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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

Injury to a blood vessel's endothelial lining is repaired through the hemostatic response, or the formation of a blood clot. When blood permeates into the extracellular matrix, tissue factors activate platelets in the blood stream that facilitate initiation of the coagulation cascade. The key mechanical component of this healing process is the fibrin matrix, composed of fibrin fibers that are highly elastic, and can sustain large forces 1-4. Many researchers have studied the formation structure, and function of fibrin extensively in the past decades 5-13.

Patients with diseases such as diabetes mellitus and sickle cell have an increased risk of developing thrombotic complications such as myocardial infarctions, ischemic heart disease, and strokes 14-19. Over 2 million people are newly diagnosed with diabetes mellitus each year in the United States. There are two types of diabetes: Type I, where the body fails to produce adequate amounts of insulin, and Type II, where the body becomes resistant to insulin. Among diabetic patients, cardiovascular disease (CVD) is the cause for 80% of the morbidity and mortality associated with the disease 20,21.

Sickle cell disease (SCD) is a genetic blood disorder that affects more than 100,000 people in the United States22. SCD is a point-mutation disease that causes red blood cells to become crescent-shaped, making it difficult for the cells to pass through the blood vasculature23. Both of these disease states increase the chances of developing atherothrombotic conditions in the body. One of the reasons for this is a result of altered fibrin structure and function in diseased states14,24-26.

In both diabetes and sickle cell disease, there is hypercoagulation and hypofibrinolysis activity that induces atherothrombosis and cardiovascular disease (CVD) as compared to healthy patients 17,27,28. It is known that hypofibrinolysis promotes atherosclerosis progression and engenders recurrent ischemic events for patients with premature coronary artery disease29. In the current manuscript, we investigated the role of fibrin physical properties in this particular setting. Fibrin clot structures in non-diseased patients are composed of thin fibers, larger pores, and generally less dense14,24. The increased porosity and less dense fibrin clots in healthy patients have been found to facilitate fibrinolysis16. In hyperthrombotic conditions such as diabetic and sickle cell disease, there is an increase in fibrinogen production, causing the fibrinogen concentration to increase from normal levels of 2.5 mg/ml in healthy patients 30-33. Fibrin clots formed in diabetic patients have been found to be less porous, more rigid, have more branch points, and denser when compared to healthy, non-diabetic patients14,24,33-35. The altered fibrin structure is a result of glycation mechanisms that occur in the proteins involved in clot formation. Nonenzymatic (irreversible) glycation occurs when glucose molecules bind to lysine residues on the fibrinogen molecule, which inhibits human factor XIIIa (FXIIIa) from properly cross-linking glutamine and lysine residues33,36,37.

The structural analysis of fibrin networks has been studied extensively recently. In particular, researchers have utilized electron microscopy and 3D reconstruction of fibrin networks38, investigated how both intravascular (endothelial) cells and extravascular (fibroblasts and smooth muscle) cells affect fibrin structure39, utilized viscoelastic and spectral analysis to analyze fibrin structures40 , and developed correlations between fibrin structure and mechanical properties using experimental and computational approaches41. The focus of the current study was to formulate clot structures under simulated diabetic and sickle cell thrombosis conditions and to use confocal microscopy for examination of the structure and function of the clots in diseased states. Fibrin clots were formed from human fibrinogen, human thrombin, and FXIIIa. The clots were lysed using plasmin. To simulate diabetic conditions, increased concentration of fibrinogen was incubated in glucose solution to induce in vitro fibrinogen glycation. To simulate sickle cell disease clotting conditions, increased fibrinogen concentrations were mixed with sickle cell hematocrit collected from patients as done previously by our group42. These methods were used to examine the structure and functions involved in fibrin clot formation and fibrinolysis under diseased conditions, as well as the mechanisms that induce CVD. Based on current information about these diseases, the glycated fibrin clot structures were denser with fewer and smaller pores. The fibrin clots with red blood cells from sickle cell patients (RBCs) were also denser and displayed aggregation of the RBCs and agglomerated fibrin clusters. This is a well-established phenomenon that has been determined previously43. It was also hypothesized that the fibrinolysis rate would be significantly lower in glycated fibrin clots with and without reduced plasmin compared to healthy, normal fibrin. The results showed that for glycated fibrin clots, significantly different lysis rate results were observed only under conditions of reduced plasmin concentration. This experimental technique of using confocal microscopy offers significant advantages over other imaging methods because the cells and proteins remain in their native state, which enables the capturing of real-time video of the clotting activity. This method of synthetically inducing clotting is also cheaper and more time efficient than obtaining patient samples and filtering out individual proteins and enzymes. Furthermore, by using separated proteins and enzymes to synthesize clots, the clots were standardized so that there was not variability between samples as a result of other proteins in the plasma.

