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
Summary
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.
Abstract
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.
Introduction
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>。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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| PBS | Life Technologies | 10010031 | |
| Ficoll-Paque (hydrophilic polysaccharide) | GE Healthcare | 45-001-749 | |
| 10 ml heparinized vacutainer tubes | BD Biosciences | 366643 | |
| Human Fibrinogen | Enzyme Research Laboratories | N/A | |
| Alexa Fluor 488 human fibrinogen conjugate | Molecular Probes | F13191 | |
| 0.5 mL graduated microcentrifuge tube | Fisher Scientific | 05-408-120 | |
| Glucose powder | Life Technologies | 15023-02 | |
| FXIIIa | Enzyme Research Laboratories | N/A | |
| VIS Confocal Microscope | Zeiss LSM 510 | LSM 510 | |
| 50 mM Tris | Lonza | S50-642 | |
| Calcium Chloride (CaCl2) | Sigma Aldrich | 449709-10G | |
| Vybrant DiD cell-labeling solution | Life Technologies | L7781 | |
| Plasmin | Enzyme Research Laboratories | N/A | |
| Sodium Chloride (5 M NaCl) | Life Technologies | AM9759 | |
| Statistical Modeling Software | IBM | SPSS Statistics 22 |
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