Name: experimental and imaging techniques for examining fibrin clot structures in normal and diseased states
Uploaded: 2015-04-01T13:00:00
Description: Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

The authors would like to thank the Lam Lab at Georgia Tech for many helpful discussions in developing the experimental assays. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL115486 and by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTA: Il seguente protocollo aderisce alle linee guida stabilite dal Institutional Review Board (IRB) presso la Georgia Tech. 1. Raccolta di sangue e Blood Red isolamento delle cellule Procedura <ol><li> Raccogliere 40-120 ml di sangue da donatori in 10 ml vacutainer eparina. Inizia PBMC (cellule mononucleate del sangue periferico) isolamento entro 4 ore di raccolta. Durante questo tempo, mantenere il sangue a RT. NOTA: O / N stoccaggio di sangue in RT o 4 ° C non ...

<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>

Per ottenere dati significativi sulla struttura dei meccanismi di coagulazione in stati di malattia, è importante isolare i fattori coinvolti nella coagulazione per determinare gli effetti delle proteine ​​e cellule in queste condizioni. Questo protocollo è stato sviluppato per i fini della valutazione della struttura del coagulo di fibrina in stati diabetici e SCD in vitro. E &#39;necessario comprendere i meccanismi coinvolti nella formazione di fibrina e fibrinolisi in stati...

Lesioni a rivestimento endoteliale di un vaso sanguigno viene riparato attraverso la risposta emostatico, o la formazione di un coagulo di sangue. Quando il sangue permea nella matrice extracellulare, fattori tissutali attivano piastrine nel sangue che facilitano l&#39;iniziazione della cascata coagulativa. Il componente chiave meccanica di questo processo di guarigione è la matrice di fibrina, composto da fibre di fibrina che sono altamente elastica, e può sostenere le grandi forze 1-4. Molti ricercatori hanno studiato la struttura di formazione, e la funzione di fibrina ampiamente negli ultimi decenni 5-13. I pazienti con malattie come il diabete mellito e falciforme hanno un aumentato rischio di sviluppare complicanze trombotiche, come infarto miocardico, cardiopatia ischemica, e colpi 14-19. Oltre 2 milioni di persone sono di nuova diagnosi di diabete mellito ogni anno negli Stati Uniti. Esistono due tipi di diabete: tipo I, dove lacorpo non riesce a produrre quantità sufficienti di insulina, e tipo II, dove il corpo diventa resistente all&#39;insulina. Tra i pazienti diabetici, malattie cardiovascolari (CVD) è la causa per l&#39;80% della morbilità e la mortalità associate con la malattia 20,21. Anemia falciforme (SCD) è una malattia genetica del sangue che colpisce più di 100.000 persone negli Stati Uniti 22. SCD è una malattia mutazioni puntiformi che induce le cellule rosse del sangue per diventare a forma di mezzaluna, il che rende difficile per le cellule di passare attraverso il sistema vascolare del sangue 23. Entrambi questi stati di malattia aumentano le probabilità di sviluppare condizioni aterotrombotici nel corpo. Uno dei motivi per questo è il risultato di strutture di fibrina alterata e funzione negli stati malati 14,24-26. In entrambi diabete e anemia falciforme, c&#39;è ipercoagulazione e l&#39;attività hypofibrinolysis che induce aterotrombosi e malattie cardiovascolari (CVD) rispetto ai pazienti sani 17,27,28. E &#39;noto che hypofibrinolysis promuove la progressione dell&#39;aterosclerosi e genera eventi ischemici ricorrenti per i pazienti con malattia coronarica precoce 29. Nel manoscritto attuale, abbiamo studiato il ruolo di fibrina proprietà fisiche in questo ambiente particolare. Strutture coagulo di fibrina nei pazienti non malati sono composti da fibre sottili, pori più grandi, e in genere meno densa 14,24. La maggiore porosità e coaguli di fibrina meno fitta in pazienti sani sono stati trovati per facilitare fibrinolisi 16. In condizioni hyperthrombotic quali malattia a cellule falciformi diabetica e, vi è un aumento della produzione di fibrinogeno, causando la concentrazione di fibrinogeno aumentare da livelli normali di 2,5 mg / ml in pazienti sani 30-33. Coaguli di fibrina formata in pazienti diabetici sono stati trovati per essere meno porosa, più rigida, avere più punti di ramificazione, e più densa rispetto a sano, non diapazienti Betica 14,24,33-35. La struttura di fibrina alterata è il risultato di meccanismi di glicazione che si verificano nelle proteine ​​coinvolte nella formazione del coagulo. Non enzimatica (irreversibile) glicazione si verifica quando le molecole di glucosio si legano a residui di lisina sulla molecola fibrinogeno, che inibisce il fattore XIIIa umana (FXIIIa) da glutamina e lisina residui opportunamente incrociati collegano 33,36,37. L&#39;analisi strutturale delle reti di fibrina è stato ampiamente studiato di recente. In particolare, i ricercatori hanno utilizzato la microscopia elettronica e la ricostruzione 3D delle reti di fibrina, 38 indagati come sia intravascolari (endoteliali) cellule e extravascolari (fibroblasti e muscolari lisce) cellule influenzano la struttura di fibrina 39, viscoelastico utilizzati e l&#39;analisi spettrale per analizzare le strutture di fibrina 40, e correlazioni sviluppati tra struttura di fibrina e le proprietà meccaniche utilizzando approcci sperimentali e computazionali 41 </sup&gt;. Il focus di questo studio è stato quello di formulare strutture coagulo in condizioni trombosi cellulare diabetico e falce simulati e di utilizzare la microscopia confocale per l&#39;esame della struttura e funzione dei coaguli in stati malati. Coaguli di fibrina sono formati da fibrinogeno umano, trombina umana, e FXIIIa. I coaguli sono stati lisati con plasmina. Per simulare le condizioni di diabetici, aumento della concentrazione di fibrinogeno è stato incubato in soluzione di glucosio di indurre in vitro fibrinogeno glicazione. Per simulare malattie cella condizioni coagulazione falce, un aumento delle concentrazioni di fibrinogeno sono stati mescolati con ematocrito falciforme raccolti da pazienti, come fatto in precedenza dal nostro gruppo 42. Questi metodi sono stati usati per esaminare la struttura e le funzioni coinvolte nella formazione di coaguli di fibrina fibrinolisi e in condizioni malate, nonché i meccanismi che inducono CVD. Sulla base delle informazioni attuali su queste malattie, le strutture di coagulo di fibrina glicata erano più denso con meno di unnd piccoli pori. I coaguli di fibrina con globuli rossi di pazienti con anemia falciforme (RBC) erano anche più denso e visualizzati aggregazione dei globuli rossi e di cluster di fibrina agglomerati. Questo è un fenomeno ben noto che è stato determinato in precedenza 43. E &#39;stato anche ipotizzato che il tasso di fibrinolisi sarebbe significativamente più bassa nel coaguli di fibrina glicata con e senza riduzione plasmina rispetto ai sani, di fibrina normale. I risultati hanno mostrato che per coaguli di fibrina glicate, risultati molto diversi tassi lisi stati osservati solo in condizioni di concentrazione di plasmina ridotta. Questa tecnica sperimentale mediante microscopia confocale offre vantaggi significativi rispetto ad altri metodi di imaging perché le cellule e proteine ​​rimangono nel loro stato nativo, che permette la cattura di video in tempo reale dell&#39;attività coagulazione. Questo metodo di coagulazione sinteticamente Induzione è anche più economico e più tempo efficiente di ottenere campioni di pazienti e filtrando individualeproteine ​​ed enzimi. Inoltre, utilizzando proteine ​​ed enzimi separati per sintetizzare coaguli, i coaguli sono stati standardizzati in modo che non ci fosse la variabilità tra i campioni a causa di altre proteine ​​del 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.

