CISMM has a long and productive history of collaboration with investigators studying the mechanical processes related to blot clotting [Campbell 2010, Falvo 2010, Falvo 2008, Guthold 2007, Houser 2010, Hudson 2013, Hudson 2010, Liu 2006, O'Brien 2008]. We are currently embarking on two related projects utilizing the Panoptes system to provide high-throughput permeability measurements of clots.
Permeability of Blood Clots:
Plasma hypercoagulability is an established risk factor for thrombosis; however, it is unknown whether abnormal plasma protein levels are a biomarker of pro-inflammatory disease, a causative mechanism in the etiology and therefore a therapeutic target, or both. Using in vitro and in vivo thrombosis models, we have shown that elevated fibrinogen (hyperfibrinogenemia) directly promotes thrombosis by increasing thrombus fibrin content [Machlus 2011]. Importantly, we further showed that hyperfibrinogenemia increases thrombus resistance to intravenous thrombolytics [Hogan 2001, Machlus 2011], demonstrating a novel mechanism to explain the high rate of failure of thrombolytic therapy. These novel findings have important implications for the use of thrombolytic agents in stroke, myocardial infarction, and deep vein thrombosis and the importance of clot permeability relative to these conditions. We hypothesize network structural changes affect clot permeability. Fibrin permeability is used as an explicit measure of fluid transport through the clot and has been correlated with heart disease, stroke, diabetes, and venous thromboembolism.
(Left) Experimental plate layout for a murine clotting experiment. (Right) Permeability data for the layout depicted at left. Permeability was determined in each well from bead diffusion data (dozens of beads in multiple locations within each well).
- Alisa Wolberg, Assistant Professor, Department of Pathology and Lab. Medicine, The University of North Carolina at Chapel Hill. Prof. Wolberg has worked most recently with CISMM in the development of methods assessing structure and permeability of fibrin gels on cell cultures using confocal microscopy and passive microbead diffusion techniques.
- Frank Church, Professor of Pathology and Laboraotory Medicine, UNC-CH.
Mechanics of Fibrin molecules, Fibers, and Networks:
The mechanical properties of blood clots are of central importance to normal hemostasis. A normal clot is sufficiently strong to seal the site of vessel injury and prevent blood loss from a circulatory system under pressure. A normal clot is sufficiently flexible to deform in response to the shear forces of pulsatile flowing blood. Abnormal hemostasis can cause either bleeding or thrombotic diseases. Although bleeding diseases, such as hemophilia, are relatively rare, thrombotic diseases-myocardial infarction, ischemic stroke, venous thrombosis, pulmonary embolism-are the leading cause of morbidity and mortality in the Western world. Large epidemiological studies correlate clot structure to thrombotic disease, and biochemical studies indicate this correlation reflects the mechanical properties of clots. The studies proposed within this collaboration cluster will focus on molecular mechanisms and physiological conditions that control the structural and mechanical properties of clots. Our findings will thus provide insight into the basis of pathological clots, which may permit design of new treatments for thrombotic diseases. To date we have completed several successful investigations lending insight to fibrin mechanics [1-9].
Constant force unfolding of single molecule fibrinogen through the ‘A-a’ knob-hole interaction
Fibrin, the polymerized form of the soluble plasma protein fibrinogen, plays a critical role in hemostasis as the structural scaffold of blood clots. The primary functions of fibrin are to withstand the shear forces of blood flow and provide mechanical stability to the clot, protecting the wound. As a viscoelastic material, fibrin networks possess unique mechanical properties critical to its physiological function. Studies show that fibrin clots are extensible and elastic. Fibrin fibers are the most highly extensible biological polymers, with strains of up to five fold before breaking. How the fibrin fibers accommodate such high strain is not completely understood but studies show that extensibility of fibrin fiber is due, at least in part, to unfolding of individual fibrinogen monomers. As a multifunctional tool capable of force spectroscopy, the atomic force microscope (AFM) has enabled examination of single molecule protein-protein interactions. Using the AFM to preform constant force pulling experiments, extension-time traces show the unfolding of fibrin molecules. These force curves provide mechanical information on the probability of unfolding and forced rupture of the ‘A-a’ knob-hole interaction. Direct measurements of the stability of the interaction and the kinetics of unfolding the fibrin molecule as a function of force can be obtained and applied to understanding full fiber extensibility.
