Characterisation of Cellular Fibrinogen Phosphorylation and its Functional Implications in Clot Formation

Fibrinogen is a vital component of coagulation; cleavage of fibrinogen yields fibrin monomers that polymerise to form a network of fibres, constituting the blood clot. Human fibrinogen is secreted from hepatocytes in its phosphorylated form, with 20-25 % of circulating fibrinogen phosphorylated exclusively at Aα chain Ser3 and Ser345. Phosphorylation of fibrinogen is elevated in acute phase conditions, venous thrombosis and ovarian cancer, but little is known about the regulation and effects of this modification. The aims of this PhD project were to characterise the cellular mechanism and functional role of fibrinogen phosphorylation in vivo. Human hepatoma cells were incubated in the presence and absence of IL-6 and the phosphate content of secreted fibrinogen was analysed by western blotting. Interleukin-6 caused a 3.1-fold increase in fibrinogen phosphorylation, demonstrating for the first time that the up-regulation of this modification in acute phase conditions is regulated at the cellular level. Using real-time PCR, IL-6 was found to significantly enhance (6.0-fold) the expression of Golgi casein kinase Fam20A, whose recognition sequence matches the Ser3 and Ser345 phosphorylation sites. Expression of other potential fibrinogen kinases, including CK2, Fam20B and Fam20C, were unchanged. This finding suggests that Fam20A plays an important role in the hepatocellular response to acute phase conditions and may phosphorylate fibrinogen in vivo. Chromatographic enrichment of phosphorylated human plasma fibrinogen was conducted for functional analyses. Binding and activity assays found no effect of fibrinogen phosphorylation on FXIII cross-linking of fibrin α and γ chains, plasmin(ogen) binding to fibrinogen, or α2-antiplasmin incorporation. Analysis by SDS-PAGE revealed a small decrease in the rate of fibrinogen degradation by plasmin with increasing phosphorylation, indicating a possible role in protection from fibrinolysis. Scanning electron microscopy and turbidimetric assays revealed thinner fibres and more extensive branching in clots with a higher phosphate content, which typically represents a pro-thrombotic structure. This work highlights the importance of fibrinogen phosphorylation in maintaining the balance between clot formation and lysis. Investigations have shown that increased intracellular kinase activity leads to elevated fibrinogen phosphorylation in acute phase conditions. The observed alterations to clot phenotype with elevated fibrinogen phosphorylation suggest this modification may help to stem bleeding following trauma. Furthermore, it may have important implications in the development of thrombosis, which would make it a valuable target for therapeutic intervention in associated pathologies.