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Protocol

NOTE: The following protocol adheres to the guidelines set by the Institutional Review Board (IRB) at Georgia Tech.

1. Blood Collection and Red Blood Cell Isolation Procedure

  1. Collect 40-120 ml of blood from donors in 10 ml heparinized vacutainer tubes. Start PBMC (peripheral blood mononucleated cell) isolation within 4 hr of collection. During this time, keep blood at RT.
    NOTE: O/N storage of blood in RT or in 4 °C is NOT recommended as this will result in lower PBMC yield.
  2. Dilute whole blood 1:1 in ice-cold (4 °C) sterile PBS.
  3. Pipette 10 ml of ice-cold hydrophilic polysaccharide into an empty, sterile 50 ml conical tube. Gently transfer diluted whole blood on top of the hydrophilic polysaccharide.
  4. Use a 25 ml pipette in slow setting and pipette blood along the side of the tube very slowly. Ensure that the blood is not mixed with the saccharide prior to centrifugation because the hydrophilic polysaccharide is toxic to cells and otherwise, the PBMC yield will be lower.
  5. Centrifuge blood samples for 30 min at 400 x g at 4 °C. If available, reduce the brake speed of the centrifuge to half of maximum for better separation after centrifugation.
  6. Aspirate off the plasma/platelet fraction of the centrifuged blood sample. Carefully aspirate off the hydrophilic polysaccharide layer directly above the packed RBC layer.
  7. Collect 8-10 ml of packed RBCs and dilute 1:1 with unsterile 0.9% NaCl saline.

2. Confocal Microscopy Evaluation of Glycated Clot Structures (Simulated Diabetic Clots)

  1. Defrost human fibrinogen and fluorescently labeled human fibrinogen conjugate at 37 °C.
  2. Pipette the following into a 0.5 ml graduated microcentrifuge tube.
    1. For the healthy, normal fibrinogen clots, mix human fibrinogen with 10% fluorescently labeled human fibrinogen conjugate in a 50 mM Tris/100 mM NaCl buffer at a concentration of 5 mg/ml.
    2. For artificially glycated fibrinogen (to simulate diabetic clots), incubate human fibrinogen and 10% fluorescently labeled fibrinogen conjugate in a 5 mM solution of glucose dissolved in 50 mM Tris/100 mM NaCl for a resulting concentration of 6.8 mg/ml.
  3. Cover the micro-centrifuge tubes using an opaque material to avoid exposure to light. Incubate the tubes in a 37 °C water bath for 48 hr.
  4. Make a chamber on a glass microscope slide by affixing a thin adhesive on 2 sides of the slide.
  5. After the 48 hr incubation period, prepare reagents for fibrin formation by defrosting FXIIIa and human alpha thrombin at RT.
  6. Dilute FXIIIa to 80 U/ml in 5 mM CaCl2 buffer.
  7. Dilute thrombin to 4 U/ml in 5 mM CaCl2 buffer.
    1. For normal clots, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 2.5 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 μl.
    2. For the glycated clots, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 3.4 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 μl.
  8. Immediately pipette 30 μl of the fibrin solution into the chamber formed by the adhesive.
  9. Place a 22 mm x 22 mm glass cover slip with thickness of 0.15 mm on top of the 2 layers of adhesive. Seal the open sides with clear adhesive to prevent the fibrin clot from drying out. Ensure that the adhesive does not interact with the fibrin clot.
  10. Allow the fibrin clots to polymerize at 21-23 °C for 2 hr before confocal imaging.
  11. Take confocal microscopy images of the clots using a confocal microscope with a 40X/1.30 Oil M27 lens with a 488 nm Argon laser.

