Name: turbidimetry on human washed platelets: the effect of the pannexin1-inhibitor brilliant blue fcf on collagen-induced aggregation
Uploaded: 2017-04-06T13:00:00.000+00:00
Duration: 9 min 13 s
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柠檬酸三钠二水合物（的Na 3 C 6 H 5制备酸-柠檬酸盐-葡萄糖（ACD）的100毫升水溶液O 7•2 H 2 O，85毫摩尔）和2克无水D（+） -对葡萄糖。该溶液的pH为约4.5。 </li><li> 为蒂罗德缓冲准备股票方案如下<ol><li>通过溶解80克的NaCl，2g的氯化钾，10g的碳酸氢钠和0.58克的NaH 2 PO 4 * H 2 O在500ml的蒸馏水H 2 O的各自的最终浓度为2.73男，53.6毫米，238制备储备溶液1 mM和8.4毫米。保持在4℃下该溶液中。 </li><li>通过在500毫升10.15克的MgCl 2 * 6 H 2 O（100毫摩尔）蒸馏水H 2 O溶液保持在4℃下溶解制备储备溶液2。 </li><li>通过溶解10.95克（100毫米）的CaCl 2 * 6 H在500ml蒸馏水H 2 2 O O.保持溶液在4℃下制备储备溶液3。 </li></ol></li><li>通过在50毫升蒸馏水H 2 O的最终体积。这对应于136.5毫氯化钠，2.68毫米氯化钾，终浓度稀释2.5毫升的储备溶液1准备蒂罗德缓冲液11.9毫米的NaHCO 3和0.42毫摩尔的NaH 2 PO 4 * H 2 O.将pH调节至7.35，并通过与0.22μm的过滤˚F消毒ilters。 </li><li> 通过稀释5毫升储备溶液1准备台氏白蛋白0.35％缓冲液（TA缓冲器7），1毫升的储备溶液2,2毫升的储备溶液3，将0.5mL 1M HEPES，1.8毫升200g / L的人血清白蛋白的和0.1克无水D（+） -对在100毫升的最终体积葡萄糖蒸馏H 2 O. <ol><li>将pH调节至7.35，用1N的HCl，并通过加入蒸馏水H 2 O（总体积的10％）设定的摩尔渗透压浓度至295毫渗量/ L。最终浓度在该溶液中有:124毫米的NaCl，2.44毫摩尔的KCl，10.82毫的NaHCO 3，0.38毫摩尔的NaH 2 PO 4 * H 2 O，0.91毫摩尔MgCl 2 * 6 H 2 O，1.82毫摩尔CaCl 2的* 6 H 2 O ，保持在37℃TA缓冲器在整个实验期间。 </li></ol></li></ol> 2.采血车<ol><li> 收集45-50毫升静脉血，从使用19号针和无或低的止血带肘前静脉，到50 mL管CON泰宁ACD抗凝剂（1个体积ACD为6个体积的血液）。丢弃的血液第一1-2毫升，以避免凝血酶和组织因子的存在。 <ol><li>收集后，轻轻颠倒混合管与ACD血。在37℃下孵育10分钟的样品。 </li></ol></li></ol> 3.人的制备洗涤血小板<ol><li>预加热离心机至37℃。所有离心步骤如下在此温度下进行。 </li><li>调度所采集的血液成15 mL管（每管5mL）和离心机在250×g离心13分钟，得到PRP。 注意:此离心步骤导致产生的三个层的样品中:1）上层，血浆，血小板组成，和白血细胞的一小部分。 2）中间层，富含白细胞的部分。 3）底层，其基本上由红血细胞。 </li><li>通过吸取上层LA收集PRP揭掉仔细到一个新的15ml管中，以最大程度地防止污染与红和白血细胞，并在37℃下孵育10分钟。 </li><li>离心PRP在2200×g离心12分钟（5毫升PRP）。 注意:此离心步骤应与低速制动器或无制动来执行。 </li><li>除去上清液（贫血小板血浆），并使用塑料巴斯德吸管小心地重悬沉淀，用10毫升含2μL/ mL肝素的TA缓冲液（5000 U / mL）和25μMPGI 2的2.5μL/毫升。