Visualization of the Dynamics of Fibrin Clot Growth One Molecule at a Time by Total Internal Reflection Fluorescence Microscopy

Department of Cell and Developmental Biology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, United States
Blood (Impact Factor: 10.45). 12/2012; 121(8). DOI: 10.1182/blood-2012-08-451518
Source: PubMed


Individual fluorescently-labeled fibrin(ogen) molecules and their assembly to make a clot were observed by total internal reflection fluorescence microscopy (TIRFM). We used the bleaching of the fluorescent labels to determine the number of active fluorophores attached non-specifically to each molecule. From the total intensity of bleaching steps, as single-molecule signature events, and the distribution of active labeling, we developed a new single-molecule intensity calibration which accounts for all molecules, including those "not seen". Live observation of fibrin polymerization in TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics of individual fibrin fibers quantitatively at molecular level. Some fibers thickened in time to thousands of molecules across equivalent to hundreds of nm in diameter, whereas others reached an early stationary state at smaller diameters. This new approach to determine the molecular dynamics of fibers growth provides information important for understanding clotting mechanisms and the associated clinical implications.

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Available from: Alina Hategan, Dec 13, 2013
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