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.

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
<ol>
	<li>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 &#176;C is NOT recommended as this will result in lower PBMC yield.</li>
	<li>Dilute whole blood 1:1 in ice-cold (4 &#176;C) sterile PBS.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge blood samples for 30 min at 400 x g at 4 &#176;C. If available, reduce the brake speed of the centrifuge to half of maximum for better separation after centrifugation.</li>
	<li>Aspirate off the plasma/platelet fraction of the centrifuged blood sample. Carefully aspirate off the hydrophilic polysaccharide layer directly above the packed RBC layer.</li>
	<li>Collect 8-10 ml of packed RBCs and dilute 1:1 with unsterile 0.9% NaCl saline.</li>
</ol>
2. Confocal Microscopy Evaluation of Glycated Clot Structures (Simulated Diabetic Clots)
<ol>
	<li>Defrost human fibrinogen and fluorescently labeled human fibrinogen conjugate at 37 &#176;C.</li>
	<li>Pipette the following into a 0.5 ml graduated microcentrifuge tube.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Cover the micro-centrifuge tubes using an opaque material to avoid exposure to light. Incubate the tubes in a 37 &#176;C water bath for 48 hr.</li>
	<li>Make a chamber on a glass microscope slide by affixing a thin adhesive on 2 sides of the slide.</li>
	<li>After the 48 hr incubation period, prepare reagents for fibrin formation by defrosting FXIIIa and human alpha thrombin at RT.</li>
	<li>Dilute FXIIIa to 80 U/ml in 5 mM CaCl2 buffer.</li>
	<li>Dilute thrombin to 4 U/ml in 5 mM CaCl2 buffer.
	<ol>
		<li>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 &#956;l.</li>
		<li>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 &#956;l.</li>
	</ol>
	</li>
	<li>Immediately pipette 30 &#956;l of the fibrin solution into the chamber formed by the adhesive.</li>
	<li>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.</li>
	<li>Allow the fibrin clots to polymerize at 21-23 &#176;C for 2 hr before confocal imaging.</li>
	<li>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.</li>
</ol>
3. Confocal Microscopy Evaluation of Fibrin Clots Containing RBCs from Sickle Cell Patients
<ol>
	<li>Defrost human fibrinogen and fluorescently labeled human fibrinogen conjugate at 37 &#176;C.</li>
	<li>Pipette the following into a 0.5 ml graduated microcentrifuge tube.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Label the isolated RBCs (both normal and those from sickle cell patients obtained from the RBC isolation protocol) using a cell-labeling solution.
	<ol>
		<li>Suspend cells at a density of 1 x 106 per ml in any chosen serum-free culture medium.</li>
		<li>Add 5 &#956;l/ml of the cell suspension to the cell-labeling solution. Mix well by gentle pipetting gently.</li>
		<li>Incubate for 20 min at 37 &#176;C.</li>
		<li>Centrifuge the labeled suspension tubes at 1,500 rpm for 5 min at 37 &#176;C.</li>
		<li>Remove the supernatant and gently re-suspend the cells in 37 &#176;C medium.</li>
		<li>Repeat the wash procedure (3.3.4 and 3.3.5) two more times.</li>
	</ol>
	</li>
	<li>Dilute FXIIIa to 80 U/ml in 5 mM CaCl2 buffer.</li>
	<li>Dilute thrombin to 4 U/ml in 5 mM CaCl2 buffer.
	<ol>
		<li>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 &#956;l.</li>
		<li>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 &#956;l.</li>
	</ol>
	</li>
	<li>Pipette 10 &#956;l of the labeled RBCs into the fibrin solution. Pipette 30 &#956;l of the fibrin with RBCs onto the microscope glass (refer to slide preparation for glycated clots).</li>
	<li>Allow the fibrin to polymerize at 21-23 &#176;C for 2 hr before confocal imaging.</li>
	<li>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.</li>
</ol>
4. Confocal Microscopy Evaluation of Fibrinolysis Rates in Glycated Clot Structures
<ol>
	<li>Repeat the confocal microscopy protocol of glycated clots (Protocol Step 2) for the fibrinolysis rate evaluation in glycated clots.</li>
	<li>Do not seal the ends of the chamber with clear adhesive. Allow the fibrin clots to polymerize for 1.5 hr at 21-23 &#176;C in a sealed enclosure containing 250 ml of water to prevent dehydration.</li>
	<li>After polymerization, obtain images of the clots using the confocal microscope.</li>
	<li>Pipette plasmin into the open end of the chamber and disperse the plasmin through the clot via capillary action.
	<ol>
		<li>For the normal and glycated fibrinolysis experiments with normal concentrations, pipette 25 &#956;l at 200 &#956;g/ml of plasmin into the opening of the chamber.</li>
		<li>For the glycated clots with reduced concentrations, pipette 25 &#956;l at 50 &#956;g/ml into the opening of the chamber.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Click acquisition mode and then select &#8220;Time Series.&#8221; Select the number of cycles and the recording time interval.</li>
	</ol>
	</li>
</ol>
5. Calculating Fibrinolysis Rates
<ol>
	<li>Determine the lysis rate by setting an area (2,500 &#181;m2) and calculating the elapsed time required to dissolve a fixed area of the clot. Calculate the lysis rate using the following equation (Figure 2): 
	<img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/52019/52019eq1.jpg" /></li>
</ol>
6. Statistical Analysis of Fibrinolysis Rates
<ol>
	<li>Analyze the lysis data using statistical analysis software. Perform a one-way ANOVA and a post hoc Tukey-Kramer test44,45.</li>
</ol>