在37℃下孵育10分钟。 </li><li>添加2.5μL/ mL的25μMPGI 2和离心机在1900×g离心8分钟（与低速制动器或无制动）。 </li><li>除去上清，重悬与使用塑料巴斯德吸管含有2.5μL/ mL的25μMPGI 2的5毫升TA缓冲液中的沉淀物。在37℃下孵育10分钟。 </li><li>在温育期间，吸管的血小板悬浮液的150μL在到1.5mL管中并计数血小板，使用自动化的细胞计数器（即通过测量直流电阻的变化来检测血细胞的尺寸）。 </li><li> 10分钟温育后，加入2.5μL/ mL的25μMPGI 2与血小板悬浮液，并立即在1900×g下离心8分钟。 </li><li>去除上清，重悬沉淀至250,000血小板/μL的浓度与TA缓冲区的适当体积（ 即 ，如果细胞数为50万每μL，在10毫升TA缓冲液重悬）含有腺苷三磷酸双的32μL/ mL，在为0.01U /毫升（最终浓度0.32 U / mL）中。 注:腺苷三磷酸双高浓度使用，以避免在不存在激动剂的ATP 10,11自发分泌诱导P2X1受体的脱敏作用。这是重要的，因为胶原蛋白诱导的反应是通过快速旁分泌诱导/三磷酸腺苷P2X1的分泌激活发布FROM活化血小板。如果血小板信号通路不严重要求P2X1功能的保存，使用0.02 U / mL的腺苷三磷酸双。一些研究（在Mahaut-Smith 等人的综述10）证明了0.02 U / ml的腺苷三磷酸双避免了与可忽略的响应P2X1 ADP受体脱敏P2Y1。 </li><li>执行aggregometric测量之前孵育细胞悬浮液在37℃下至少30分钟。制剂是5至8小时是稳定的。 </li></ol> 4.集合度<ol><li>准备在蒂罗德缓冲液的纤维蛋白原（56毫克/毫升）。 </li><li>吸管260μL的血小板悬浮液在玻璃试管（ 图1A;左试管）含有10微升的纤维蛋白原（56毫克/毫升）和磁力搅拌棒，然后孵育2的悬浮液- 3分钟，在37℃在温育的孔本在凝集（ 图1B和1C）。 </li><li>与Panx1预孵化通过在37℃下加入2.8 mM或28 mM储备溶液（最终浓度为100μM和1mM，分别地）进行7分钟的10μL抑制剂亮蓝FCF。 </li><li> 通过测量含有10微升血纤维蛋白原（56毫克/毫升），10微升亮蓝FCF（2.8 mM或28毫摩尔）和TA缓冲无血小板的比色皿的OD校准凝集到一个假设100％的聚集值。 <ol><li>放置反应杯在聚合以及下自动搅拌和按键盘链接到凝集的计算机上的相应按钮（ 即按F1如果聚合孔1时）。 注意:下面描述的该实验包括亮蓝FCF。用于校准的化合物必须被调整到实验条件。 </li></ol></li><li> 通过使用将被用于下自动stirrin实验相同的血小板样品校准凝集到一个假设0％的聚集值G。 <ol><li>放置在聚集比色皿井和按键盘连接到凝集计算机上的相应按钮。继续之前等待约20-30秒。这种延迟供应，以确保没有聚集加入激动剂之前发生。 注意:如血小板数的任何差异可能对测量的OD的效果，0％校准步骤需要重复为每个单独的测量。 </li></ol></li><li>加入20μL所需激动剂，如15微克/毫升的胶原蛋白（1微克/毫升终浓度）或1.125毫花生四烯酸（75μM终浓度），放入试管中。立即通过按下键盘链接到凝集的计算机上的相应按钮来启动下连续搅拌下自动记录。 注:除了激动剂诱导血小板激活。血小板聚集体可清楚地在实验（ 图1A的端部的玻璃比色皿来区分;右击牛逼比色皿）。 </li><li>记录6分钟后自动停止。在这一点上，通过点击计算机的保存图标保存数据。 注:聚集速率的计算是由计算机，它体现了聚集过程以百分比的最终结果进行。 </li><li> 分析数据。 <ol><li>有关为洗涤血小板悬浮液和血小板聚集的浊测量的制备方案附加的广泛信息，指的是由该领域的专家12,13撰写其他论文。 </li></ol></li></ol>

