Visualization of fibrinogen αC regions and their arrangement during fibrin network formation by high-resolution AFM.
BACKGROUND
Fibrinogen has been intensively studied with transmission electron microscopy and x-ray diffraction. But until now, a complete 3D structure of the molecule has not yet been available because the two highly flexible αC regions could not be resolved in fibrinogen crystals. This study was aimed at determining whether the αC regions can be visualized by high-resolution atomic force microscopy.


METHODS
Atomic force microscopy with super high resolution was used to image single molecules of fibrinogen and fibrin associates. The key approach was to use a graphite surface modified with the monolayer of amphiphilic carbohydrate-glycine molecules and unique supersharp cantilevers with 1 nm tip diameter.


RESULTS
Fibrinogen αC regions were visualized along with the complete domain structure of the protein. In almost all molecules at pH 7.4 the D domain regions had one or two protrusions of average height 0.4 ± 0.1 nm and length 21 ± 6 nm. The complex, formed between thrombin and fibrinogen, was also visualized. Images of growing fibrin fibers with clearly visible αC regions have been obtained.


CONCLUSIONS
Fibrin αC regions were visible in protofibrils and large fibers; αC regions intertwined near a branchpoint and looked like a zipper. These results support the idea that αC regions are involved in the thickening of fibrin fibers. In addition, new details were revealed about the behavior of individual fibrin molecules during formation of the fibrin network. Under the diluted condition, the positioning of the αC regions could suggest their involvement in long-range interactions between fibrin but not fibrinogen molecules.
Scientific Research Institute of Physical-Chemical Medicine, Moscow, Russia.
A D
AD
Protopopova
N A
NA
Barinov
E G
EG
Zavyalova
A M
AM
Kopylov
V I
VI
Sergienko
D V
DV
Klinov
D065966:Absorption, Physicochemical; D001777:Blood Coagulation; D005337:Fibrin / Q000378:metabolism / Q000648:ultrastructure*; D005340:Fibrinogen / Q000378:metabolism / Q000648:ultrastructure*; D006108:Graphite / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism / Q000648:ultrastructure*; D066329:Protein Aggregates; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013499:Surface Properties
D010446:Peptide Fragments; D066329:Protein Aggregates; C098000:fibrinogen alphaC; D006108:Graphite; D005337:Fibrin; D005340:Fibrinogen
atomic force microscopy; blood coagulation; fibrin; fibrinogen; graphite