The authors would like to thank the Lam Lab at Georgia Tech for many helpful discussions in developing the experimental assays. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL115486 and by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

注:以下协议遵循由机构审查委员会（IRB）在佐治亚理工学院设置指引。 1.采血车和红血细胞分离过程<ol><li>收集40-120毫升血液捐赠者在10毫升肝素真空采血管。 4小时内收集的开始PBMC（外周血单核细胞）分离。在这段时间内，保持血液在RT。 注:O / N储存血RT还是在4℃下不建议，因为这将导致较低的PBMC产量。 </li><li>在冰冷的（4℃）无菌PBS 1:稀释全血1。 </li><li>移取10毫升的冰冷的亲水性多糖的到一个空的，无菌的50ml锥形管中。轻轻转移的亲水性多糖顶部稀释全血。 </li><li>用25毫升移液管在缓凝和吸管血液沿管的侧面非常缓慢。保证血液不与以CENTRI之前糖混合fugation因为亲水性多糖是有毒的细胞和否则，PBMC收率将会更低。 </li><li>离心血液样品30分钟，在400×g离心在4℃下。如果有的话，减少离心机的制动速度的一半最大的用于离心后更好的分离。 </li><li>抽吸掉离心血样的血浆/血小板组分。小心吸脱亲水性多糖层填充红细胞层的正上方。 </li><li>收集8-10毫升填充红细胞和稀释1:1与未消毒0.9％NaCl的盐水。 </li></ol>糖化共聚焦显微镜评价2.血块结构（模拟糖尿病凝块） <ol><li>除霜人纤维蛋白原和荧光标记的人纤维蛋白原结合物在37℃。 </li><li>吸取以下到0.5毫升刻度离心管中。 <ol><li>对于健康的，正常的纤维蛋白原血栓，混合人纤维蛋白原与10％的荧光标记的人纤维蛋白原缀合物在50mM的Tris / 100mM NaCl的缓冲液以5毫克/毫升的浓度。 </li><li>对于人工糖化血纤维蛋白原（以模拟糖尿病凝块），孵育人纤维蛋白原和10％荧光标记的纤维蛋白原结合物中的葡萄糖溶解在50mM Tris / 100mM NaCl的要得到的6.8毫克/毫升浓度5mM溶液。 </li></ol></li><li>盖采用不透明的材料，以避免暴露在光线下微离心管。孵育在37℃的水浴的管子48小时。 </li><li>使在玻璃显微镜载片的腔室由固定用的薄粘合剂在滑动的2边。 </li><li>在48小时温育期后，通过除霜FXIIIA和人α-凝血酶在RT制备试剂的血纤维蛋白的​​形成。 </li><li>稀释FXIIIA 80 U / ml的5毫米氯化钙2缓冲区。 </li><li>凝血酶稀释到4 U / ml于5毫米氯化钙2缓冲区。 <ol><li>对于正常的凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶是2.5米微克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li><li>为糖化凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶的是3.4毫克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li></ol></li><li>立即吸取30微升的血纤维蛋白溶液到由粘合剂形成的腔室。 </li><li>放置22毫米×22毫米的玻璃盖玻片为0.15毫米的2层的粘合剂的顶部的厚度。密封该开口侧用透明的粘合剂，以防止纤维蛋白凝块干燥。确保该粘合剂不与血纤蛋白凝块交互。 </li><li>允许纤维蛋白凝块聚合在21-23℃下进行共焦成像前2小时。 </li><li>采取利用共聚焦显微镜用40X / 1.30石油M27镜头采用了488纳米氩激光的凝块共聚焦显微镜图像。 </li></ol>纤维蛋白凝块的镰状细胞患者红细胞含有3.