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C., Holmsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+gel-filtered+human+platelets:+isolation+and+characterization+in+a+medium+containing+no+added+Ca2+,+Mg2+,+or+K+.">Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca2+, Mg2+, or K+.</a> J Lab Clin Med. 85 (5), 811-825 (1975).</li></ol>

作者什么都没有透露。

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，以避免任何类型的过早活化。采血过程中，也可能发生血小板预活化和洗涤血小板制剂，导致自发聚集。因此，应血小板制品完整的过程中，要采取谨慎的措施。 比浊法是基于Øn中的血小板悬浮液的OD的测量。这使得该技术不适合于在全血中测定的聚集由于红血细胞的存在。因此，比浊法只能在PRP或从PRP分离洗涤血小板来进行。虽然容易处理和相当便宜，比浊法已被公认为不敏感的微团聚体14，15的存在。当微团聚预计是至关重要的，阻抗集合度，当血小板粘附于浸渍一柠檬酸化的全血样品中的电极，其测量电阻的变化，更适合16。血小板聚集过程也很大程度上依赖于在血小板悬浮液17血小板计数;流式细胞术的患者使用来自血小板减少患18，从他们的血小板的最大数量能够得到不足以比浊法。然而，比浊法的一般优点在于，它允许血小板反应的详细分析，如形状变化和分类，这是不能全血样品中进行评估。 要认识到，在人血小板聚集自然变异存在因年龄，性别和生活方式，以及其血小板功能受到挑战19，20，21主题的健康状况的差异是很重要的。执行和解释这些实验时，在获得来自无关的健康志愿者的足够数量的血小板，让可靠的统计数据，自然变异可能是比浊结果10-20％左右，因此应小心。此限制是由以下事实:浊度测量是高度可再现的，至少当聚合然而平衡重程序是整个实验室以及标准化。标准化工作已经由血栓与止血22国际协会提出。出于这个原因，浊度仍是血小板聚集的临床和基础科学实验室测量最常遇到的考验和仍然是首选的技术来研究药物对血小板反应的影响。 在本研究中，我们已经证明，用比浊法测定人洗涤血小板反应，一个常用的食品色素亮蓝FCF影响血小板聚集。优雅的电生理研究已通过这种染料9,23透露Panx1通道的特异性阻断。事实上，这种染料的高浓度（1mM）的显示在两个胶原诱导的形状变化和最大凝集但低10倍的浓度抑制作用（100μM）染料不影响血小板聚集反应。据对作为食品添加剂亮蓝FCF（E133）的重新评价的科学意见，由欧洲食品安全局24日公布的，亮蓝FCF（分子量=792.84克/摩尔）的最大容许浓度为500毫克/公斤食物。为H 2 O，这将对应于0.63毫米，这是一个比在我们的实验中抑制胶原诱导的血小板聚集一个1.59倍浓度较低。假定只有染料的一小部分将在口服后肠道被吸收，并且该染料将终于在成年人的血液中的5-L体积稀释，每天摄入这个蓝色食用染料可能具有大幅超出正常水平，可以预期对血小板聚集的任何副作用之前。在我们对血小板的可行性实验的染料浓度最高的可能的副作用，例如，是通过的固体聚合反应存在下另一激动剂（AA）排除在外。这些影响AA反应也证实，亮蓝FCF的抑制作用似乎特别涉及胶原蛋白信号通路。这是符合我们先前的研究7详述通过胶原蛋白诱导的血小板聚集反应Panx1渠道ATP释放的特定位置。 通过调整洗涤血小板隔离的协议这项研究中，我们描述了从人血中分离血小板的简单且直接的方法。经过多次洗涤步骤通过离心执行基本上，血小板重新悬浮在尊重精确的生理条件包括pH，工作温度和二价离子的浓度，但保证了不存在的血浆蛋白的一个平台。这是这一技术优于其它方法如凝胶过滤的优点，其中等离子体COMPON的消除经济需求不会发生25。 在洗涤程序的所有步骤都是至关重要的使用洗涤血小板时获得可重复的结果。药物登塔可能会影响血小板反应性，因而是必不可少的健康志愿者被要求不要服用任何药物采血前至少10天。此外，采血必要的是着眼于避免静脉外伤或过慢流，因为这可能会导致凝血酶和随后的血小板活化22的产生。沿着相同的线，在离心和洗涤步骤潜在的血小板活化可以通过重复使用的PGI 2的被避免。由于其非常低的稳定性，PGI 2应该被添加到每个离心前立即每个洗涤步骤。在TA缓冲血小板含有腺苷三磷酸双重悬浮（在生理pH下）确保血小板反应保持完整。腺苷三磷酸双在血小板悬浮液中的存在是必要的，以允许以从它们的嘌呤能受体的脱敏保存血小板的ATP和ADP的降解。作为Panx1信号途径涉及在胶原受体活化人血小板P2X1受体活化，我们为了避免P2X1脱敏7加入腺苷三磷酸双的相对高浓度（0.32 U / mL）添加到该血小板悬浮液。为了避免在血小板其它嘌呤能受体的densensitization，腺苷三磷酸双较低浓度就足够了10。 虽然串行离心和洗涤步骤的过程是劳动密集的，并且需要更多的时间，使用这个协议提供更高的稳定性，在37℃（5-8 h）上的益处相比，直接从PRP使用血小板如（1得到的洗涤的血小板-3小时）。然而，使用PRP的选择或洗涤血小板应作出CAUtiously并应包括考虑诸如可用于实验血液数量，激动剂被用作以及将要解决的问题。例如，纤维蛋白原的活化α2β3整联作为结合介导是血小板聚集，不存在在洗涤血小板可能影响纤维蛋白原反应的主要机制;特别是，当弱激动剂（如ADP）用于13。此问题是不太关键的，当一个功能强大的激动剂时，如凝血酶或胶原蛋白，其可通过本身能够诱导纤维蛋白原从α-颗粒的释放。尽管如此，我们经常外源性纤维蛋白原，以增加由胶原诱导的聚集应答的振幅添加至经洗涤的人血小板悬浮液。 总之，使用人洗涤血小板和比浊法，我们发现，食用色素亮蓝FCF，通过对Panx1的抑制作用，表示血小板功能的潜在抑制剂。

