Name: experimental and imaging techniques for examining fibrin clot structures in normal and diseased states
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Description: Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

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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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+fibrin.">Fibrinogen and fibrin.</a> Advances in protein chemistry. 70, 247-299 (2005).</li><li>Collet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Fibrin+Network+Conformation+and+Fibrin+Fiber+Diameter+on+Fibrinolysis+Speed+Dynamic+and+Structural+Approaches+by+Confocal+Microscopy.">Influence of Fibrin Network Conformation and Fibrin Fiber Diameter on Fibrinolysis Speed Dynamic and Structural Approaches by Confocal Microscopy.</a> Arteriosclerosis, thrombosis, and vascular biology. 20, 1354-1361 (2000).</li><li>J&#246;rneskog, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+properties+of+the+fibrin+gel+structure+in+patients+with+IDDM.">Altered properties of the fibrin gel structure in patients with IDDM.</a> Diabetologia. 39, 1519-1523 (1996).</li><li>Svensson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+and+glycation+of+fibrinogen+in+vitro+occur+at+specific+lysine+residues+in+a+concentration+dependent+manner:+A+mass+spectrometric+and+isotope+labeling+study.">Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study.</a> Biochemical and Biophysical Research Communications. 421, 335-342 (2012).</li><li>Pieters, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+of+fibrinogen+in+uncontrolled+diabetic+patients+and+the+effects+of+glycaemic+control+on+fibrinogen+glycation.">Glycation of fibrinogen in uncontrolled diabetic patients and the effects of glycaemic control on fibrinogen glycation.</a> Thrombosis Research. 120, 439-446 (2007).</li><li>Baradet, T. C., Haselgrove, J. C., Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+fibrin+clot+networks+from+stereoscopic+intermediate+voltage+electron+microscope+images+and+analysis+of+branching.">Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching.</a> Biophysical journal. 68, 1551-1560 (1995).</li><li>Campbell, R. A., Overmyer, K. A., Selzman, C. H., Sheridan, B. C., Wolberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+extravascular+and+intravascular+cells+to+fibrin+network+formation,+structure,+and+stability.">Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability.</a> Blood. 114, 4886-4896 (2009).</li><li>Curtis, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+microstructural+templating+in+fibrin-thrombin+gel+networks+by+spectral+and+viscoelastic+analysis.">A study of microstructural templating in fibrin-thrombin gel networks by spectral and viscoelastic analysis.</a> Soft Matter. 9, 4883-4889 (2013).</li><li>Kim, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+fibrin+network+structure+and+mechanical+properties:+an+experimental+and+computational+analysis.">Correlation between fibrin network structure and mechanical properties: an experimental and computational analysis.</a> Soft Matter. 7, 4983-4992 (2011).</li><li>Keegan, P. M., Surapaneni, S., Platt, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+disease+activates+peripheral+blood+mononuclear+cells+to+induce+cathepsins+k+and+v+activity+in+endothelial+cells.">Sickle cell disease activates peripheral blood mononuclear cells to induce cathepsins k and v activity in endothelial cells.</a> Anemia. 2012, 201781 (2012).</li><li>Allison, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+sickle-cell+haemoglobin.">Properties of sickle-cell haemoglobin.</a> Biochemical Journal. 65, 212 (1957).</li><li>Kuehl, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design of experiments : statistical principles of research design and analysis. , (2000).</li><li>Lindman, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of variance in experimental designs. , (1992).</li><li>Pan, W., Galkin, O., Filobelo, L., Nagel, R. L., Vekilov, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastable+Mesoscopic+Clusters+in+Solutions+of+Sickle-Cell+Hemoglobin.">Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin.</a> Biophysical Journal. 92, 267-277 (2007).</li><li>Wootton, D. M., Popel, A. S., Alevriadou, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+theoretical+study+on+the+dissolution+of+mural+fibrin+clots+by+tissue-type+plasminogen+activator.">An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator.</a> Biotechnology and bioengineering. 77, 405-419 (2002).</li><li>Sazonova, I. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=127+Mathematic+model+of+fibrin+clot+lysis+by+plasmin.">127 Mathematic model of fibrin clot lysis by plasmin.</a> Fibrinolysis and Proteolysis. 12, 45 (1998).</li></ol>

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?...

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）</str...

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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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

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

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在体外溶栓模型中跟踪剪切流下钱德勒环形成的全血凝块的纤维蛋白溶解