艾莉森州是来自加拿大卫生研究院 (研究院) 的弗雷德里克班廷和查尔斯最佳加拿大研究生奖学金获得者。劳拉· l · Swystun 是研究院奖学金获得者。戴维 Lillicrap 是一个加拿大研究椅子分子止血的接受者。这项研究部分由研究院的营运补助金 (MOP-97849) 资助。

这些研究得到了加拿大皇后大学研究伦理学委员会的批准。
1. 内皮细胞刺激
<ol><li>
胶原蛋白涂层一个6良好的组织培养板。
	<ol>
<li>提前24小时, 在37° c 的情况下, 用1毫升胶原蛋白缓冲液 (50 µg/mL 鼠尾胶原 1, 0.02 米冰醋酸) 涂上6层组织培养板。</li>
		<li>用2毫升的汉克平衡盐溶液 (HBSS) 冲洗两次井。 注: 板材可以用锡纸包裹, 并存放在4° c 的密闭塑料袋中。</li>
	</ol>
</li>
	<li>在含有10% 胎牛血清 (FBS) 的2毫升生长培养基中, 以 1 x 106细胞的密度对内皮细胞进行种子。孵育细胞为24小时在37° c (5% CO2)。 注: 在这里, 血液的副产物内皮细胞 (BOECs) 之间的5和15的使用。BOECs 使用斯塔克et al.中引用的方法从野生 VWF 序列中分离出健康志愿者22的方式与以前由朱庇特23描述的方法类似。</li>
	<li>24小时后取出培养基, 用 HBSS 冲洗细胞。</li>
	<li>添加1毫升无血清培养基 (不含 FBS 的最低基本生长培养基), 含有佛波 12-酸13乙酸酯 (PMA; 100 nM), 普通组蛋白 (UH), 和 HK 或 HR (全部在25µg/毫升) 到细胞为 2 h 在37° c (5% CO2)。 注: 这种组蛋白的浓度是基于以前的体外, 由许et al.的细胞毒性报告24和艾布拉姆斯et al。25 , 使用50µg/毫升。</li>
	<li>2小时后收集培养基, 并在-20 ° c 贮存, 直至分析。</li>
</ol>2. 酶联免疫吸附试验 (ELISA) VWF 定量
<ol><li>用100µL 涂层缓冲剂 (10 毫米二元磷酸磷酸钠和145毫米氯化钠, pH 7.2), 在10° c 的夜间涂上4µg/毫升多克隆抗 VWF 抗体的微。</li>
	<li>洗涤盘三次用250µL 洗涤缓冲 (10 毫米二元磷酸钠, 500 毫米氯化钠, 和0.1% 吐温 20, pH 7.2)。</li>
	<li>用洗涤缓冲器稀释培养基样品1:2 和 1:4, 并使用标准曲线的普通参考等离子体, 从1:10 稀释开始。</li>
	<li>100µL 稀释样品和标准隔夜在4° c。</li>
	<li>洗盘三次, 用250µL 洗涤缓冲器。</li>
	<li>添加100µL 稀释抗 VWF, HRP 共轭检测抗体 (1.1 µg/µL 在洗涤缓冲) 到板和孵化1小时室温。</li>
	<li>根据制造商的说明, 用邻苯二胺盐酸盐 (门诊) 试剂清洗钢板并孵育10分钟。</li>
	<li>停止反应与100µL 1 M H2, 因此4并且读板材在492毫微米。</li>
</ol>3. 固相组蛋白-VWF 结合试验
<ol><li>微与组蛋白稀释在0.5% 钠胆酸50毫米碳酸盐缓冲 (pH 9.6) 到10µg/毫升过夜在4° c。</li>
	<li>洗涤板三次与250µL PBS 补充0.1% 吐温 20, 孵化10分钟之间的洗涤。</li>
	<li>在室温下, 用0.1% 吐温20和1% 牛血清白蛋白 (BSA) µL 200 的 PBS 为 1 h 块的平板。</li>
	<li>重复步骤3.2。</li>
	<li>从200亩/毫升到25亩/毫升的盐水缓冲液 (10 毫米二元磷酸磷酸钠, 500 毫米氯化钠, 0.1% 吐温 20, pH 7.2) 连续稀释血浆衍生的人 VWF/因子 VIII 复合物。在室温下, 在2小时内将100µL 添加到盘中。</li>
	<li>重复步骤3.2。</li>
	<li>使用100µL PBS-0.1%tween 20 和 1:1, 000 兔多克隆抗 VWF 抗体结合在室温下 1 h, 检测 VWF 对固定组蛋白的束缚。</li>
	<li>重复步骤3.2。</li>
	<li>孵育板为30分钟与100µL 门诊试剂和停止反应与100µL 1 M H2, 因此4。测量 492 nm 的光学密度。</li>
</ol>4. 将内皮细胞播种到流室
<ol><li>将0.5 毫升胶原蛋白涂层缓冲液 (在步骤2.1 中描述) 装入口锁1毫升注射器。</li>
	<li>将注射器的口锁末端拧到流室滑动的储水池中。</li>
	<li>慢慢按压活塞, 直到整个车道被涂上, 每个蓄水池都满了。对每条车道重复, 在37° c 下孵育24小时。</li>