共聚焦显微镜评价<ol><li>除霜人纤维蛋白原和荧光标记的人纤维蛋白原结合物在37℃。 </li><li>吸取以下到0.5毫升刻度离心管中。 <ol><li>为健康，正常的血纤维蛋白凝块，混合人纤维蛋白原与在50mM的Tris / 100mM NaCl的缓冲液的10％荧光标记的人纤维蛋白原结合物以5毫克/毫升的浓度。 </li><li>为含有红细胞的SCD的凝块，混合人纤维蛋白原和10％的荧光标记的人纤维蛋白原结合物在50mM的Tris / 100mM NaCl的，​​使得所得的纤维蛋白原的浓度为10毫克/毫升。 </li></ol></li><li>标签使用细胞标记溶液的分离的红细胞（正常的和那些从红细胞分离协议获得镰状细胞的患者）。 <ol><li>暂停细胞以1×10 6每毫升中任何选择的无血清培养基中的密度。 </li><li>添加5微升/毫升的细胞悬浮液至细胞标记溶液。轻柔吹打拌匀平缓。 </li><lI&gt;孵育20分钟，在37℃。 </li><li>离心标记悬浮液的管在1,500rpm离心5分钟，在37℃。 </li><li>除去上清液，并轻轻重悬浮细胞在37℃的介质。 </li><li>重复清洗过程（3.3.4和3.3.5）两次以上。 </li></ol></li><li>稀释FXIIIA 80 U / ml的5毫米氯化钙2缓冲区。 </li><li>凝血酶稀释到4 U / ml于5毫米氯化钙2缓冲区。 <ol><li>对于正常的凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶的是2.5毫克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li><li>为含有红细胞的SCD的凝块，确保最终的浓度纤维蛋白原，FXIIIA和凝血酶的是5毫克/毫升，20单位/毫升，和1单位/毫升，分别以50微升的体积。 </li></ol></li><li>吸管将10μl标记的RBCs入血纤维蛋白溶液。吸管30微升与红细胞上的显微镜玻璃纤维蛋白（参照滑动准备GLycated血块）。 </li><li>允许纤维蛋白聚合，在21-23℃下进行共焦成像前2小时。 </li><li>取使用共焦显微镜的凝块的共聚焦图像，并利用激发激光器的488纳米和633纳米的波长，以激发荧光标记的血纤维蛋白和红细胞。 </li></ol>纤溶价格在糖化血块结构4.共聚焦显微镜评价<ol><li>重复糖化血凝块（协议第2步）在糖化血栓纤维蛋白溶解率评价的共聚焦显微镜协议。 </li><li>不要封明确胶粘剂室的两端。允许纤维蛋白凝块聚合1.5小时，在21-23℃下在含有250ml水，以防止脱水密封外壳。 </li><li>聚合后，获得使用共焦显微镜的凝块的图像。 </li><li>吸管纤溶酶到腔室的开口端，并通过经由毛细管作用凝块分散纤溶酶。 <ol><li>为正常和糖化纤溶实验正常浓度，吸管25微升在纤溶酶200微克/毫升到腔室的开口。 </li><li>为糖化凝块具有减小浓度，吸管25微升，在50微克/毫升到腔室的开口。 </li></ol></li><li>使用共焦显微镜软件时间序列的特征（ 见图1）捕获的纤维蛋白溶酶裂解凝血块的实时录像。 <ol><li>点击捕获模式，然后选择"时间序列"。选择周期的数量和记录时间间隔。 </li></ol></li></ol> 5.计算纤溶价格<ol><li>通过设置的区域（2500微米2），并计算溶解血栓的一个固定区域所需的经过时间确定裂解速率。计算使用下面的等式（ 图2）的裂解速率: <img alt="式（1）" fo:content-width="2in" srC ="/文件/ ftp_upload / 52019 / 52019eq1.jpg"/&gt; </li></ol>价格纤溶6.统计分析<ol><li>分析使用统计分析软件裂解的数据。执行单向ANOVA和事后杜克-克莱默试验44,45。 </li></ol>

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The authors have nothing to disclose.

