Inhibition of γ′ Fibrinogen Production in Liver Cells by D-Dimers

γ′ fibrinogen contains a variant γ chain that arises from an alternative splicing event within the γ gene mRNA. While the more abundant γA chain contains the platelet-binding AGDV sequence at its carboxyl terminus, the γ′ chain carboxyl terminus has an extended VRPEHPAETEYDSLYPEDDL amino acid sequence that is highly negatively charged and contains two sulfated tyrosine residues. The γ′ chain contains binding sites for factor XIII zymogen and thrombin. This extended γ chain variant confers several unique properties on clots formed with γ′ fibrinogen, including resistance to fibrinolysis. γ′ fibrinogen is also a newly-emerging cardiovascular disease biomarker that shows an inverse association with deep venous thrombosis and thrombotic microangiopathy, and a significant positive association with coronary artery disease, myocardial infarction, and stroke. However, the underlying factors that regulate its production remain unknown. A recent study investigating the role of γ′ fibrinogen in stroke showed that the ratio of γ′ fibrinogen to total fibrinogen was higher in patients during the acute phase of a stroke and lower in the convalescent phase following a stroke. We therefore hypothesized that the end products of fibrinolysis may feed back to regulate γ′ fibrinogen expression. Fibrin(ogen) degradation products, including fibrinopeptides A and B, E-domains, and D-dimers, were therefore assayed for their effects on the production of γ′ fibrinogen in HepG2 human liver cells. The results showed that fibrinopeptide A, fibrinopeptide B, and E-domains had no effect on γ′ fibrinogen production by HepG2 cells, as measured by ELISA. However, the production of γ′ fibrinogen decreased in a dose-dependent manner when incubated with purified cross-linked D-dimers. RT-PCR demonstrated an 85.1 ± 4.6% inhibition in γ′ mRNA levels by 31 nM D-dimers after 24 hours of exposure. For comparison, the lower limit of the pathophysiologic cutoff of D-dimer concentration in disseminated intravascular coagulation is in the range of 41 nM. To further investigate the interactions of the D-dimers with the cells, D-dimers were labeled with fluorescein, incubated with HepG2 cells, and examined by fluorescence microscopy. Fluorescein-labeled D-dimers entered the cells within 20 minutes of incubation and displayed a diffuse cytoplasmic distribution. The D-dimers showed an increasing peri-nuclear localization with increasing time. By 60 minutes, the D-dimers had largely been cleared from within the cells. D-dimer uptake displayed a very different staining pattern than the non-receptor-mediated uptake of fluorescein-labeled albumin, which showed only a weak, diffuse cytoplasmic staining. These results suggest that D-dimers regulate γ′ fibrinogen production in liver cells in a process that involves binding to a cell-surface receptor, internalization, and degradation.