	<li>用200μ l 吸管抽吸胶原蛋白涂层缓冲液。加载另一个注射器与1毫升 HBSS, 并按下柱塞慢慢地洗涤每个车道的流室。用200μ l 吸管在气流室的相对端吸吸洗涤液。重复两次。</li>
	<li>吸管大约100µL 3 x 106细胞/mL 内皮细胞 (BOEC) 溶液在生长介质中与 10% FBS 进入流室的车道。 注: 内皮细胞的播种密度超过了腔室的容量。这是为了确保最大的渠道覆盖面;在随后的清洗步骤中移除换药单元格。</li>
	<li>孵育室在37° c 为 24 h. 使用口锁注射器 (如步骤4.4 所示) 每8-12 小时更换培养基。确保车道上没有可见气泡。</li>
</ol>5. 从人的全血中分离血小板
<ol><li>在一个30克针的生物安全柜中, 加入300µL 酸-柠檬酸葡萄糖缓冲液 (85 毫米柠檬酸三钠, 111 毫米 d-葡萄糖, 71 mm 柠檬酸, pH 4.5) 到无添加剂3毫升血液收集采血。</li>
	<li>通过静脉穿刺 (每三管3毫升) 从健康志愿者 (无血小板和甾抗炎药七天) 中收集全血, 遵循与博纳博et al26所发布的协议类似的方法。</li>
	<li>丢弃第一管, 因为与静脉穿刺相关的初始组织损伤会引起自发的血小板活化。</li>
	<li>轻轻倒置 vacutainers 三次, 将血液与抗凝剂混合。</li>
	<li>离心 vacutainers 在 150 x g 为15分钟在24° c (室温)。</li>
	<li>用吸管在生物安全柜中慢慢取出富含血小板的血浆 (上清), 并小心地将其转移到15毫升的锥形管上。将每个采血的富含血小板的体积转移到单个锥形管上, 以避免潜在的 cross-activation。 注: 应在生物安全柜中进行血小板隔离, 以防止细菌污染和潜在的人工血小板和/或内皮细胞活化。</li>
	<li>离心圆锥管在 900 x g 为 10 min 在24° c (室温), 以颗粒的血小板。</li>
	<li>吸管和丢弃血小板--贫上清。</li>
	<li>用1毫升柠檬酸-葡萄糖钠 (厘米) 缓冲液 (13 毫米柠檬酸钠, 30 毫米 d-葡萄糖, 120 毫米氯化钠, pH 7.0) 仔细清洗颗粒。 注: 当添加悬浮时, 血小板会出现在类似于一缕状的形态中, 而颗粒也会被轻轻地放在上面。在受血小板活化污染的样本中, 血小板不会轻易重, 并会出现笨。</li>
	<li>一旦完全悬浮, 离心样品在 900 x g 为10分钟在24° c (室温)。</li>
	<li>取出厘米洗涤缓冲液, 重3毫升 Tyrode 的缓冲液 (138 毫米氯化钠, 5.5 毫米 d-葡萄糖, 12 毫米碳酸氢钠, 2.9 毫米氯化钾, 0.36 毫米二元磷酸钠, pH 7.4)。确保血小板完全悬浮, 如果观察到聚合, 则丢弃样本。</li>
	<li>添加1.5 µL 从20毫米 DiOC6 库存溶液 (溶解57.25 毫克在5毫升的甲醇) 到每3毫升锥形管洗涤血小板的最后浓度1µM。 注: DIOC6 是一种绿色荧光线粒体染料, 用于本实验中对血小板进行标记。</li>
	<li>分1毫升的洗涤血小板溶液, 以三1.5 毫升离心管。 注: 本实验所需的血小板体积是根据流室的大小、所需的剪切应力和实验的长度 (步骤 6.5) 计算的。这一协议可以调整, 因为条件的变化, 以确保有足够的体积流。</li>
	<li>在隔离2小时内使用冲洗过的血小板。</li>
</ol>6. 流动装置总成
<ol><li>将一根70厘米的无菌硅胶油管 (内径1.6 毫米) 连接到男性肘部口连接一端和一个女性口锁接头。</li>
	<li>将女性口锁接头连接至20毫升注射器。</li>
	<li>重复步骤6.1 和6.2 两次, 以产生三相同的注射器油管连接。</li>
	<li>
使用注射器泵产生流体流动。输入以下设置到注射器泵浦: 注射器的内部直径和流速。 注:20 毫升注射器的内部直径为19.55 毫米。根据所需的剪切速率/剪切应力和流室的尺寸计算流量。对于当前研究中使用的流室, 要达到 1 dyn/厘米2 (在低静脉切变中观察), 需要9.37 µL/分钟的流速。由于证据连接组蛋白与静脉血栓形成, 4.45 dyn/cm2的适度静脉切变被选择。这也是基于这样的假设, 即缓冲液的粘度与水相似。流速等于41.7 µL/分钟. 用于计算不同流室尺寸的流速的公式可以在以下方面找到:<ol>
<li>使用注射器泵上的按钮选项设置泵来提取液体。</li>
	</ol>
</li>
	<li>在注射器泵上装上油管的三注射器, 并在注射器和活塞周围紧紧地固定支架。</li>
	<li>将男性肘口到第二20厘米的无菌硅胶管的两端。</li>
	<li>连接一个肘口到一个钝的18口径针。</li>