以获得有关的疾病状态的凝血机制结构有意义的数据，以隔离参与凝血，以确定在这些条件下，蛋白质和细胞的效应的因素是很重要的。这个协议是用于在体外研究了血纤维蛋白凝块的结构在糖尿病和SCD状态的目的而开发的。 有必要了解涉及纤维蛋白形成和纤维蛋白溶解中的疾病状态，因为改变的条件下会引起高凝，动脉粥样硬化，和CVD的机制。从本实验所得到的共焦图像，显而易见的是，高凝活动发生在糖尿病和SCD的状态，这会导致纤维蛋白原浓度的增加。增加的纤维蛋白原浓度的结果在一个致密的纤维蛋白网络与多个分支点和较小的孔在整个网络。在SCD患者的纤维蛋白网络，不仅有更多的纤维蛋白目前，卜吨从镰状细胞的患者也改变了网络的结构，因为它俘获纤维蛋白成簇红细胞的聚集性质。从镰状细胞的患者的红细胞的聚集也减少了纤维蛋白簇在网络中的孔隙率，但似乎增加了总的网络中空隙的浓度。糖化凝块的纤维蛋白溶解分析的结果所支持的裂解速率糖化血块是低级具有比健康，正常凝块减少纤溶酶的假说​​。然而，结果并没有提供有关糖化与血栓纤维蛋白溶酶的正常浓度的溶解率的确凿证据。由此可以得出结论，对于糖尿病患者的正常纤溶酶浓度，可以存在足够纤溶活性降解纤维蛋白凝块以有效的方式。 在此研究中进行的实验，分离的蛋白质和红细胞来代替全血和血浆。通过使用分离的蛋白，合成的纤维蛋白凝块，其原因为正常之间改变的结构与患病凝块可确定。这大大降低了具有其它血浆蛋白干扰血纤维蛋白凝块的结构和形态学的可能性。然而，通过使用分离的蛋白质，这是至关重要的，以存储和妥善处理的细胞和蛋白质，因为它们更容易受到细胞死亡和蛋白质降解。 本研究选择的共焦显微镜的成像技术，是由于在保持细胞和蛋白质的天然血栓性质，得到的纤维蛋白凝块的结构的图像的能力。共焦显微镜也已经显示，得到改善的对比度和降低的噪声比传统的光显微术检体的光学拆分。该激光从进入引导到针孔，阻止未聚焦的光。共焦显微镜的利用率免除了对另外的固定剂的化学物质;因此细胞和蛋白质能够维持成形的纤维蛋白凝块的天然蛋白质的活性。其结果是，细胞，蛋白质，和酶发挥作用在其天然状态，允许使用共聚焦显微镜的用于捕获实时活动的图像和视频。用共焦显微镜，在纤维蛋白凝块的三维图像被捕获和纤维蛋白凝块的纤维蛋白溶酶消化的视频进行实时获得的。当添加纤溶酶对血块捕捉凝块溶解过程的时间序列的视频，为使用毛细管作用通过纤溶酶通过血纤蛋白基质，以均匀地分散酶是重要的。这样做是为了确保在添加纤溶酶浓度为准确，并允许所述酶裂解整个凝块，而相比之下，该纤溶酶溶液中加入该地区。 的方法和在本研究中得到的结果是类似于由Dunn 等人 ，谁发现凝块溶解做了研究率分别比对照组25糖尿病患者显著降低。在两项研究中，有对糖尿病（糖化）凝块溶解率显著的差异;然而，目前的协议可以精确地表示，因为减少纤溶酶水平的糖尿病状态。另外，在此研究纤溶连续实时使用共焦显微镜，在对比分析离散部分在不同的时间间隔捕获。伍顿等人 47已经调查纤维蛋白凝块溶解并使用的裂解前速度（厘米/秒），以调查裂解为凝块的速率。他们利用共聚焦图像分析在他们的研究，以确定前的速度。代替裂解前速度在目前的研究中，一个裂解区速度，使用（因为图像上的共聚焦分析的方式）。为了进一步验证我们的研究中开发的方法，在48中的作者描述的数学模型<html>纤维蛋白凝块溶解由纤维蛋白溶酶，在其规定的溶解速度前作为纤维蛋白溶解的有效速率常数的功能。