Morphometric characterization of fibrinogen's αC regions and their role in fibrin self-assembly and molecular organization

Abstract

The flexible C-terminal parts of fibrinogen's Aα chains named the αC regions have been shown to play a role in fibrin self-assembly, although many aspects of their structure and functions remain unknown. To examine the involvement of the αC regions in the early stages of fibrin formation, we used high-resolution atomic force microscopy to image fibrinogen and oligomeric fibrin. Plasma-purified full-length human fibrinogen or des-αC fibrinogen lacking most of the αC regions, untreated or treated with thrombin, was imaged. Up to 80% of the potentially existing αC regions were visualized and quantified; they were highly heterogeneous in their length and configurations. Conversion of fibrinogen to fibrin was accompanied by an increase in the incidence and length of the αC regions as well as transitions from more compact conformations, such as a globule on a string, to extended and more flexible offshoots. Concurrent dynamic turbidimetry, confocal microscopy, and scanning electron microscopy revealed that trimming of the αC regions slowed down fibrin formation, which correlated with longer protofibrils, thinner fibers, and a denser network. No structural distinctions, except for the incidence of the αC regions, were revealed in the laterally aggregated protofibrils made of the full-length or des-αC fibrinogens, suggesting a pure kinetic effect of the αC regions on the fibrin architecture. This work provides a structural molecular basis for the promoting role of the αC regions in the early stages of fibrin self-assembly and reveals this stage of fibrin formation as a potential therapeutic target to modulate the structure and mechanical properties of blood clots.

Fibrinogen αC‐regions are not directly involved in fibrin polymerization as evidenced by a “Double‐Detroit” recombinant fibrinogen mutant and knobs‐mimic peptides

“…Additional studies showed concentration‐dependent aggregation of recombinant αC‐region fragments, and optical tweezers experiments demonstrated αC‐region interactions with the central E‐region and, more weakly, between them . More recently, AFM and AFM/turbidity studies further investigated the issue . The consensus picture was that the αC‐regions following FpB cleavage extend further and help the lateral aggregation of fibrils by binding to each other.…”

“…51 More recently, AFM and AFM/turbidity studies further investigated the issue. 52,53 The consensus picture was that the αC-regions following FpB cleavage extend further and help the lateral aggregation of fibrils by binding to each other. Furthermore, a very recent molecular dynamics simulation 54 investigated the role of a particular residue, AαM476, located in the β-hairpin present within the only (partially) structured domain so far identified in the αC-region.…”

Factor XIII topology: organization of B subunits and changes with activation studied with single‐molecule atomic force microscopy

“…Here, we visualized and characterized quantitatively the morphology of the entire pFXIII‐A 2 B 2 molecules and individual A and B subunits before and after proteolytic activation using high‐resolution single‐molecule atomic force microscopy (AFM) . We showed that pFXIII consisted of a globular A 2 dimer and thin flexible strands of B subunits that typically protrude from the globular core.…”

Revealing the molecular origins of fibrin’s elastomeric properties by in situ X-ray scattering

“…The rationale for this mechanism is that the αC-region consists of a natively unfolded αC-connector of 170 amino acids that can be modelled as a wormlike chain with a persistence length of 0.6 nm, and a folded αC-domain of 218 amino acids 11,63 . The αC-region as a whole can accommodate large elongations because it is only 45-50 nm long at rest 70 and can be stretched to 148 nm. If the fiber elongates through stretching of the αC-tethers, we expect no shift of the Bragg peak until the strain reaches a point where the αC-tethers are pulled taut and the protofibrils start to feel the strain.…”