Lack of a 5.9 kDa Peptide C-Terminal Fragment of Fibrinogen α Chain Precedes Fibrosis Progression in Patients with Liver Disease

<div><p>Early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. This study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, HCV-positive patients who underwent liver transplantation (LT). We analyzed 93 LT patients with HCV recurrence, 41 non-LT patients with liver disease showing a fibrosis stage F≥1 and 9 patients without HCV recurrence who received antiviral treatment before LT, as control group. Blood obtained from 16 healthy subjects was also analyzed. Serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by SELDI-TOF-MS. Characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. Marked differences were observed between the serum proteome profile of LT patients with early fibrosis recurrence and non-recurrent LT patients. A robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent LT patients. However, the same peak was barely detected in recurrent LT patients. Similar results were found when comparing samples of healthy subjects with those of non-LT fibrotic patients, indicating that our findings were not related to either LT or HCV infection. Using tandem mass-spectrometry, we identified the protein peak as a C-terminal fragment of the fibrinogen α chain. Cell culture experiments demonstrated that TGF-β reduces α-fibrinogen mRNA expression and 5905 m/z peak intensity in HepG2 cells, suggesting that TGF-β activity regulates the circulating levels of this protein fragment. In conclusion, we identified a 5.9 kDa C-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease.</p></div

Isolation, separation and identification of the 5.9 kDa protein peak.

<p>A/ Spectra obtained by SELDI-TOF-MS showing the isolated protein peak after the purification process. B/ The isolated band indicated with arrows, after running two tris-tricine gels, one stained with sypro (I) and the other with oriole (II) staining. C/ Fragment of the Fibrinogen α-Chain identified as the differential protein peak by the amino acid sequencing. The two sequences identified are shown in bold.</p

Baseline characteristics of healthy subjects and HCV-infected and non-infected fibrotic patients.

<p>*p<0.05, ***p<0.001 in comparison to healthy subjects and ^bp<0.01 in comparison to HCV infected patients (Kruskal-Wallis test with the Dunn pos hoc test). Results are given as mean±SEM.</p><p>Baseline characteristics of healthy subjects and HCV-infected and non-infected fibrotic patients.</p

TGF-β reduces the fibrinogen α-chain expression in HepG2 cells.

<p>Fold regulation in Fibrinogen Alpha Chain (FGA), Beta Chain (FGB) and Gamma Chain (FGG) genes regulation in HepG2 cells after 6 hours of treatment with TGF-β (10 ng/ml). Results are given as mean ± SE; ***p<0.001 vs. basal. Statistical analysis was calculated by unpaired Student’s t test. The insert shows the intensity values of the 5.9 kDa peak detected in the cellular supernatant of HepG2 cells after 48 hours of treatment with TGF-β (10 ng/ml). Results are given as mean ± SE; *p<0.05 vs. basal. Statistical analysis was calculated by Mann-Whitney U test.</p

Comparison of the 5.9 kDa protein peak intensity between all the groups analyzed.

<p>SELDI-TOF-MS intensity values of the 5.9 kDa peak of the different groups of patients studied. Intensity of the peak was markedly suppressed in all patients under an active fibrogenic process.</p

Intensity values (µA) of peaks showing different patterns of expression in the two groups of patients.

a<p>p<0.05, ^bp<0.01, ^cp<0.001, ^dp<0.0001; in comparison to non recurrent HCV subjects (Mann-Whitney U test). Results are given as mean±SEM.</p><p>Intensity values (µA) of peaks showing different patterns of expression in the two groups of patients.</p

Differential proteomic profile between HCV− and HCV+ patients submitted to LT.

<p>Fragment ranging from 3000 to 11000 m/z of the differential SELDI-TOF-MS spectra of all serum samples obtained from patients 6 months after LT. The upper figure shows all overlapped spectra from HCV-negative patients (n = 9). The bottom figure shows the spectra from HCV-infected patients (n = 10). Arrows indicate peaks showing significantly different intensities between HCV− and HCV+ patients. Upper letters correspond to the identification peak noted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109254#pone-0109254-t002" target="_blank">table 2</a>.</p