的方程给定为: <img alt="公式2" fo:content-width="1.5in" src="/files/ftp_upload/52019/52019eq2.jpg" />这里， <img alt="公式3" fo:content-width=".1in" src="/files/ftp_upload/52019/52019eq3.jpg" />表示纤溶的有效速率常数K a是吸附常数，C 点是纤溶酶浓度，ρFN是纤维蛋白的密度。如果测得的量<img alt="公式4" fo:content-width=".1in" src="/files/ftp_upload/52019/52019eq4.jpg" /> （从在当前的研究中的共焦分析测定）利用和链规则的情况下，这将导致: <img alt="公式5" fo:content-width="1.25in" src="/files/ftp_upload/52019/52019eq5.jpg" />T"&gt;假设只有一个维度每单位时间的变化会导致: <img alt="方程6" fo:content-width=".75in" src="/files/ftp_upload/52019/52019eq6.jpg" /><img alt="公式7" fo:content-width=".75in" src="/files/ftp_upload/52019/52019eq7.jpg" />因而纤溶用于实验的有效速率常数导致: <img alt="方程8" fo:content-width="1.5in" src="/files/ftp_upload/52019/52019eq8.jpg" />这种关系推断纤维蛋白溶解，有效速率常数（ <img alt="公式9" fo:content-width=".75in" src="/files/ftp_upload/52019/52019eq9.jpg" /> ）为纤溶酶浓度（C 点 ）和区域裂解速率的函数<img alt="10式" fo:content-width=".1in" src="/files/ftp_upload/52019/52019eq10.jpg" /> 。 这种技术也用于血栓形成研究中的重要SCD的患者，因为很少有知道关于血纤维蛋白和凝血机制相对于这种疾病。目前已经对SCD对纤维蛋白凝块结构的影响进行了很少的研究;因此，这项研究收集到的共聚焦图像提供了血块结构与整合镰状红细胞罕见的可视化。 为了比较，在群体之间装置的差异，使用单向ANOVA和事后杜克-克莱默测试。单向方差是用于确定在独立组之间的装置的统计学显著差的统计检验。在杜克-克莱默后试验用于比较来自各组独立的手段，并确定哪些组的装置是彼此统计学差异。之前执行一单向ANOVA，存在必须得到满足，以确保该结果是有效的6假设。六个假设是:一个连续的因变量（裂解率），一个独立的杂物竹叶提取由两个或多个独立的分类组（正常，糖化，糖化和具有减少纤溶酶），并独立地各组中观察到的，使得一个事件发生，不影响另一事件数据（这保证了存在于数据没有重叠。）不含有任何显著离群数据集，正态分布由夏皮罗 - 威尔克统计检验验证数据，并且在由列文统计检验验证数据方差的同质性。一旦这些假设都成立，单向ANOVA和事后杜克-克莱默试验进行以0.05的p值作为阈用于确定统计显着性。为单向ANOVA和事后图基-克莱默测试的严格的细节，请参阅44,45。 总体而言，该方法允许在其天然状态的正常和异常（由于疾病状态）的纤维蛋白凝块成像。该方法还提供了细节凝块溶解的实时成像，以及作为溶解率进行统计分析。与该血栓可以产生并成像在体外容易是大范围的细胞和组织工程应用中是有用的。