	<li>重复两次, 创造三相同的进气系统的三车道的流室。 注: 硅管和 Luers 可以用10% 漂白水冲洗后再用蒸馏水。不建议重新使用流室。</li>
</ol>7. 显微镜设置
<ol><li>使用装有全自动舞台的旋转圆盘显微镜。执行照明与激光发射, 提供 405, 458, 491, 543, 和 633 nm 激光线。使用20X 目标捕获所有图像。使用分析软件 (http://www.well.ox.ac.uk/_asset/file/metamorph-basic-commands.pdf) 调整所有参数。</li>
	<li>选择通道设置 (发射过滤器: 520/35), 与励磁在491毫微米, 为承购。将电子倍增电荷耦合器件设置为电子倍增增益为 150, 曝光时间为200毫秒, 以获得完整的动态数据范围。 注: 血小板被贴上绿色荧光染料, DiOC6, 波长最大值 (激发, 482 nm; 发射, 504 nm)。</li>
	<li>调整 491 nm 激光功率到20%。</li>
	<li>标记中心所有三车道和选择1μ m 为 z 范围和0.5 μ m 为步大小。选择每个十年代捕获图像的时间点 (即,工期) 和图像之间的时间 (即间隔) 的数量;选择每张幻灯片的 "60 时间点"。设置显微镜捕捉三车道的图像, 并将它们保存到指定的实验文件夹中。 注: 由于流室有三车道, 显微镜将需要在三位置之间切换以捕获图像。</li>
</ol>8. VWF-血小板串形成
<ol><li>使用与步骤4.4 相同的技术, 从流程室 (从步骤 4) 中删除培养基。</li>
	<li>添加100µL 或组胺 (25 µM), PMA (100 nM), UH, 香港, 或 HR (所有在25µg/毫升) 稀释无血清培养基 (如步骤 1.4), 以刺激 BOEC 单层在室温和环境 O2/CO2的15分钟。 注意: 虽然没有必要的 VWF 释放, 执行这种刺激在37° c 将改善 VWF 胞27。</li>
	<li>确保流室的三条单独的车道中的一个不被作为实验性的控制。</li>
	<li>使用1毫升注射器取出培养基, 用 100-200 µL 的 PBS 将其替换成内皮层的促。</li>
	<li>使用血管夹将流室安装到滑动支架上, 以确保腔室到位。</li>
	<li>将可用的雄性口连接器安装在 70 cm 油管 (连接到注射器) 的末端, 到流室的蓄水池。重复两个其他流室车道。</li>
	<li>启动注射器泵, 除去 PBS 清洗。将额外的 PBS 移到相反的流室中, 以确保车道不会干涸。</li>
	<li>观察所有三车道的等效流量流系统, 然后暂停注射器泵。</li>
	<li>选择一个区域来捕捉每条车道中间的图像, 在实验之间大致相同的区域。 注意: 操作显微镜的人可以对实验条件视而不见。这一步确保显微镜是在血小板流前聚焦 (参见步骤 7)。</li>
	<li>将雌性口耦合器连接到5毫升注射器, 然后到自由雄性口接头 (步骤 6.8), 并在含有洗涤血小板溶液的1.5 毫升离心管中浸入钝针 (步骤 5.13).<a> </a>通过油管慢慢地绘制血小板溶液, 确保系统内没有气泡。</li>
	<li>取出5毫升注射器, 并将雄性口附着在流动室的开口蓄水池上。重复三车道</li>
	<li>重新启动注射器泵和捕捉图像在血小板流动期间使用显微镜和协议设置从步骤7为10分钟在黑暗中。</li>
	<li>10分钟后, 将口从流动室的进水管上拔下, 并用 PBS 填充油层, 以维持流在内皮单层上。</li>
</ol>9. VWF-血小板串量化
注: PBS 的连续流动需要保持 VWF-血小板柱伸长率以进行图像分析。停止流动将导致 VWF 成为球状, 并将损害 VWF-血小板串量化。在图像捕获过程中, 必须用 PBS 将每个水库顶起来。HBSS 可以取代 PBS, 如果细胞萎缩或分离在这一步观察。
<ol><li>从第一条车道开始, 每张幻灯片捕获10幅图像, 在幻灯片中间使用20X 物镜, 不重叠。对每个车道重复此操作。 注: 由于紊流剪应力, 应避免储层周围的区域。</li>
	<li>使用图像处理软件, 通过单击 "图像"、"调整" 和 "亮度/对比度" 来调整亮度。拖动滚动条, 直到被黏附的血小板可以被看得足够好, 以量化。</li>
	<li>在图像中观察到三或更多对齐的血小板时, 记录一个 VWF 的血小板串。计算图像中所有的 VWF-血小板字符串。重复所有10图像和平均值的实验。 注: 执行量化的人可以对实验条件视而不见。没有必要为 VWF co-stain, 因为这已经被独立研究证实了8,28,29。</li>
	<li>重复实验至少三次, 以评估每个促。</li>
</ol>