3. Confocal Microscopy Evaluation of Fibrin Clots Containing RBCs from Sickle Cell Patients

  1. Defrost human fibrinogen and fluorescently labeled human fibrinogen conjugate at 37 °C.
  2. Pipette the following into a 0.5 ml graduated microcentrifuge tube.
    1. For the healthy, normal fibrin clot, mix human fibrinogen with 10% fluorescently labeled human fibrinogen conjugate in a 50 mM Tris/100 mM NaCl buffer at a concentration of 5 mg/ml.
    2. For the clots containing SCD RBCs, mix human fibrinogen and 10% fluorescently labeled human fibrinogen conjugate in a 50 mM Tris/100 mM NaCl such that the resulting concentration of the fibrinogen is 10 mg/ml.
  3. Label the isolated RBCs (both normal and those from sickle cell patients obtained from the RBC isolation protocol) using a cell-labeling solution.
    1. Suspend cells at a density of 1 x 106 per ml in any chosen serum-free culture medium.
    2. Add 5 μl/ml of the cell suspension to the cell-labeling solution. Mix well by gentle pipetting gently.
    3. Incubate for 20 min at 37 °C.
    4. Centrifuge the labeled suspension tubes at 1,500 rpm for 5 min at 37 °C.
    5. Remove the supernatant and gently re-suspend the cells in 37 °C medium.
    6. Repeat the wash procedure (3.3.4 and 3.3.5) two more times.
  4. Dilute FXIIIa to 80 U/ml in 5 mM CaCl2 buffer.
  5. Dilute thrombin to 4 U/ml in 5 mM CaCl2 buffer.
    1. For normal clots, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 2.5 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 μl.
    2. For the clots containing SCD RBCs, ensure that the final concentrations of fibrinogen, FXIIIa, and thrombin are 5 mg/ml, 20 U/ml, and 1 U/ml, respectively at a volume of 50 μl.
  6. Pipette 10 μl of the labeled RBCs into the fibrin solution. Pipette 30 μl of the fibrin with RBCs onto the microscope glass (refer to slide preparation for glycated clots).
  7. Allow the fibrin to polymerize at 21-23 °C for 2 hr before confocal imaging.
  8. Take confocal images of the clots using the confocal microscope, and utilize excitation lasers with wavelengths of 488 nm and 633 nm to excite the fluorescently labeled fibrin and RBCs.

4. Confocal Microscopy Evaluation of Fibrinolysis Rates in Glycated Clot Structures

  1. Repeat the confocal microscopy protocol of glycated clots (Protocol Step 2) for the fibrinolysis rate evaluation in glycated clots.
  2. Do not seal the ends of the chamber with clear adhesive. Allow the fibrin clots to polymerize for 1.5 hr at 21-23 °C in a sealed enclosure containing 250 ml of water to prevent dehydration.
  3. After polymerization, obtain images of the clots using the confocal microscope.
  4. Pipette plasmin into the open end of the chamber and disperse the plasmin through the clot via capillary action.
    1. For the normal and glycated fibrinolysis experiments with normal concentrations, pipette 25 μl at 200 μg/ml of plasmin into the opening of the chamber.
    2. For the glycated clots with reduced concentrations, pipette 25 μl at 50 μg/ml into the opening of the chamber.
  5. Use the time series feature (see Figure 1) in the confocal microscope software to capture real-time video footage of the plasmin lysing the clot.
    1. Click acquisition mode and then select “Time Series.” Select the number of cycles and the recording time interval.

5. Calculating Fibrinolysis Rates

  1. Determine the lysis rate by setting an area (2,500 µm2) and calculating the elapsed time required to dissolve a fixed area of the clot. Calculate the lysis rate using the following equation (Figure 2):
    figure-protocol-7954

6. Statistical Analysis of Fibrinolysis Rates

  1. Analyze the lysis data using statistical analysis software. Perform a one-way ANOVA and a post hoc Tukey-Kramer test44,45.

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Results

Confocal Microscopy Analysis of Glycated Fibrin Clot Structures

The confocal microscopy images of normal and glycated clots are presented in Figure 3. Confocal microscopy analysis of the normal and glycated clots reveals that glycated clots are denser with smaller pores than the normal clots both with and without the addition of FXIIIa during clot polymerization. In Figure 3A and 3B, there is a lower fibrin concentration which created a less d...

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Discussion

To obtain meaningful data about the structure of clotting mechanisms in disease states, it is important to isolate the factors involved in clotting to determine the effects of the proteins and cells in these conditions. This protocol was developed for the purposes of investigating the structure of the fibrin clot in diabetic and SCD states in vitro.

It is necessary to understand the mechanisms involved in fibrin formation and fibrinolysis in disease states since altered conditions cau...

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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.

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Materials

NameCompanyCatalog NumberComments
PBSLife Technologies10010031
Ficoll-Paque (hydrophilic polysaccharide)GE Healthcare45-001-749
10 ml heparinized vacutainer tubesBD Biosciences366643
Human FibrinogenEnzyme Research LaboratoriesN/A
Alexa Fluor 488 human fibrinogen conjugateMolecular ProbesF13191
0.5 ml graduated microcentrifuge tubeFisher Scientific05-408-120
Glucose powderLife Technologies15023-02
FXIIIaEnzyme Research LaboratoriesN/A
VIS Confocal MicroscopeZeiss LSM 510LSM 510
50 mM TrisLonzaS50-642
Calcium Chloride (CaCl2)Sigma Aldrich449709-10G
Vybrant DiD cell-labeling solutionLife TechnologiesL7781
PlasminEnzyme Research LaboratoriesN/A
Sodium Chloride (5 M NaCl)Life TechnologiesAM9759
Statistical Modeling SoftwareIBMSPSS Statistics 22

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Keywords Fibrin ClotClot StructureClot MorphologyConfocal MicroscopyClot LysisDiabetesSickle Cell AnemiaThrombosisExperimental TechniquesImaging

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