Ultrathin self-assembled fibrin sheets

Abstract

Fibrin polymerizes into the fibrous network that is the major structural component of blood clots and thrombi. We demonstrate that fibrin from three different species can also spontaneously polymerize into extensive, molecularly thin, 2D sheets. Sheet assembly occurs in physiologic buffers on both hydrophobic and hydrophilic surfaces, but is routinely observed only when polymerized using very low concentrations of fibrinogen and thrombin. Sheets may have been missed in previous studies because they may be very short-lived at higher concentrations of fibrinogen and thrombin, and their thinness makes them very difficult to detect. We were able to distinguish fluorescently labeled fibrin sheets by polymerizing fibrin onto micropatterned structured surfaces that suspended polymers 10 m above and parallel to the cover-glass surface. We used a combined fluorescence/atomic force microscope system to determine that sheets were Ϸ5 nm thick, flat, elastic and mechanically continuous. Video microscopy of assembling sheets showed that they could polymerize across 25-m channels at hundreds of m 2 /sec (Ϸ10 13 subunits/s⅐M), an apparent rate constant many times greater than those of other protein polymers. Structural transitions from sheets to fibers were observed by fluorescence, transmission, and scanning electron microscopy. Sheets appeared to fold and roll up into larger fibers, and also to develop oval holes to form fiber networks that were ''preattached'' to the substrate and other fibers. We propose a model of fiber formation from sheets and compare it with current models of end-wise polymerization from protofibrils. Sheets could be an unanticipated factor in clot formation and adhesion in vivo, and are a unique material in their own right.clot ͉ fiber ͉ monomolecular sheet ͉ network ͉ thrombus B ecause of the central role of fibrin in the clotting of blood during wound healing and in the formation of pathogenic vascular thrombi, the polymerization of fibrin has been actively studied since the mid-19th century (1). The parent protein, fibrinogen, is an elongated, sausage-shaped dimeric molecule with symmetry around a central globular domain (2-6). Cleavage and removal of the small peptides-fibrinopeptide A and later B-from fibrinogen by the enzyme thrombin expose specific binding sites (''knobs'') on fibrin that allow binding to corresponding regions (''holes'') on neighboring fibrin molecules (7-11). Fibrin can then self-associate to form elongated, sometimes twisted, often highly branched fibers and fiber networks that, together with platelets and blood cells, form clots in response to vascular injury (1,(12)(13)(14)(15). Fibrin fibers are elastic and adhesive and show very high extensibility (16,17). Recent studies suggest that these properties derive, at least in part, from the properties of the fibrin molecule itself (18-21). Exhaustive studies of fibrin polymerization in vitro have been carried out with purified components since the 1940s (e.g., 22, 23), but several details of fibrin assembly into fibers and fibe...

Fibrin Formation, Structure and Properties

“…An alternative model of fibrin formation and unusual structure is based on the formation of ultrathin fibrin sheets spanning channels on a plastic substrate, but the physiological relevance of these results is not yet known (O’Brien et al 2008). Another proposed mechanism of the early stages of fibrin assembly implies that short thin fibrin branches form at an initial phase of polymerization, in which single-bonded “Y-ladder” polymers rapidly elongate before undergoing a delayed transition to the double-stranded fibrils (Rocco et al 2014).…”

Fibrin Association at Hybrid Biointerfaces Made of Clot‐Binding Peptides and Polythiophene

“…Tumor vessels are less robust and stable than normal vasculature, favoring the release of some molecules such as fibrinogen (Fg), which is the fibrin precursor . Leaked Fg is converted into a fibrin meshwork by the action of thrombin (THR), which cleavages and removes fibrinopeptides allowing the self‐assembly of fibrin monomers into elongated fibers . CREKA linked to amino‐dextran coated superparamagnetic iron oxide nanoparticles not only binds to clotted plasma proteins blood and plasma but also induces further localized tumor clotting .…”

Keeping it clean: clot biofilm to wall out bacterial invasion

“…On a very superficial level, these fibrin films might be suspected to have some structural similarities to the ultra‐thin fibrin sheets formed over channels that were described about 10 years ago . However, these fibrin sheets occur under quite different circumstances.…”