作者声明没有竞争的金融利益。

虽然 VWF-血小板串的生理相关性仍然存在争议, 由于它们在 VWF 裂解蛋白酶 ADAMTS13 中的迅速溶解, 它们可以作为一个可量化的体外模型, 通过 VWF 的方法在现场进行血小板招募。在组蛋白水平增加的情况下, 血栓可能形成于5。此外, 在缺乏 ADAMTS13 活动的病理-如血栓性血小板减少紫癜 (或炎症环境-ADAMTS13 活动是抑制-VWF-血小板字符串可以观察到在体内32 ...

血栓形成是导致全球死亡的主要原因1 , 可以在反应 todysregulated 血小板活化和凝血酶生成两 veinsand 动脉。血浆水平 VWF 是血液凝固的一个关键的调节器, 藉以低水平 (< 50%) 导致被称为血管性血友病病 (VWD)2和高水平 (> 150%) 的出血性疾病与增加的风险静脉的3和动脉4血栓形成。
VWF 是巨和内皮细胞合成的 multimeric 糖蛋白, 分别保存在血小板α颗粒和 WPBs 中。在止血的挑战, VWF 可以释放从内皮 WPBs 到绳循环血小板的活化内皮细胞5或暴露的胶原蛋白在血管壁上的6。VWF 对内皮细胞的锚定是由 p-选择素7和整合因子αvβ38所介导的。随后释放的血小板α颗粒存储可以进一步增加局部 VWF 浓度, 稳定血小板与血小板的相互作用, 为血小板堵塞的形成, 需要支架的传播凝血叶栅和纤维蛋白沉积.VWF 的血小板结合活动受其 multimeric 结构的调节, 高分子重量体具有较大的止血活性9,10。在循环中, VWF 也充当凝血因子 VIII. 的载体。
流体剪切应力是 VWF 生理的重要调节因子。在没有剪切应力的情况下, VWF 存在于球状形态中, 为血小板糖蛋白 Ib 黏附力的粘附域11。当剪切应力存在时, 金属、整合和金属与凝血酶母模 (ADAMTS13) 的劈裂部位被暴露。ADAMTS13 克里夫斯裸和血小板修饰的 VWF 弦来调节 multimer 的大小, 从而减少其止血活性12。
VWF 是一种急性相蛋白, 许多刺激, 包括缺氧13, 感染的14, 和促炎细胞因子, 已被证明是调解 VWF 释放内皮细胞。类似于其他炎症因子, 胞外组蛋白也被证明在小鼠体内诱发全身 VWF 释放15,16和血小板活化,体外17,18, 19. 这被证明取决于组蛋白亚型, 因为赖氨酸和精氨酸含量的差异可能影响功能15。我们的研究目的是建立一个流室模型来研究富含赖氨酸 (HK) 和精氨酸 (HR) 组蛋白亚型和促对内皮 VWF 释放和 real-time 血小板捕获的影响, 潜在的早期事件炎症引起的血栓。
这种流室方法重述在体内的内胶原蛋白, 内皮细胞, VWF 和血小板之间的相互作用, 在一个体外系统, 是可视化, 重现性和可量化的。它允许对调节 VWF-血小板相互作用的通路的所有方面进行 real-time 评估, 包括 WPB 分泌、血小板活化和 VWF 蛋白水解。在受控剪应力条件下的 VWF 的研究已被用来评价 VWD 突变 , 影响 VWF 释放和血小板结合功能20, WPB 生理学21, VWF ADAMTS135。我们使用这种方法来量化 VWF-血小板串形成的结果, 炎症刺激: 胞外组蛋白。

<table><tbody><tr><td>Calf-thymus unfractionated histones (UH)</td><td>Worthington Biochemical</td><td>HLY</td><td>Reconstituted in serum-reduced media (5 mg/mL)</td></tr><tr><td>Calf-thymus lysine-rich histones (HK)</td><td>Sigma-Aldrich</td><td>H5505</td><td>Reconstituted in serum-reduced media (5 mg/mL)</td></tr><tr><td>Calf-thymus arginine-rich histones (HR)</td><td>Sigma-Aldrich</td><td>H4830</td><td>Reconstituted in serum-reduced media (5 mg/mL)</td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma-Aldrich</td><td>P8139</td><td>Reconstituted in DMSO (20 mM)</td></tr><tr><td>Histamine</td><td>Sigma-Aldrich</td><td>H7125-1G</td><td>Reconstituted in water (50 mg/mL)</td></tr><tr><td>3,3&#039; Dihexyloxacarbocyanine Iodide (DiOC6)</td><td>Invitrogen</td><td>D273</td><td>Reconstituted in methanol (20 mM)</td></tr><tr><td>Rabbit Anti-VWF Coating Antibody</td><td>DAKO</td><td>A0082</td><td>For VWF ELISA</td></tr><tr><td>Rabbit Anti-VWF Detection Antibody, HRP conjugated</td><td>DAKO</td><td>P0026</td><td>For VWF ELISA and histone-VWF binding assay</td></tr><tr><td>Nunc