<ol><li>Averett, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Modular+Fibrinogen+Model+that+Captures+the+Stress-Strain+Behavior+of+Fibers%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">A Modular Fibrinogen Model that Captures the Stress-Strain Behavior of Fibers.</a> Biophysical Journal. 103, 1537-1544 (2012).</li>
<li>Carlisle, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanical+properties+of+individual%2C+electrospun+fibrinogen+fibers%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The mechanical properties of individual, electrospun fibrinogen fibers.</a> Biomaterials. 30, 1205-1213 (2009).</li>
<li>Falvo, M. R., Gorkun, O. V., Lord, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+origins+of+the+mechanical+properties+of+fibrin%5BTitle%5D)+AND+%22Biophysical+Chemistry%22%5BJournal%5D)">The molecular origins of the mechanical properties of fibrin.</a> Biophysical Chemistry. 152, 15-20 (2010).</li>
<li>Guthold, M., Cho, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrinogen+Unfolding+Mechanisms+Are+Not+Too+Much+of+a+Stretch%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Fibrinogen Unfolding Mechanisms Are Not Too Much of a Stretch.</a> Structure. 19, 1536-1538 (2011).</li>
<li>Gerth, C., Roberts, W. W., Ferry, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheology+of+fibrin+clots+II%3A+Linear+viscoelastic+behavior+in+shear+creep%5BTitle%5D)+AND+%22Biophysical+Chemistry%22%5BJournal%5D)">Rheology of fibrin clots II: Linear viscoelastic behavior in shear creep.</a> Biophysical Chemistry. 2 (74), 208-217 (1974).</li>
<li>Nelb, G. W., Kamykowski, G. W., Ferry, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheology+of+fibrin+clots.+v.+shear+modulus%2C+creep%2C+and+creep+recovery+of+fine+unligated+clots%5BTitle%5D)+AND+%22Biophysical+Chemistry%22%5BJournal%5D)">Rheology of fibrin clots. v. shear modulus, creep, and creep recovery of fine unligated clots.</a> Biophysical Chemistry. 13 (81), 15-23 (1981).</li>
<li>Roberts, W. W., Kramer, O., Rosser, R. W., Nestler, F. H. M., Ferry, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheology+of+fibrin+clots.+I.%3A+Dynamic+viscoelastic+properties+and+fluid+permeation%5BTitle%5D)+AND+%22Biophysical+Chemistry%22%5BJournal%5D)">Rheology of fibrin clots. I.: Dynamic viscoelastic properties and fluid permeation.</a> Biophysical Chemistry. 1, 152-160 (1974).</li>
<li>Ryan, E. A., Mockros, L. F., Weisel, J. W., Lorand, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+Origins+of+Fibrin+Clot+Rheology%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Structural Origins of Fibrin Clot Rheology.</a> Biophysical Journal. 77, 2813-2826 (1999).</li>
<li>Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrin+assembly.+Lateral+aggregation+and+the+role+of+the+two+pairs+of+fibrinopeptides%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides.</a> Biophysical Journal. 50, 1079-1093 (1986).</li>
<li>Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanical+properties+of+fibrin+for+basic+scientists+and+clinicians%5BTitle%5D)+AND+%22Biophysical+Chemistry%22%5BJournal%5D)">The mechanical properties of fibrin for basic scientists and clinicians.</a> Biophysical Chemistry. 112, 267-276 (2004).</li>