伤到血管的内皮细胞衬里穿过止血反应，或血液凝块的形成修复。当血液渗透到细胞外基质，组织因子激活在血流中血小板，以促进凝血级联的引发。这个愈合过程中的关键机械部件是纤维蛋白基质中，血纤维蛋白纤维是高弹性的组成，并可以承受较大的力1-4。许多研究人员广泛，在过去几十年的研究5-13形成结构纤维蛋白和功能。 患者的疾病，如糖 ​​尿病和镰状细胞具有显影血栓性并发症，如心肌梗塞，缺血性心脏疾病的危险性增加，和笔画14-19。超过200万人新近每年在美国诊断为糖尿病。有两种类型的糖尿病:I型，其中身体不能产生胰岛素，和II型，其中身体变得耐胰岛素的足量的。糖尿病患者中，心血管疾病（CVD）是原因的80％与疾病20,21相关的发病率和死亡率的。 镰状细胞病（SCD）是一种遗传性血液疾病，影响超过10万人在美国22。 SCD是一个点突变的疾病，导致红血细胞成为月牙形，使其难以使细胞通过血液脉管系统23。这两种疾病状态增加显影在体内动脉粥样硬化病症的几率。其中的一个原因为，这是改变的血纤维蛋白的结构和功能在疾病状态14,24-26的结果。 在糖尿病和镰状细胞病，有高凝和诱导动脉粥样硬化和心血管疾病的低纤溶活性（℃VD）相比，患者的健康17,27,28。已知的是低纤溶促进动脉粥样硬化的进展和滋生患者过早冠状动脉疾病29复发缺血事件。在当前的手稿，我们研究在这个特定设置中的血纤维蛋白的​​物理性质的作用。纤维蛋白凝块的结构在非患病患者是由细纤维，毛孔变大，并且通常较少稠密14,24的。在健康患者增加的孔隙率和密度较小的纤维蛋白凝块已发现，以促进纤维蛋白溶解16。在hyperthrombotic条件如糖尿病和镰状细胞病，有一个增加纤维蛋白原的生产，导致纤维蛋白原浓度从2.5毫克/毫升的正常水平在健康患者30-33增加。形成在糖尿病患者的血纤维蛋白凝块已被发现是不太多孔的，更硬，有更多的分支点，并且较密时相比健康的，非直径betic患者14,24,33-35。改变的血纤维蛋白结构是发生在涉及凝块形成的蛋白质糖基化机制的结果。当葡萄糖分子结合，以赖氨酸残基上的纤维蛋白原分子，其抑制从适当交联的谷氨酰胺和赖氨酸残基33,36,37人类XIIIa因子（FXIIIA）发生非酶（不可逆的）糖基化。 血纤维蛋白网的结构分析已被广泛研究最近。特别是，研究人员利用电子显微镜和三维重建纤维网38，这两个调查血管（内皮细胞）细胞和血管外（成纤维细胞和平滑肌细胞）如何影响纤维蛋白结构39，利用粘弹性和频谱分析来分析纤维结构40，和使用纤维蛋白的结构和机械性能之间发达的相关性的实验和计算方法41 </sup&gt;。目前研究的重点是制定血块结构模拟糖尿病和镰状细胞血栓形成的条件下，并利用共聚焦显微镜检查的结构的疾病状态的凝块和功能。纤维蛋白凝块从人血纤维蛋白原，人凝血酶，和FXIIIA形成。凝块用纤维蛋白溶酶裂解。为了模拟糖尿病病症，纤维蛋白原的浓度增加温育在葡萄糖溶液中以诱导体外纤维蛋白原糖化。为了模拟镰状细胞病凝血条件，增加纤维蛋白原浓度与从患者收集以前我们组做42镰状细胞比容。这些方法被用来研究的结构和病变的条件下参与纤维蛋白凝块形成和纤维蛋白溶解功能，以及诱导CVD法的机制。基于关于这些疾病的最新信息，糖化血纤维蛋白凝块的结构是致密的具有较少一第二毛孔变小。纤维蛋白凝块与红血细胞从镰状细胞的患者（红细胞）也更致密并显示在红细胞的聚集和凝聚的纤维蛋白簇。这是一个公认的现象，即先前已43确定。也有人推测，纤维蛋白溶解率会在糖化血纤蛋白凝块有和没有减少纤溶酶相比健康，正常的血纤维蛋白显著降低。结果表明，对于糖化血纤维蛋白凝块，显著不同的裂解率的结果，观察只减少纤溶酶浓度的条件下进行。使用共焦显微镜的这种实验技术提供显著优于其它成像方法，因为细胞和蛋白质保持在它们的天然状态，这使的凝血活性的实时视频捕捉。合成诱导凝血的这种方法也更便宜，更有效的时间比获得患者样品并过滤掉个别蛋白质和酶。此外，通过使用分离的蛋白质和酶的合成凝块，该凝块被标准化，以便有没有样品之间的变异如在血浆中的其它蛋白质的结果。