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><tbody><tr><td>citric acid monohydrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;*H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Roth</td><td>5949-29-1</td><td>danger of eye damage/irritation</td></tr><tr><td>trisodium citrate dihydrate (Na&lt;sub&gt;3&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O)</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 (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td>-</td></tr><tr><td>Sodium dihydrogenophosphate monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;*H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>S9638</td><td>-</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;*6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>M9272</td><td>-</td></tr><tr><td>Calcium chloride hexahydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;*6H&lt;sub&gt;2&lt;/sub&gt;O)</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&amp;nbsp;</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&amp;nbsp;</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。滞后时间和形状变化也可以图形可视化。 控制的条件下洗涤的人血小板（H 2 O）的聚合曲线的一个典型的例子示于图2A中 。在一个的加入gonist（胶原），诱导（在短暂延迟后）引起的血小板的形状变化的聚集曲线的凹陷。然后，聚集随着时间的推移逐渐增加，直到最大值的百分比为大约3-4分钟达到。被朝向所述6分钟记录，这反映了一些解聚的端部观察到聚集的百分比略有下降。 如图2A-B所示，预孵育的人用的Panx1通道抑制剂亮蓝FCF血小板，以1mM的浓度，减慢或完全废除了初始的血小板形状改变和阻止从5得到洗涤的血小板的胶原诱导的聚集不同的无关的健康志愿者。当其中具有亮蓝FCF较低浓度（100μM）一起预温育的血小板，中不再观察到染料的对胶原诱导的反应和形状变化的抑制效果（参见，例如， 图2A）。 Quantif由1μg/ mL的胶原诱导的血小板聚集反应的ication表明与对照相比，条件以及对Panx1抑制剂较低浓度（100μM）（ 图2C）1mM的亮蓝FCF显著降低最大凝集反应。血小板聚集的抑制是特异性的胶原诱导的应答，而不是由于亮蓝FCF的不期望的副作用（对细胞活力，例如）为相同的浓度的染料（1毫米）不影响由诱导的聚集反应另一激动剂， 即 75μMAA， 如图2D所示。这些结果证实，我们和其他人先前Panx1的其它药理学抑制剂例如丙磺舒，甲氟喹示于人血小板的胶原诱导凝集反应Panx1通道的具体作用，具体10种 Panx1肽7,8。 <img alt="图1" src="/files/ftp_upload/55525/55525fig1.jpg" /> 图1:集合度。用于集合度（A）的玻璃比色皿的代表性图像（包含搅拌磁铁）。左侧的反应杯示出静止的血小板，而右侧的反应杯示出胶原诱导的活化后的血小板聚集物。 （B）对于比浊测量的8孔凝集的代表性图像。将孔用（星号）在37℃下孵育存在于玻璃试管血小板悬浮液，以及那些用于测量（白色箭头）以C表示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55525/55525fig1large.jpg" target="_blank">请点击此处以查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55525/55525fig2.jpg" /><br/&gt; 图2:亮蓝FCF块血小板活化的响应于胶原蛋白的高浓度。 （A）人的胶原诱导的聚集的代表性迹线洗涤从对照条件下的同一健康志愿者（V1）得到的血小板（H 2 O;深蓝色）或后7分钟预温育与Panx1抑制剂亮蓝FCF以1mM（浅蓝色）或在100μM（中间蓝色）。聚合痕迹记录6分钟。 （B）人的胶原诱导的聚集的代表性迹线洗涤后7分钟预温育与Panx1抑制剂亮蓝FCF（1毫摩尔）从4名健康志愿者（V2-V5）中获得的血小板。的人的最大凝集反应（C）的量化控制条件（白色柱）或下后7分钟预温育与亮蓝FCF，在100μM（灰色条）或以1mM（黑色条）洗涤血小板。 N = 5，**** P0 0.0001; ANOVA然后Bonferroni事后检验，多重比较;结果表示为平均值±SEM。 （D）人的AA诱导的聚集的代表性迹线洗涤后7分钟预温育与Panx1抑制剂亮蓝FCF（1毫摩尔）从5名健康志愿者（V1-V5）得到的血小板。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55525/55525fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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 ...