<li>Wolberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin+generation+and+fibrin+clot+structure%5BTitle%5D)+AND+%22Blood+Reviews%22%5BJournal%5D)">Thrombin generation and fibrin clot structure.</a> Blood Reviews. 21, 131-142 (2007).</li>
<li>Wolberg, A. S., Campbell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombin+generation%2C+fibrin+clot+formation+and+hemostasis%5BTitle%5D)+AND+%22Transfusion+and+Apheresis+Science%22%5BJournal%5D)">Thrombin generation, fibrin clot formation and hemostasis.</a> Transfusion and Apheresis Science. 38, 15-23 (2008).</li>
<li>Yeromonahos, C., Polack, B., Caton, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanostructure+of+the+Fibrin+Clot%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Nanostructure of the Fibrin Clot.</a> Biophysical Journal. 99, 2018-2027 (2010).</li>
<li>Dunn, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrinogen+and+fibrin+clot+structure+in+diabetes%5BTitle%5D)+AND+%22Herz%22%5BJournal%5D)">Fibrinogen and fibrin clot structure in diabetes.</a> Herz. 29, 470-479 (2004).</li>
<li>Gladwin, M. T., Sachdev, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiovascular+abnormalities+in+sickle+cell+disease%5BTitle%5D)+AND+%22Journal+of+the+American+College+of+Cardiology%22%5BJournal%5D)">Cardiovascular abnormalities in sickle cell disease.</a> Journal of the American College of Cardiology. 59, 1123-1133 (2012).</li>
<li>Collet, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+Fibrin+Architecture+Is+Associated+With+Hypofibrinolysis+and+Premature+Coronary+Atherothrombosis%5BTitle%5D)+AND+%22Arteriosclerosis%2C+thrombosis%2C+and+vascular+biology%22%5BJournal%5D)">Altered Fibrin Architecture Is Associated With Hypofibrinolysis and Premature Coronary Atherothrombosis.</a> Arteriosclerosis, thrombosis, and vascular biology. 26, 2567-2573 (2006).</li>
<li>Carr, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+mellitus%3A+a+hypercoagulable+state%5BTitle%5D)+AND+%22Journal+of+Diabetes+and+its+Complications%22%5BJournal%5D)">Diabetes mellitus: a hypercoagulable state.</a> Journal of Diabetes and its Complications. 15, 44-54 (2001).</li>
<li>Ajjan, R. A., Ariëns, R. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiovascular+disease+and+heritability+of+the+prothrombotic+state%5BTitle%5D)+AND+%22Blood+Reviews%22%5BJournal%5D)">Cardiovascular disease and heritability of the prothrombotic state.</a> Blood Reviews. 23, 67-78 (2009).</li>
<li>Standeven, K. F., Ariëns, R. A. S., Grant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+physiology+and+pathology+of+fibrin+structure%2Ffunction%5BTitle%5D)+AND+%22Blood+Reviews%22%5BJournal%5D)">The molecular physiology and pathology of fibrin structure/function.</a> Blood Reviews. 19, 275-288 (2005).</li>
<li>American Diabetes Association. , Available from:
 <a target="_blank" href="http://www.diabetes.org/">http://www.diabetes.org/</a> (2014).</li>
<li>Alzahrani, S., Ajjan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+article%3A+Coagulation+and+fibrinolysis+in+diabetes%5BTitle%5D)+AND+%22Diabetes+and+Vascular+Disease+Research%22%5BJournal%5D)">Review article: Coagulation and fibrinolysis in diabetes.</a> Diabetes and Vascular Disease Research. 7, 260-273 (2010).</li>
<li>Sickle Cell Disease Association of America, Inc. , 
 Available from:
 