<table><tbody><tr><td>PBS</td><td>Life Technologies</td><td>10010031</td><td></td></tr><tr><td>Ficoll-Paque (hydrophilic polysaccharide)</td><td>GE Healthcare</td><td>45-001-749</td><td></td></tr><tr><td>10 ml heparinized vacutainer tubes</td><td>BD Biosciences</td><td>366643</td><td></td></tr><tr><td>Human Fibrinogen</td><td>Enzyme Research Laboratories</td><td>N/A</td><td></td></tr><tr><td>Alexa Fluor 488 human fibrinogen conjugate</td><td>Molecular Probes</td><td>F13191</td><td></td></tr><tr><td>0.5 ml graduated microcentrifuge tube</td><td>Fisher Scientific</td><td>05-408-120</td><td></td></tr><tr><td>Glucose powder</td><td>Life Technologies</td><td>15023-02</td><td></td></tr><tr><td>FXIIIa</td><td>Enzyme Research Laboratories</td><td>N/A</td><td></td></tr><tr><td>VIS Confocal Microscope</td><td>Zeiss LSM 510</td><td>LSM 510</td><td></td></tr><tr><td>50 mM Tris</td><td>Lonza</td><td>S50-642</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>449709-10G</td><td></td></tr><tr><td>Vybrant DiD cell-labeling solution</td><td>Life Technologies</td><td>L7781</td><td></td></tr><tr><td>Plasmin</td><td>Enzyme Research Laboratories</td><td>N/A</td><td></td></tr><tr><td>Sodium Chloride (5 M NaCl)</td><td>Life Technologies</td><td>AM9759</td><td></td></tr><tr><td>Statistical Modeling Software</td><td>IBM</td><td>SPSS Statistics 22</td><td></td></tr></tbody></table>

