Visualization of fibrinogen αC regions and their arrangement during fibrin network formation by high‐resolution AFM

Abstract

To cite this article: Protopopova AD, Barinov NA, Zavyalova EG, Kopylov AM, Sergienko VI, Klinov DV. Visualization of fibrinogen aC regions and their arrangement during fibrin network formation by high-resolution AFM. J Thromb Haemost 2015; 13: 570-9.See also Rocco M, Weisel JW. Exposed: the elusive aC regions in fibrinogen, fibrin protofibrils and fibers. This issue, pp 567-9.Summary. 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 aC regions could not be resolved in fibrinogen crystals. This study was aimed at determining whether the aC 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 aC 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 AE 0.1 nm and length 21 AE 6 nm. The complex, formed between thrombin and fibrinogen, was also visualized. Images of growing fibrin fibers with clearly visible aC regions have been obtained. Conclusions: Fibrin aC regions were visible in protofibrils and large fibers; aC regions intertwined near a branchpoint and looked like a zipper. These results support the idea that aC 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 aC regions could suggest their involvement in long-range interactions between fibrin but not fibrinogen molecules.

Programming the mechanics of cohesive fiber networks by compression

“…We conclude that FXIII induced cross-linking is not responsible for bond formation. Instead, we propose that bond formation is facilitated by noncovalent interactions, most likely mediated by the unstructured αC-regions, two long and flexible chains attached to the distal ends of each fibrin monomer 44 . Specific interactions between these chains mediate lateral association of protofibrils into fibers as well as interactions between fibrin fibers 45 (see schematic in Fig.…”

Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII

“…28,29 The aC region of fibrinogen contributes its own critical roles in the assembly and properties of clot structure. 8,[30][31][32][33] This region has also been directly correlated with adhesion events. [34][35][36] Fibrinogen aC region (221-610) is composed of a flexible aC connector (221-391) and a more structured aC domain (392-610).…”

“…4,7 During fibrin polymerization, crosslinking in the aC region has been shown to promote lateral aggregation and protofibril staggering. 8,30,37 Studies with native fibrinogen vs the mutant (gQ398N, gQ399N, and gK406R) have revealed independent contributions coming from a-a crosslinks. 16,38 Such crosslinks play major roles in promoting clot stiffness, fiber straightening, and hindering fibrinolysis.…”

Characterizing reactive glutamines in fibrinogen and elucidating factor XIII substrate specificity.

“…26,31,58,87,88 This region has also been directly correlated with adhesion events. [89][90][91] Fibrinogen αC region (221-610) is composed of a flexible αC connector (221-391) and a more structured αC domain (392-610).…”

“…Prior to fibrin polymerization, the αC region is tethered to the central E region. 30,31 Fibrinogen is converted to active fibrin upon thrombin cleavage of fibrinopeptides A and B, on the Aα and Bβ chains respectively. Cleavage of fibrinopeptides A and B initiates fibrin polymerization by exposing knobs "a" and "b" in the central E region which interact with the complementary holes "A" and "B" in the D region of another fibrin molecule.…”