This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Investigating the molecular origins of these mechanical properties, has led us to question the traditional understanding of the fiber structure. Briefly, the fibrinogen monomer is a symmetric protein consisting of three polypeptide chains (known from crystal structure). They form a dumbbell-type shape with two outer globular 'D'domains connected to a central 'E'domain. The E domain contains four 'knobs.' These knobs, when activated by thrombin, connect to corresponding sites in the D domains of nearby monomers, forming a half-staggered structure. This structure elongates as more monomers are recruited, forming a protofibril. Traditionally the ~100nm thick fibers are thought to be composed of laterally aggregated fibrin protofibrils. In addition to these interactions, each monomer contains an unstructured alpha-C domain that may play a role in the lateral aggregation of protofibrils. The fibers are thought to contain up to 80% water based on index of refraction matching measurements. The mechanical properties of fibrin are now known, but reconciling these properties with the internal structure of the fiber poses many questions, specifically: 1) If the protofibril hypothesis is correct, do protofibrils span the length of the fiber, or is each of finite length inside the fiber? (2) Are the protofibrils stiff? If protofibrils are of finite length (not spanning the entire fiber), could they slide/shear against one another during fiber elongation, or are there other lateral interactions between adjacent protofibrils? If they are not finite length, then all stretching must come from within the protofibril. (3) If fibers are 80% water, does this mean that most protofibibrils are separated from other lateral protofibrils in space, or does most of the water volume exist within the protofibril substructure (based on crystal packing, there's room for 50% water within a fibrin monomer crystal). We along with many other groups have imaged fibrin fibers with traditional TEM and SEM techniques. These data indicate that fibers are highly ordered with a 23nm spacing across the fiber seen in TEM images (23 nm is half the length of a fibrin monomer). What is not clear is whether this order within the fiber exists within the native state, when the fiber has not been fixed and dried. To our knowledge, no one has used CryoEM tomography to push the understanding of the fiber structure forward. Fibrin sheets are a recently discovered form of fibrin. They appear to be molecularly thin and mechanically continuous. Sheets have been observed rolling into fibers and shredding into fibrin networks, but it is not known whether they play a role in fibrin network formation in vivo. The internal structure of sheets is completely unknown. Are they formed by a series of laterally aggregated protofibrils, or do they come from a completely different set of interactions? We feel that cryoET, with its ability to freeze a macromolecular assembly in its native conformation could resolve many of these questions and give new insight into the molecular origins of fibrin's mechanical properties. These are important questions that need to be addressed in order to understand, at the macroscopic level, the deformation mechanisms in blood clots. We have experience forming fibrin fibers and sheets on a variety of substrates and conditions, and preparing fibers for SEM and traditional TEM imaging. We believe that we can achieve high resolution images that will provide a critical breakthrough in the understanding of these fibrin structures.
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