experimental and imaging techniques for examining fibrin clot structures in normal and diseased states

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

糖化纤维蛋白凝块结构共聚焦显微镜分析的正常和糖化凝块的共焦显微镜图像示于图3中 。在正常和糖化凝块的共聚焦显微镜分析表明，糖化血块与较小孔隙比正常凝块都具有和不具有凝块聚合过程中加入FXIIIA的致密。在图3A和3B中 ，存在其中在一个不太致密的结构比糖化凝块具有较高的血纤维蛋白浓度（ 图3C和3D）的下纤维蛋白浓度。 在糖化凝块越大密度观察（每单位面积的纤维的根数）是一种发生在糖尿病患者中增加的纤维蛋白原浓度的结果。在正常的凝块，血纤维蛋白纤维是更线性和聚合时用FXIIIA，形成长纤维的是有可能的FXIIIA形成纤维间的交联的结果。在糖化凝块的情况下，纤维似乎是更短，即使附加FXIIIA的。这是可能的改变引起的糖化区域上的纤维蛋白原分子的存在下聚合过程的结果。另外，从分析图3中 ，它很可能是该纤维的厚度也减少为糖化的结果，这可能是为什么从糖化纤维的荧光似乎具有较低的强度。 纤维蛋白凝块的镰状细胞患者含红细胞的共焦显微镜分析与正常凝块和红细胞从镰状细胞的患者的血纤维蛋白凝块结构的共焦显微镜图像示于图4中 ，作为预测的，含有选自镰状细胞的患者的红细胞的血纤维蛋白凝块的共焦图象有更致密结构比那些健康的血纤维蛋白Ç拍。 图4A示出了纤维蛋白和正常红细胞的均匀分布。然而，在图4B中有在没有血纤维蛋白网形成纤维蛋白和空隙的团块。从镰状细胞的患者的红细胞没有均匀地分散，而是包埋于纤维蛋白团块。增加的密度（每单位面积的纤维的根数）为发生如高凝在SCD患者的结果增加的纤维蛋白原浓度的结果。此外，从镰状细胞的患者的红细胞的自然趋势，以形成聚集体主义及其，成为交织成的纤维蛋白网状红细胞的团块。这导致异常凝块结构，是在形态比正常患者明显不同的形成。从图4B中 ，可以观察到，从镰状细胞的患者的红细胞的血纤维蛋白样品中造成更大的异质性，也导致红细胞的血纤维蛋白样品，在更广阔的空间分布时COM相比于健康的纤维蛋白凝块正常的红细胞。与此相反，在健康的血纤维蛋白凝块与正常红细胞显示细胞的更均匀的分散在整个纤维蛋白网状。 它是未知的多的HbS（镰刀血红蛋白）如何的聚合从镰状细胞的患者的红细胞和被包含在样品中。什么是已知的，但是，是对的HbS任何量的存在导致细胞的每单位体积的HbS分子与正常的HbA分子数更大的聚集。 Pan 等人46已经证明这种现象在一项研究中，他们比较脱氧HBS的聚合/聚类效果，并且，为了比较，氧的HbS及氧-正常成人血红蛋白，糖化的。溶液的制备和它们的大小和数量后几秒钟内形成的簇保持相对稳定长达3小时。一个从研究的结论是，这两个氧哈佛商学院和脱氧哈佛商学院的分子聚集在一个较大的数字书房sity比氧的HbA（正常）分子作为温度的函数。关于目前的研究，这意味着，从镰状细胞的患者的红细胞有可能聚集在一起，并捕获纤维蛋白，因为它是被从纤维蛋白原的前体聚合。 正常和糖化凝块共聚焦显微镜裂解率分析据推测，所述纤维蛋白溶解率将是更大的对于正常的血纤维蛋白凝块相比糖化血纤维蛋白凝块具有降低纤溶酶的浓度。为了检验这一假设，共聚焦显微镜来评估纤维蛋白凝块的溶解率在添加纤溶酶。平均（平均值）用加入了纤溶酶200微克/毫升正常的血纤维蛋白的溶解率是43.2±26.5微米2 /秒，平均（均值）裂解糖化血纤维蛋白的速率通过添加纤溶酶200微克/毫升29.7±11.2微米2 /秒。对于糖化纤维蛋白凝块溶解与纤溶酶50微克/毫升，平均裂解率为12.8±3.1微米2 /本身。共有各（30个样品）10个样品用于测定的平均值和标准偏差值。这些值在图5中给出。0.05的p值被用来确定独立的实验组之间的显着性。的纤维蛋白溶解率分析表明，对于正常的血块的值相比，所述糖基（模拟糖尿病）凝块具有降低血纤维蛋白溶均显著不同。 <img alt="图1" src="/files/ftp_upload/52019/52019fig1.jpg" /> 从共聚焦显微镜的软件，详细方法获得的实时图1.样本截图，纤维蛋白凝块溶解的连续影像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52019/52019fig1large.jpg" target="_blank">请点击此处查看THI的放大版本。</a>的身影。 <img alt="图2" src="/files/ftp_upload/52019/52019fig2.jpg" /> 图2.示例与用于计算纤维蛋白凝块样品溶解率固定区域共聚焦图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52019/52019fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52019/52019fig3.jpg" /> 图3。的健康和糖化（糖尿病）的血纤维蛋白凝块共聚焦图像。绿色荧光代表的纤维蛋白网状。所述unglycated纤维蛋白表示形成在健康患者血栓，而糖化血纤维蛋白表示形成在糖尿病患者中的血块。 （A）不加FXIIIA的Unglycated纤维蛋白。 （B）Unglycated纤维蛋白与FXIIIA。 （C </STRONG&gt;）纤维蛋白糖化无FXIIIA和（D）纤维蛋白糖化与FXIIIA。红色的比例尺为50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52019/52019fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52019/52019fig4.jpg" /> 图健康纤维蛋白凝块与正常红细胞和纤维蛋白凝块与镰状细胞贫血患者的红细胞4.共聚焦图像。绿色荧光代表的纤维蛋白网络和品红色标记代表健康的红细胞，从镰状细胞贫血患者。 （A）在正常纤维蛋白浓度FXIIIA和（B）增加纤维蛋白与FXIIIA和红细胞从镰状细胞贫血患者的健康的红细胞。白色的比例尺为50微米。白色圆圈图像（B）表示辨认镰状红细胞。 <aHREF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52019/52019fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/52019/52019fig5.jpg" /> 图的正常的血纤维蛋白凝块5.纤溶率相比糖化（糖尿病）的纤维蛋白凝块和糖化凝块具有减少纤溶酶的浓度。统计意义的基础上的单向ANOVA和事后杜克-克莱默多重比较试验来确定。方差分析表明，存在的裂解速率装置之间的显著差异。在图基 - 克莱默测试表明，在正常的血块和血块与减少纤溶酶与AP &lt;0.05之间装置的显著差异。 *表示与正常凝块一个显著差异。

Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

对于检查纤维蛋白凝块结构在正常和疾病状态的实验和成像技术

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

experimental-imaging-techniques-for-examining-fibrin-clot-structures

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Cardiovascular Drugs: Anticoagulants and Antianginal Agents

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11.4K 次观看.  Georgia Institute of Technology & Emory University School of Medicine. In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

视频: Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

科研

生物化学

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

生物工程

一种基于纤溶酶的研究脑血栓细胞成分的方法

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

作者聚焦：一种脑血栓综合细胞成分分析的新方法 

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

一种分析脑血凝块细胞成分的综合方法

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

医学

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 ...

免疫与感染

A Method to Study the Correlation Between Local Collagen Structure and Mechanical Properties of Atherosclerotic Plaque Fibrous Tissue

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture mechanics of the heterogeneous, highly collagenous plaque tissue, and how this is related to the tissue's fibrous structure, are not known yet. Existing pipelines to study plaque mechanics are limited to obtaining only gross mechanical characteristics of the plaque tissue, based on the assumption of structural homogeneity of the tissue. However, fibrous plaque tissue is structurally heterogeneous, arguably mainly due to local variation in the collagen fiber architecture.
The mechano-imaging pipeline described here has been developed to study the heterogeneous structural and mechanical plaque properties. In this pipeline, the tissue's local collagen architecture is characterized using multiphoton microscopy (MPM) with second-harmonic generation (SHG), and the tissue's failure behavior is characterized under uniaxial tensile testing conditions using digital image correlation (DIC) analysis. This experimental pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue. The obtained knowledge is key to better understand, predict, and prevent atherosclerotic plaque rupture events.

We have developed a mechano-imaging pipeline to study the heterogeneous structural and mechanical atherosclerotic plaque properties. This pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue.

一种研究动脉粥样硬化斑块纤维组织局部胶原结构与力学性能相关性的方法

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture ...

结合 Chandler Loop 和 RT-FluFF 进行离体血凝块裂解监测

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

作者聚焦:通过将流动动力学集成到体外模型中来推进溶栓测试

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

对于检查纤维蛋白凝块结构在正常和疾病状态的实验和成像技术

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