Impact of Genetic and Post-Translational Modifications on Structure and Function of Fibrinogen

Introduction: Fibrinogen is a highly heterogeneous protein that is a result of several types of alteration, including genetic polymorphisms, alternative mRNA processing, posttranslational modifications, proteolytic cleavage and environmental factors. The different combinations of these alternations cause a formation of abnormal variants of fibrinogen that may affect the molecule. Fibrinogen is more vulnerable to oxidative stress than other plasmatic proteins. The oxidative post-translational modifications can be caused by different reactive oxygen species (ROS) and change the spatial structure, thus, impaired protein function.

Here, we characterized 48 patients, belonging to 30 unrelated families, with fibrinogen mutation. This study aims to understand the behavior of these alternated fibrinogens in blood coagulation.

Methods: We investigated these fibrinogens using a wide range of biochemical procedures, including fibrin polymerization, fibrinolysis, fibrinopeptides (Fps) release measurement, tandem mass spectrometry, thromboelastography, protein modeling, and scanning electron microscopy (SEM).

Results: Dysfibrinogenemia was diagnosed in 35 cases, mostly in exon 2 of FGA gene, around thrombin cleavage site in N-termini of Aα chain. Hypofibrinogenemia was found in 13 remaining cases. Novel mutations were identified in 7 cases - heterozygous missense BβY416C and BβA68S, homozygous nonsense BβY345*, heterozygous nonsense BβW403*, heterozygous missense FGG c.8G>A, heterozygous missense γT34A, and heterozygous missense γG284E mutations. All patients had decreased levels of functional and/or plasmatic fibrinogen. Functional tests showed abnormal fibrin development in all patients with lower maximal absorbance. Moreover, fibrinopeptide cleavage measurements revealed differences between the release of Fps in patients with mutation in position close or in the binding site od thrombin. The results obtained from SEM were significantly different in fiber thicknesses or average number of fibers per 1µm². Homology modeling showed the impaired protein structure of BβA68S and γG284E. Mass spectrometry detected several post-translational oxidations of fibrinogen.

Conclusion: The results indicate that detected post-translational modifications, induced very likely by ROS, may affect the overall clottability of fibrinogen and clot morphology. Consequently, our study demonstrates that the identification, characterization, and modeling of novel fibrinogen variants brings new knowledge to fibrinogen domain structures and interactions that influence the assembly, secretion and function of this molecule.

This work was supported by the project of the Ministry of Health, Czech Republic (00023736).

No relevant conflicts of interest to declare.