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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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?...

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?...

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

Universidad de Cartagena UDEC

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.

<meta charset="utf8"></head><body>Kinetx：流式细胞术检测和分析软件框架，用于实时定量测量和分类血小板活化。

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.

Kinetx：流式细胞术检测和分析软件框架，用于实时定量测量和分类血小板活化。

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

<meta charset="utf8"></head><body>使用三层涂层系统对单血小板迁移进行成像

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.

<meta charset="utf8"></head><body>使用 RGD 功能化亲和素-生物素系绳研究血小板迁移的体外测定

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.

使用 RGD 功能化亲和素-生物素系绳研究血小板迁移的体外测定

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

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

摘要

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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：流式细胞术检测和分析软件框架，用于实时定量测量和分类血小板活化。

Kinetx：流式细胞术检测和分析软件框架，用于实时定量测量和分类血小板活化。

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

使用 RGD 功能化亲和素-生物素系绳研究血小板迁移的体外测定

使用 RGD 功能化亲和素-生物素系绳研究血小板迁移的体外测定

使用 RGD 功能化亲和素-生物素系绳研究血小板迁移的体外测定