Molecular structural characterization of fibrinogen and fibrin provide a basis for prophylaxis and treatments of thrombotic and hemostatic disorders. The flexible C-terminal parts of fibrinogen's Aα chains (αC regions) have been shown to play a role in fibrin formation, cross-linking by factor XIIIa, fibrinolysis, etc. The αC regions comprise about ¼ of the whole molecule by mass, but, because of their extreme flexibility, their structure is missing in the X-ray crystallography of fibrinogen and fibrin fragments. Here, we used high-resolution atomic force microscopy to examine the structure of the αC regions in fibrinogen monomers and in fibrin oligomers formed at the early stages of fibrin polymerization.
The surface of freshly cleaved graphite was rendered hydrophilic with an amphiphilic hydrocarbon-glycine modifier (Klinov et al., Nanotechnology, 2007, 18, 225102). The modified surface provides favorable conditions for protein absorption and allows obtaining high-resolution imaging of single protein molecules in air. To further improve the resolution, super sharp homemade cantilevers were also used.
Plasma-purified full-length human fibrinogen and fibrinogen sub-fraction I-9 lacking most of the αC regions were imaged before and after thrombin treatments. As judged from SDS-PAGE, the I-9 fibrinogen lacked the C-terminal portions of its Aα chains and contained the Aα chain core remnants from 46.5 kDa to 22.6 kDa (or ~66% to 32% of the mass of intact Aα chain). As estimated from these measurements, the sub-fraction lacks virtually all of the αC-domains (Aα392-610) and variable lengths of the αC-connector (Aα221-391). The Bβ and γ chains of the I-9 fibrinogen were intact.
We analyzed the incidence of intact αC regions, their overall shape, and measured the contour length of the αC regions in the full-length and truncated (I-9) fibrinogen variants as well as their derivatives. In addition, we compared fibrin polymerization kinetics and the final clot structures. The incidence of the αC regions was 50% in the full-length fibrinogen compared to 19% in fibrinogen I-9. In the full-length fibrinogen, we could identify compact globular C-terminal αC-domains linked to the bulk molecule via flexible filamentous αC-connectors, confirming the previously proposed structure of the αC regions. The residual αC regions in fibrinogen I-9 were significantly shorter than in the full-length fibrinogen.
To see if the structure of the αC regions changed after conversion to fibrin, we compared the structures of fibrin oligomers formed from the full-length and I-9 fibrinogen variants by thrombin. Up to 80% of the potentially existing αC regions were visualized and quantified in the full-length fibrin; they were highly heterogeneous in their length and configurations. Importantly, conversion of the full-length fibrinogen to fibrin was accompanied by an increase of 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. On the other hand, in fibrinogen I-9 more αC regions were visualized after conversion to fibrin, but the contour length and morphology of shortened αC regions did not change upon fibrin oligomerization.
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 laterally aggregated protofibrils made of the full-length or I-9 fibrinogens, suggesting a pure kinetic effect of the αC regions on the fibrin architecture.
Our results confirmed that after conversion of fibrinogen to fibrin, the αC regions become exposed, undergo partial unfolding and/or conformational elongation. However, the effects of the αC regions on fibrin formation have a kinetic nature due to reduction of the threshold length of protofibrils, leading to accelerated lateral aggregation. The final structure and macroscopic properties of fibrin clots are affected by kinetics of the earliest stages of fibrin self-assembly. Therefore, these early polymerization steps comprise a good potential therapeutic target to modulate the structure and mechanical properties of blood clots.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.