MaxiSorp flat-bottom 96-well microplates</td><td>eBioscience</td><td>44-2404-21</td><td>For histone-VWF binding assay</td></tr><tr><td>Immulon 4 HBX Flat Bottom Microtiter 96-Well Plates</td><td>Thermo Scientific</td><td>3855</td><td>For VWF ELISA</td></tr><tr><td>Humate-P</td><td>CSL Behring</td><td>N/A</td><td>Plasma-derived human von Willebrand factor/factor VIII complex</td></tr><tr><td>Normal Reference Plasma</td><td>Precision BioLogic</td><td>CCNRP-05</td><td>For VWF ELISA standard curve</td></tr><tr><td>O-Phenylenediamine dihydrochloride (OPD) reagent</td><td>Sigma-Aldrich</td><td>P8287</td><td>Equivalent product available through ThermoFisher Scientific (Catalogue Number: 34006)</td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td>For culturing and initial seeding of BOEC</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>ThermoFisher Scientific</td><td>14025092</td><td></td></tr><tr><td>Rat-tail Collagen Type 1</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Gibco Opti-MEM I Reduced Serum Media</td><td>ThermoFisher Scientific</td><td>31985070</td><td>For endothelial cell stimulations</td></tr><tr><td>METAMORPH Microscopy Automation and Image Analysis Software</td><td>Molecular Devices</td><td>N/A</td><td></td></tr><tr><td>BD Vacutainer Blood Collection Tubes, No Additive</td><td>BD Biosciences</td><td>366703</td><td></td></tr><tr><td>&amp;micro;-Slide III 0.1 (flow chambers)</td><td>Ibidi</td><td></td><td>This product has been discontinued. We suggest using &amp;micro;-Slide VI 0.1 (#80661) or 0.4 (# 80601) and recalculating flow rate and platelet volume needed to maintain a shear stress of 4.45 dyn/cm2</td></tr><tr><td>Silicone Tubing 1.6 mm ID: 5 m, sterilized</td><td>Ibidi</td><td>10842</td><td></td></tr><tr><td>Luer Lock Connector Female: natural Polypropylene, sterilized</td><td>Ibidi</td><td>10825</td><td></td></tr><tr><td>Elbow Luer Connector Male: white Polypropylene, sterilized</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>Blunted 18G Needle</td><td>BD Biosciences</td><td>305180</td><td></td></tr><tr><td>20 mL syringes</td><td>BD Biosciences</td><td>302830</td><td></td></tr><tr><td>Syringe Pump</td><td>New Era Pump Systems Inc. NE-1600 Multi-PhaserTM</td><td>N/A</td><td></td></tr><tr><td>Quorum WaveFX- 4X1 spinning disk microscope</td><td>Quorum Technologies</td><td>N/A</td><td></td></tr><tr><td>Image Processing Software</td><td>ImageJ</td><td>N/A</td><td></td></tr></tbody></table>

