FITC-linked Fibrin-Binding Peptide and real-time live confocal microscopy as a novel tool to visualize fibrin(ogen) in coagulation

Background and Aim: Although fibrinogen has been established as a key player in the process of coagulation, many questions about the role of fibrinogen under specific conditions remain. Confocal microscopic assessment of clot formation, and in particular the role that fibrinogen plays in this process, is commonly investigated using pre-labeled fibrinogen. This has a number of drawbacks, primarily that it is impossible to stain fibrin networks after their formation. The aim of the present study is to present an alternative for conveniently post-staining fibrin in a wide range of models/situations, in real time and with high resolution. Methods: We combined a peptide known to bind fibrin and linked it to fluorescein isothiocyanate (FITC), creating the FITC-Fibrin-Binding Peptide (FFBP). We subsequently tested its fibrin-binding capability in vitro under static conditions, as well as under simulated flow, using real-time live confocal microscopy. Results: Fibrin stained with FFBP overlaps with fibrin stained with fibrinogen pre-labeled with Alexa Fluor 647 following coagulation induction. In contrast to pre-labeled fibrinogen, FFBP also stains already formed fibrin networks. By combining FFBP with real-time live confocal microscopy even the visualization of single fibrin fibers is possible. Conclusions: These data indicate that FFBP is a valid and valuable tool for real-time live confocal assessment of clot formation. Relevance for patients: Our findings present a valuable alternative for the visualization of fibrin even after its formation, and we believe this approach will be particularly valuable for future investigations of important, but previously overlooked, aspects of clot formation.