Counting 1 fibrin molecule at a time

In this issue of Blood, Hategan et al report on the development of a novel method to study single molecule kinetics of fibrin polymerization.¹ 

Using total internal reflection fluorescence microscopy (TIRFM), Hategan and colleagues are able to follow in real time the addition of individual fibrin molecules to growing fibrin fibers during clot formation. In essence, the authors base their calculations on steps of fluorescence bleaching during the TIRFM experiments, which they relate back to single fibrin molecules. On the basis of these bleaching experiments the authors developed what they call a molecular calibrator, with which they can count individual fibrin molecules during clot formation using TIRFM. Therefore, accurate quantitative data at the single molecule level is provided, leading for the first time to new insights into the number of fibrin molecules involved in fiber formation, over the entire time course of clot formation. In doing so, the authors calculate that fibrin fibers on average contain several thousand molecules in diameter (see figure). In other words, because fibrin fibers are composed of parallel protofibril strands of half-staggered, overlapping fibrin molecules,²  this means that fibrin fibers are on average composed of thousands of protofibrils arranged side-by-side.

Armed with TIRFM, Hategan et al first developed a molecular calibration of the method with clots made from well-defined systems using purified proteins. The authors tested 2 different fluorescent probes, which produced similar results, to exclude potential artifacts caused by a particular dye. Several molar ratios of fluorescent probe over fibrinogen in the range of 0.3-4.0 dyes/molecule were investigated to evaluate the system. Clots were produced both by mixing thrombin with the fluorescently labeled fibrinogen beforehand, and by allowing diffusion of thrombin from one side of a microchamber into the opposite side, where fibrinogen was located. Then fibrin formation was studied in plasma, by spiking plasma with fluorescent fibrinogen, and it was found that fibrin fibers grew up to 25 000 molecules and 900 nm in diameter. Fibers showed a range in thickness. Fibers with a thickness of 400 nm for example consisted of around 5000 molecules in diameter. These findings show for the first time the dynamic molecular composition of fibrin fibers in a growing clot.

The study of fibrin polymerization at the molecular level has been the focus of increasing research activity. Alterations in the way fibrin molecules interact influence the structure of the final blood clot. Because fibrin clot structure has been implicated in thrombotic diseases,³  elucidation of molecular mechanisms in fibrin polymerization is important for our understanding of clot formation and stability. While investigators have previously used light scattering,⁴  electron microscopy,^5,6  and atomic force microscopy^7,8  to study the qualitative behavior of fibrin molecules during clot formation, none of these previous studies were able to provide an accurate quantitative assessment from single molecules to mature fibers during the process of fibrin clot formation as presented here. Hategan et al for the first time elucidate the fibrin formation process at a single molecule level, and it is truly fascinating to see the live growth of fibrin fibers in terms of the number of molecules that constitute the diameter of a fiber.

There is another reason why the study of fibrin polymerization at the molecular level is so important. Traditional biophysical methods to study fibrin polymerization such as turbidity rely on macroscopic changes and are unable to provide information regarding fibrin structures before the gelpoint. Yet, protofibril formation has already occurred when fibrin reaches the gelpoint. Therefore, new nanoscale methods that analyze fibrin polymerization before gelation are expected to provide new insights into abnormalities of the clotting process, even before a macroscopically visual clot has formed. This study by Hategan et al provides an important new step forward in our understanding of the intriguing process by which individual fibrin molecules produce a 3-dimensional structure and provide the proteinaceous backbone with remarkable biochemical and biomechanical properties for the developing blood clot.