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...

<ol>
	<li>Averett, R. D., 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+Modular+Fibrinogen+Model+that+Captures+the+Stress-Strain+Behavior+of+Fibers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+properties+of+individual,+electrospun+fibrinogen+fibers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+origins+of+the+mechanical+properties+of+fibrin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+Unfolding+Mechanisms+Are+Not+Too+Much+of+a+Stretch.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+fibrin+clots+II:+Linear+viscoelastic+behavior+in+shear+creep.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+fibrin+clots.+v.+shear+modulus,+creep,+and+creep+recovery+of+fine+unligated+clots.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+fibrin+clots.+I.:+Dynamic+viscoelastic+properties+and+fluid+permeation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Origins+of+Fibrin+Clot+Rheology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+assembly.+Lateral+aggregation+and+the+role+of+the+two+pairs+of+fibrinopeptides.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+properties+of+fibrin+for+basic+scientists+and+clinicians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin+generation+and+fibrin+clot+structure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin+generation,+fibrin+clot+formation+and+hemostasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructure+of+the+Fibrin+Clot.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+fibrin+clot+structure+in+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+abnormalities+in+sickle+cell+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Fibrin+Architecture+Is+Associated+With+Hypofibrinolysis+and+Premature+Coronary+Atherothrombosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus:+a+hypercoagulable+state.">Diabetes mellitus: a hypercoagulable state.</a> Journal of Diabetes and its Complications. 15, 44-54 (2001).</li><li>Ajjan, R. A., Ari&#235;ns, R. 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=Cardiovascular+disease+and+heritability+of+the+prothrombotic+state.">Cardiovascular disease and heritability of the prothrombotic state.</a> Blood Reviews. 23, 67-78 (2009).</li><li>Standeven, K. F., Ari&#235;ns, R. A. S., Grant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+physiology+and+pathology+of+fibrin+structure/function.">The molecular physiology and pathology of fibrin structure/function.</a> Blood Reviews. 19, 275-288 (2005).</li><li>Alzahrani, S., Ajjan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+article:+Coagulation+and+fibrinolysis+in+diabetes.">Review article: Coagulation and fibrinolysis in diabetes.</a> Diabetes and Vascular Disease Research. 7, 260-273 (2010).</li><li>Stuart, M. J., Nagel, R. L. <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.">Sickle-cell disease.</a> The Lancet. 364, 1343-1360 (2004).</li><li>Dunn, E. J., Ari&#235;ns, R. A. S., Grant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+type+2+diabetes+on+fibrin+structure+and+function.">The influence of type 2 diabetes on fibrin structure and function.</a> Diabetologia. 48, 1198-1206 (2005).</li><li>Dunn, E. J., Philippou, H., Ari&#235;ns, R. A. S., Grant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+involved+in+the+resistance+of+fibrin+to+clot+lysis+by+plasmin+in+subjects+with+type+2+diabetes+mellitus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+fibrinogen+levels+in+sickle+cell+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+Changes+in+Coagulation+and+Viscosity+during+Sickle-Cell+Crisis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypercoagulability+in+sickle+cell+disease:+a+curious+paradox.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+fibrin+architecture+is+associated+with+hypofibrinolysis+and+premature+coronary+atherothrombosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Fibrinogen+Production+in+Type+2+Diabetic+Patients+without+Detectable+Vascular+Complications:+Correlation+with+Plasma+Glucagon+Concentrations.">Increased Fibrinogen Production in Type 2 Diabetic Patients without Detectable Vascular Complications: Correlation with Plasma Glucagon Concentrations.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 85, 3121-3125 (2000).</li><li>Ceriello, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+activation+in+diabetes+mellitus:+the+role+of+hyperglycaemia+and+therapeutic+prospects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+adhesiveness,+plasma+fibrinogen+and+factor+VIII+levels+in+diabetes+mellitus.">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/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>

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 border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">PBS</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ficoll-Paque (hydrophilic polysaccharide)</td>
			<td style="width:287px;">GE Healthcare</td>
			<td style="width:119px;">45-001-749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml heparinized vacutainer tubes</td>
			<td style="width:287px;">BD Biosciences</td>
			<td style="width:119px;">366643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Human Fibrinogen</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 human fibrinogen conjugate</td>
			<td style="width:287px;">Molecular Probes</td>
			<td style="width:119px;">F13191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mL graduated microcentrifuge tube</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose powder</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">15023-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FXIIIa</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VIS Confocal Microscope</td>
			<td style="width:287px;">Zeiss LSM 510</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50 mM Tris</td>
			<td style="width:287px;">Lonza</td>
			<td style="width:119px;">S50-642</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:315px;">Calcium Chloride (CaCl2)</td>
			<td style="width:287px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiD cell-labeling solution</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">L7781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Plasmin</td>
			<td style="width:287px;">Enzyme Research Laboratories</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Chloride (5 M NaCl)</td>
			<td style="width:287px;">Life Technologies</td>
			<td style="width:119px;">AM9759</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Statistical Modeling Software</td>
			<td>IBM</td>
			<td style="width:119px;">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...

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.

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

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

Cardiovascular Drugs: Anticoagulants and Antianginal Agents

SCIENTISTS IN ACTION

11.3K Aufrufe.  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.