 <a target="_blank" href="http://www.sicklecelldisease.org/">http://www.sicklecelldisease.org/</a> (2014).</li>
<li>Stuart, M. J., Nagel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sickle-cell+disease%5BTitle%5D)+AND+%22The+Lancet%22%5BJournal%5D)">Sickle-cell disease.</a> The Lancet. 364, 1343-1360 (2004).</li>
<li>Dunn, E. J., Ariëns, R. A. S., Grant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+type+2+diabetes+on+fibrin+structure+and+function%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">The influence of type 2 diabetes on fibrin structure and function.</a> Diabetologia. 48, 1198-1206 (2005).</li>
<li>Dunn, E. J., Philippou, H., Ariëns, R. A. S., Grant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+involved+in+the+resistance+of+fibrin+to+clot+lysis+by+plasmin+in+subjects+with+type+2+diabetes+mellitus%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Molecular mechanisms involved in the resistance of fibrin to clot lysis by plasmin in subjects with type 2 diabetes mellitus.</a> Diabetologia. 49, 1071-1080 (2006).</li>
<li>Famodu, A., Reid, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+fibrinogen+levels+in+sickle+cell+disease%5BTitle%5D)+AND+%22Tropical+and+geographical+medicine%22%5BJournal%5D)">Plasma fibrinogen levels in sickle cell disease.</a> Tropical and geographical medicine. 39, 36-38 (1987).</li>
<li>Richardson, S., Matthews, K., Stuart, J., Geddes, A., Wilcox, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serial+Changes+in+Coagulation+and+Viscosity+during+Sickle-Cell+Crisis%5BTitle%5D)+AND+%22British+journal+of+haematology%22%5BJournal%5D)">Serial Changes in Coagulation and Viscosity during Sickle-Cell Crisis.</a> British journal of haematology. 41, 95-103 (1979).</li>
<li>Ataga, K. I., Orringer, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypercoagulability+in+sickle+cell+disease%3A+a+curious+paradox%5BTitle%5D)+AND+%22The+American+Journal+of+Medicine%22%5BJournal%5D)">Hypercoagulability in sickle cell disease: a curious paradox.</a> The American Journal of Medicine. 115, 721-728 (2003).</li>
<li>Collet, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+fibrin+architecture+is+associated+with+hypofibrinolysis+and+premature+coronary+atherothrombosis%5BTitle%5D)+AND+%22Arteriosclerosis%2C+thrombosis%2C+and+vascular+biology%22%5BJournal%5D)">Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis.</a> Arteriosclerosis, thrombosis, and vascular biology. 26, 2567-2573 (2006).</li>
<li>Barazzoni, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+Fibrinogen+Production+in+Type+2+Diabetic+Patients+without+Detectable+Vascular+Complications%3A+Correlation+with+Plasma+Glucagon+Concentrations%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Endocrinology+%26+Metabolism%22%5BJournal%5D)">Increased Fibrinogen Production in Type 2 Diabetic Patients without Detectable Vascular Complications: Correlation with Plasma Glucagon Concentrations.</a> The Journal of Clinical Endocrinology & Metabolism. 85, 3121-3125 (2000).</li>
<li>Ceriello, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coagulation+activation+in+diabetes+mellitus%3A+the+role+of+hyperglycaemia+and+therapeutic+prospects%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Coagulation activation in diabetes mellitus: the role of hyperglycaemia and therapeutic prospects.</a> Diabetologia. 36, 1119-1125 (1993).</li>
<li>Mayne, E. E., Bridges, J. M., Weaver, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+adhesiveness%2C+plasma+fibrinogen+and+factor+VIII+levels+in+diabetes+mellitus%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Platelet adhesiveness, plasma fibrinogen and factor VIII levels in diabetes mellitus.</a> Diabetologia. 6, 436-440 (1970).</li>
<li>Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrinogen+and+fibrin%5BTitle%5D)+AND+%22Advances+in+protein+chemistry%22%5BJournal%5D)">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/pubmed?term=((Influence+of+Fibrin+Network+Conformation+and+Fibrin+Fiber+Diameter+on+Fibrinolysis+Speed+Dynamic+and+Structural+Approaches+by+Confocal+Microscopy%5BTitle%5D)+AND+%22Arteriosclerosis%2C+thrombosis%2C+and+vascular+biology%22%5BJournal%5D)">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>Jörneskog, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+properties+of+the+fibrin+gel+structure+in+patients+with+IDDM%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">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/pubmed?term=((Acetylation+and+glycation+of+fibrinogen+in+vitro+occur+at+specific+lysine+residues+in+a+concentration+dependent+manner%3A+A+mass+spectrometric+and+isotope+labeling+study%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">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/pubmed?term=((Glycation+of+fibrinogen+in+uncontrolled+diabetic+patients+and+the+effects+of+glycaemic+control+on+fibrinogen+glycation%5