止血过程。最初，血小板附着到内皮下分子，如胶原蛋白和血管性血友病因子，被暴露的损伤粘附步骤2的结果。然后，他们改变形状和分泌的化学信使，激活步骤。最后，它们通过桥接受体聚集步骤相互连接。初期止血之后是涉及凝血级联的活化与纤维蛋白沉积的二次处理，其稳定S中的初始血栓2。 急性局部缺血事件如心肌梗死3通常由血栓形成，因为动脉粥样硬化斑块的物理破坏（破裂）的结果。目前的抗血小板药物是这种普遍的疾病的治疗的基石，但它们的临床益处被出血风险增加的限制。在心血管患者中，阿司匹林和抗P2Y12化合物最处方药，靶向血栓烷A2和分别ADP通路4，这是导致血小板活化的主要途径。不过，迈向新的目标，将最大限度地平衡血栓形成及出血性风险创新性的研究仍然是必要的。 从20世纪60年代5至今天，浊度凝集起到研究了至关重要的作用，增强了我们的血小板反应性和我的知识N的人类抗血栓剂的效力的监督。比浊法最初应用于从血液样品中提取PRP。事实上，血液收集在含有柠檬酸允许快速和大生产的PRP，而无需对血小板的完整性和功能的任何影响的试管中进行。然而，PRP的短期稳定性（约3小时），并且剩余的血浆酶如凝血酶，和具有潜在假象聚合简档相关联的低钙浓度是使用PRP的主要不便。一个重要的进步已经的方法的发展为血小板隔离用另外的离心和洗涤步骤6。总之，PRP从收集在酸 - 柠檬酸盐 - 葡萄糖（ACD）全血中分离和血小板被串行离心步骤之后，分离被再悬浮于等渗磷酸缓冲液（蒂罗德缓冲液）含有葡萄糖，人血清白蛋白和的二价C前ations（CA 2+和Mg 2+）。为了避免血小板反应性的变化，台氏缓冲液的pH值仔细地保持在7.35-7.4。另外，血小板的不希望的激活是由之前的一些离心步骤加入前列环素（PGI 2）防止。最后，加入腺苷三磷酸双可防止洗涤血小板变得对ATP / ADP的作用有抗性。将所得的血小板悬浮液缺乏凝血因子和血小板的稳定性增加至少两倍相比PRP的解决方案。此外，事实上，血小板活性，但是完整权证浊度测量的再现性，并提供研究激动剂或血小板聚集的拮抗剂的作用以最佳的方式的能力。 使用这种方法，我们在最近的研究已经表明，通过遗传方法抑制Panx1通道的形成（敲除小鼠）或由药理减小Panx1通道活性接近降低胶原诱导的血小板聚集7。 Panx1形成ATP释放信道，其在多种细胞类型，包括人血小板7,8遍在表达。事实上，我们证实通过比浊法对人洗涤的血小板，一个7分钟预温育与更多或更少的特定化学阻断剂（丙磺舒，甲氟喹和10种 Panx1肽）之前加入各种激动剂的一个面板，抑制特异性胶原诱导血小板聚集而以AA和ADP的血小板反应并没有受到影响。我们表明，ATP释放通过Panx1通道在GPVI信号传导途径，导致胶原诱导的聚集特异性干扰。有趣的是，与其他疾病（丙磺舒，甲氟喹）应用的多个FDA批准的化合物影响血小板Panx1通道的活性。一方面，这会打开新的治疗前景选择结构延续修改血小板反应性。在另一方面，应该考虑这些化合物的潜在的辅助疗效。在这种背景下，安全的食品染料亮蓝FCF在多个糖果和能量饮料中使用已描述为Panx1 9的选择性抑制剂。在这里我们描述用于人洗涤的血小板和血小板聚集的浊度测量的隔离的协议适合于调查亮蓝FCF染料的血小板聚集的拮抗剂的作用。

<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

比浊法是用于测量悬浮在血浆（富含血小板的血浆，PRP）或在缓冲液（洗涤血小板）血小板聚集实验室技术，通过使用一个或激动剂的组合。使用从它们的等离子体环境和在没有抗凝血剂的分离洗涤血小板的允许研究固有血小板特性。间激动剂，花生四烯酸（AA），腺苷二磷酸（ADP）的大面板，凝血酶和胶原是最常使用的。聚合反应是由在连续搅拌下测量随血小板悬浮液的时间的相对光密度（OD）进行定量。在均匀的悬浮液血小板限制光的通过加入激动剂后，血小板形状改变发生在制造OD小的暂时性增加。以下该初始活化步骤，血小板凝块逐步形成，从而允许光通过穿过悬浮液作为水库的ULT OD降低。聚集过程最终以百分比表示，相对于贫血小板血浆或缓冲液的OD。严格校准因此在每次实验开始时是必不可少的。作为一般规则：校准到0％，通过测量非刺激的血小板悬浮液的OD同时测量不含血小板悬浮介质的OD表示的100％的值设定。血小板聚集通常显现为实时聚合曲线。比浊法是最常用的实验室技术用于血小板功能的调查之一，被认为是历史的金标准，并用于旨在抑制血小板聚集的新药物的开发。在这里，我们描述了详细方案1）制备人洗涤血小板和2）人的胶原诱导的聚集的浊分析与食品染料亮蓝FCF，这是最近的iDEN洗涤，预处理血小板tified如Pannexin1（Panx1）通道的抑制剂。

聚集仪软件自动生成聚集曲线，给人以百分比最大聚集值。这些值可以在为了进行统计分析，在条形图的形式可视化最大凝集值被复制到一个数据分析软件。任选地，聚合曲线的每个单独的点可被相继地以可视化的曲线导出到一个电子表格软件，然后统计软件（ 例如的GraphPad）。一些研究者使用聚合曲线的最大斜率计算曲线下的速度和面积，以评估血小板活性。滞?...

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

我们描述了洗涤的血小板的人血液，然后通过比浊法激动剂诱导的血小板聚集的测量隔离的简单方法。作为例子，我们申请研究的预温育与Pannexin1通道抑制剂亮蓝FCF后的人血小板聚集响应于胶原此方法。

比浊法对人洗涤血小板：所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

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

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