turbidimetry-on-human-washed-platelets-effect-pannexin1-inhibitor

Nanyang Technological University

Research

JoVE Journal

Medicine

11.9K 次观看.  University of Geneva. 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。 

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

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.

根据流量的血小板粘附和聚集，使用微流控流细胞

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

科研

生物学

Kinetx: A Combined Flow Cytometry Assay and Analysis Software Framework to Quantitatively Measure and Categorize Platelet Activation in Real-time.

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests measure endpoint responses after a defined time period and not the rate of platelet activation. However, the rate at which platelets convert extracellular stimuli into a functional response is an essential factor in determining how efficiently they can respond to injury, bind to a forming thrombus, and signal to recruit other platelets. This paper describes a flow cytometry-based platelet function assay that enables simultaneous data acquisition and sample stimulation and utilizes newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux. Kinetix was developed in R so that users can alter parameters such as degree of smoothing, identification of outlying data points, or time scales. To aid users unfamiliar with the R environment, Kinetix analysis of data can be performed by a single command. Together, this allows real-time platelet activation metrics, such as rate, acceleration, time to peak-rate, time to peak-calcium, and qualitative shape changes, to be accurately and reproducibly measured and categorized. Kinetic measurements of platelet activation give a unique insight into platelets' behavior during the first stages of activation and may provide a method of predicting the recruitment of platelets into a forming thrombus.

Platelets react rapidly to a range of stimuli. This paper describes a real-time flow cytometry-based platelet function assay and a newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux.

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests ...

生物化学

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.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

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

生物工程

<meta charset="utf8"></head><body>评估创伤和输血中血小板功能的平台

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.

<meta charset="utf8"></head><body>微流体评估血小板功能

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.

微流体评估血小板功能

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

医学

Imaging Single Platelet Migration Using a Three-Layer Coating System

An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers

Despite being anucleated cell fragments, platelets are now widely recognized for their multifaceted abilities. Not only do they form blood clots to prevent bleeding after injury, but they also fight infections and maintain vascular integrity during inflammatory diseases. While hemostatic plugs require the collective activation and aggregation of platelets, their role in protecting inflamed blood vessels is performed at the single-cell level. In this context, recent data have shown that platelets can migrate autonomously, a process dependent on the mechanosensing of their adhesive environment. Here, a detailed protocol for imaging single platelet migration is presented, utilizing a three-layer coating system consisting of a poly-L-lysine graft poly(ethylene glycol) (PLL-PEG)-biotin backbone (1), a fluorescent avidin linker (2), and biotin-cyclic Arg-Gly-Asp (cRGD) (3) as the platelet integrin-binding motif. This reductionist approach allows precise control of substrate adhesion properties and serves as a simple, standardized in vitro assay to study the mechanisms underlying platelet migration. The results indicate that migrating platelets binding to cRGD exert forces capable of disrupting the avidin-biotin bond. Furthermore, the density of biotin-cRGD significantly influences both platelet spreading and migration.

A detailed protocol for imaging single migrating platelets using RGD-functionalized avidin-biotin tethers with tunable density is provided, revealing that platelets generate enough force to rupture the avidin-biotin bond.

Despite being anucleated cell fragments, platelets are now widely recognized for their multifaceted abilities. Not only do they form blood clots to ...

<meta charset="utf8"></head><body>分析血小板磷脂酰丝氨酸和微泡释放

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

<meta charset="utf8"></head><body>通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

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

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根据流量的血小板粘附和聚集，使用微流控流细胞

Kinetx: A Combined Flow Cytometry Assay and Analysis Software Framework to Quantitatively Measure and Categorize Platelet Activation in Real-time.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

微流体评估血小板功能

微流体评估血小板功能

微流体评估血小板功能

An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers

通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板

通过测量人纯化血小板中的磷脂酰丝氨酸暴露和微泡释放来表征促凝血小板