BTitle%5D)+AND+%22Thrombosis+Research%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+reconstruction+of+fibrin+clot+networks+from+stereoscopic+intermediate+voltage+electron+microscope+images+and+analysis+of+branching%5BTitle%5D)+AND+%22Biophysical+journal%22%5BJournal%5D)">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/pubmed?term=((Contributions+of+extravascular+and+intravascular+cells+to+fibrin+network+formation%2C+structure%2C+and+stability%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((A+study+of+microstructural+templating+in+fibrin-thrombin+gel+networks+by+spectral+and+viscoelastic+analysis%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">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/pubmed?term=((Correlation+between+fibrin+network+structure+and+mechanical+properties%3A+an+experimental+and+computational+analysis%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">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/pubmed?term=((Sickle+cell+disease+activates+peripheral+blood+mononuclear+cells+to+induce+cathepsins+k+and+v+activity+in+endothelial+cells%5BTitle%5D)+AND+%22Anemia%22%5BJournal%5D)">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/pubmed?term=((Properties+of+sickle-cell+haemoglobin%5BTitle%5D)+AND+%22Biochemical+Journal%22%5BJournal%5D)">Properties of sickle-cell haemoglobin.</a> Biochemical Journal. 65, 212(1957).</li>
<li>Kuehl, R. O. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKuehl%2C+RO+%22Design+of+experiments+%3A+statistical+principles+of+research+design+and+analysis%22">Design of experiments : statistical principles of research design and analysis</a>. , 2nd ed, Cengage Learning. Pacific Grove, CA. (2000).</li>
<li>Lindman, H. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLindman%2C+HR+%22Analysis+of+variance+in+experimental+designs%22">Analysis of variance in experimental designs</a>. , Springer-Verlag Publishing. New York, N.Y., USA. (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/pubmed?term=((Metastable+Mesoscopic+Clusters+in+Solutions+of+Sickle-Cell+Hemoglobin%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">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/pubmed?term=((An+experimental+and+theoretical+study+on+the+dissolution+of+mural+fibrin+clots+by+tissue-type+plasminogen+activator%5BTitle%5D)+AND+%22Biotechnology+and+bioengineering%22%5BJournal%5D)">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/pubmed?term=((127+Mathematic+model+of+fibrin+clot+lysis+by+plasmin%5BTitle%5D)+AND+%22Fibrinolysis+and+Proteolysis%22%5BJournal%5D)">127 Mathematic model of fibrin clot lysis by plasmin.</a> Fibrinolysis and Proteolysis. 12, Suppl 1. 45(1998).</li>
</ol>

The authors have nothing to disclose.

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...

Injury to a blood vessel&#39;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.

<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.

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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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.

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

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11.4K Views.  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.

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

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.

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 ...

Bioengineering

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.

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

Biochemistry

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

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.

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

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.

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.

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

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.

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

Immunology and Infection

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.

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

Combining the Chandler Loop and RT-FluFF for Ex Vivo Clot Lysis Monitoring

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.

Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In Vitro Models

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.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test ...

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients&#39; hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, ...

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.

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

Biology

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like ...

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

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