Microscopia confocale Analisi di glicata fibrina Strutture Clot Le immagini microscopia confocale di coaguli normali e glicate sono presentati in Figura 3. Microscopia confocale dei coaguli normali e glicate rivela che coaguli glicate sono più densi con pori inferiori ai coaguli normali con e senza l&#39;aggiunta di FXIIIa durante la polimerizzazione coagulo. Nella Figura 3A e 3B, vi è una concentrazione di fibrina inferiore che crea una s...

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.

Tecniche sperimentali e di imaging per l&#39;esame Strutture coagulo di fibrina in normali e patologiche Uniti

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

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

Cardiovascular Drugs: Anticoagulants and Antianginal Agents

SCIENTISTS IN ACTION

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

Il trattamento dei difetti osteocondrali del ginocchio congiunta del Coniglio con impianto di cellule staminali mesenchimali allogeniche in coaguli di fibrina

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Ricerca

Medicina

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Una pipeline per il 3D multimodalità integrazione di immagini e progettazione assistita da computer in chirurgia dell&#39;epilessia

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

Visualizzazione dei Vascolare e parenchimale rigenerazione dopo il 70% epatectomia parziale in topi normali

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Un chiodo endomidollare di bloccaggio per standardizzata Fissazione del femore osteotomie per analizzare normale e difettoso guarigione ossea nei topi

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Tracciamento in vivo di sviluppo dell&#39;edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l&#39;utilizzo di immagini a risonanza magnetica

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Visualizzazione quantitativa di leucociti infiltrati in un modello di murina della miocardite fulminante mediante microscopia a foglio chiaro

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

In Vivo Analisi morfometrica dei nervi cranici umano usando la formazione immagine a risonanza magnetica in orecchie di malattia di Menière e orecchie udito normale

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

Risonanza magnetica cardiaca per la valutazione di un trombo cardiaco sospetto: tecniche convenzionali ed emergenti

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...