investigating von willebrand factor pathophysiology using a flow chamber model of von willebrand factor-platelet string formation

血管性血友病因子 (VWF) 是一种 multimeric 糖蛋白凝血因子, 介导血小板黏附和聚集在内皮损伤的部位, 并携带因子 VIII 在循环。VWF 是由内皮细胞合成的, 或者被释放组成到血浆中, 或者被储存在专门细胞器中, 称为 Weibel-Palade 体 (WPBs), 以满足止血的挑战而按需释放。促和促炎刺激能迅速诱导 WPB 胞和 VWF 释放。内皮细胞释放的大部分 VWF 在血浆中循环;然而, VWF 的一部分被锚定在内皮细胞表面。在生理剪切条件下, 内皮锚 VWF 可与血小板结合, 形成 VWF-血小板柱, 可能代表血栓形成的病灶。流室系统可用于视觉观察的释放 VWF 从内皮细胞和随后的血小板捕获的方式, 是重现性和相关的病理生理 VWF 介导的血栓形成。利用这种方法, 内皮细胞培养在流室, 并随后刺激与促诱导 WPB 胞。洗过的血小板在活化的内皮细胞上进行灌注。血小板被激活, 并随后绑定到细长的 VWF 弦的流体流向。利用胞外组蛋白作为促和促炎症刺激, 我们观察到, 与未处理的内皮细胞相比, VWF-血小板的形成与组织治疗的内皮细胞的增加有关。本议定书描述了定量的, 视觉的和 real-time 的评估, 活化的 VWF-血小板相互作用模型的血栓和止血。

为了直接评估组蛋白对内皮细胞 VWF 释放的影响, 我们将汇合 BOECs 暴露在含有 PMA (阳性对照)、呃、HR 和 HK 2 h 的无血清培养基中。我们发现, 在治疗的内皮细胞 (图 1) 中, 香港诱导的 VWF 蛋白 (VWF: Ag) 增加了2倍。有趣的是, 当 BOECs 被刺激与 UH 和 HR, 有较少 VWF: Ag 在媒介比在未处理的情况发现了。我们假设, 由于组蛋白可以直接绑定到 VWF30</s...

Watch this Scientific Journal Video about Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation at JoVE.com

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

本文介绍了一种方法来评估内皮血管性血友病因子释放和随后的血小板捕获在流体剪切应激反应的炎症刺激使用的体外流室系统。

血管性血友病因子-血小板串形成的流室模型研究血管血友病因子的病理生理学

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

investigating-von-willebrand-factor-pathophysiology-using-flow

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.1K 次观看.  Queen's University. 本文介绍了一种方法来评估内皮血管性血友病因子释放和随后的血小板捕获在流体剪切应激反应的炎症刺激使用的体外流室系统。

视频: Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

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

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

生物工程

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

免疫与感染

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 ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

恒流中原发性人类内皮细胞的肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

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.

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

<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 ...