Formation of blood clots: Fibrin fiber networks are the major structural framework of blood clots. Fibrinogen is a 340,000 Dalton protein, consisting of two identical halves, each comprised of three peptide chains (Aa, Bb,g), which are held together by a network of disulfide bonds. When fibrinogen monomers are exposed to thrombin, fibrinopeptides A and B are cleaved, converting fibrinogen monomers to fibrin monomers which then polymerize initially into half staggered protofibrils, then coalesce into fibers, and eventually form a gel or clot. Fibrin clot mechanics and permeability have traditionally been studied using macroscopic techniques. High resolution structural information has traditionally been collected using electron microscopy on dried samples under vacuum. In the studies proposed here, these properties will be investigated at the microscopic level using microbead techniques, atomic force microscopy (AFM) and confocal microscopy. These techniques enable efficient evaluation of large numb,ers of small volume samples. They also reveal the heterogeneity and microscopic details of mechanics, permeability and structure.
- Nikolay Dokholyan.
- Susan Lord, Professor, Department of Pathology and Lab. Medicine, The University of North Carolina at Chapel Hill. Susan Lord is an leading researcher in the role of fibrin and blood clot formation.
- Martin Guthold. Associate Professor, Department of Physics, Wake Forest University, Supported by the NSF, American Heart Association, Wake Forest University Cross-Campus grant, North Carolina Biotechnology Center. Martin Guthold served as a postdoctoral fellow in the CISMM Resource from 1998-2001. During that time he developed much of the methodology for the manipulation of biological samples and fibrin fibers in particular. Guthold has acquired two nanoManipulator system and AFM in his laboratory at Wake Forest University, and has continued to work closely with CISMM in developing instrumentation and methodology within the fibrin project. He is also developing a microscopy-based aptamer-selection system.
Though independent projects, the four collaborations within this cluster focus on the common themes of mechanical and structural properties as affected by a range of fibrinogen variants and physiological conditions. The CISMM resource offers many unique microscopy and software tools that are well positioned to address these research goals. Broadly, the collaborators seek to determine:
- The role of physiological parameters on clot structure and permeability.
- The role of physiological parameters on clot mechanics.
- The particular regions of the fibrin monomer that contribute to fibrin fiber’s exceptional elasticity and extensibility.
1. Spero, R.C., R.K. Sircar, R. Schubert, R.M. Taylor, 2nd, A.S. Wolberg, and R. Superfine, Nanoparticle diffusion measures bulk clot permeability. Biophys J, 2011. 101(4): p. 943-50.
2. Hudson, N.E., J.R. Houser, E.T. O’Brien, 3rd, R.M. Taylor, 2nd, R. Superfine, S.T. Lord, and M.R. Falvo, Stiffening of individual fibrin fibers equitably distributes strain and strengthens networks. Biophys J, 2010. 98(8): p. 1632-40.
3. Houser, J.R., N.E. Hudson, L. Ping, E.T. O’Brien, 3rd, R. Superfine, S.T. Lord, and M.R. Falvo, Evidence that alphaC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers. Biophys J, 2010. 99(9): p. 3038-47.
4. Falvo, M.R., O.V. Gorkun, and S.T. Lord, The molecular origins of the mechanical properties of fibrin. Biophys Chem, 2010. 152(1-3): p. 15-20.
5. Campbell, R.A., M. Aleman, L.D. Gray, M.R. Falvo, and A.S. Wolberg, Flow profoundly influences fibrin network structure: implications for fibrin formation and clot stability in haemostasis. Thromb Haemost, 2010. 104(6): p. 1281-4.
6. O’Brien, E.T., 3rd, M.R. Falvo, D. Millard, B. Eastwood, R.M. Taylor, 2nd, and R. Superfine, Ultrathin self-assembled fibrin sheets. Proc Natl Acad Sci U S A, 2008. 105(49): p. 19438-43.
7. Falvo, M.R., D. Millard, E.T. O’Brien, 3rd, R. Superfine, and S.T. Lord, Length of tandem repeats in fibrin’s alphaC region correlates with fiber extensibility. J Thromb Haemost, 2008. 6(11): p. 1991-3.
8. Guthold, M., W. Liu, E.A. Sparks, L.M. Jawerth, L. Peng, M. Falvo, R. Superfine, R.R. Hantgan, and S.T. Lord, A comparison of the mechanical and structural properties of fibrin fibers with other protein fibers. Cell Biochem Biophys, 2007. 49(3): p. 165-81.
9. Liu, W., L.M. Jawerth, E.A. Sparks, M.R. Falvo, R.R. Hantgan, R. Superfine, S.T. Lord, and M. Guthold, Fibrin fibers have extraordinary extensibility and elasticity. Science, 2006. 313(5787): p. 634.