Fibg^390-396A Limits Metastatic Potential Via a Mechanism Independent of Fibrin-Myeloid Cell Interactions, but Prospectively through the Disruption of FXIII-Mediated Fibrin Crosslinking

Previous studies have shown that fibrin(ogen) supports metastasis. In order to better define the mechanisms coupling fibrin(ogen) to metastasis, we compared metastatic potential in immunocompetent mice carrying specific structure/function alterations in fibrinogen and controls. Fibrinogen^AEK mice, which have a germ-line mutation in the thrombin cleavage site that essentially "locks" fibrinogen in its soluble state, exhibited diminished metastatic potential relative to Fib^WT mice. Notably, previous studies have established that factor XIII is also a significant determinant of metastasis. Taken together, these studies suggest that stable fibrin polymer formation is important in the metastatic process. However, Fib^AEK retained significant metastatic potential relative to mice with complete fibrinogen deficiency, indicating that fibrinogen possesses prometastatic properties in the absence of polymer formation.

Fibrin(ogen) has also been shown to mediate inflammatory cell functions through direct engagement of leukocyte integrins independently of its role in platelet aggregation. Given previous studies demonstrating that myeloid cells are promote metastatic potential, we hypothesized that fibrin(ogen)-mediated leukocyte engagement represents one mechanism by which fibrinogen drives metastasis. Consistent with this view, mice expressing a mutant fibrinogen lacking the y chain binding motif for the leukocyte integrin α_M^β2 (Fibγ^390-396A) had diminished metastatic potential in both spontaneous and experimental metastasis assays. Furthermore, fate analyses revealed that Fibγ^390-396A results in diminished survival of newly-formed metastatic foci. The early reduction in metastatic potential observed in Fibγ^390-396A mice suggests that fibrin(ogen) promotes metastasis by recruiting myeloid cells to the early metastatic niche. In order to explore this hypothesis, we performed experimental metastasis assays in immunocompetent mice in which macrophages or neutrophils were specifically depleted using either clodronate or an anti-Ly6G antibody. In contrast to mice carrying the Fibγ^390-396A mutation, depletion of macrophages or neutrophils had no significant impact on the early survival of metastatic foci. These studies suggest that expression Fibγ^390-396A limits metastatic potential via a mechanism independent of interactions with myeloid cells.

In addition to disrupting fibrin-α_Mβ₂ interactions, elimination of the γ chain 390-396a binding motif has also been shown to limit factor XIII binding to fibrinogen. Given the established importance of FXIII in metastasis, it is conceivable that even subtle alterations in the kinetics of fibrin cross-linking resulting from expression of Fibγ^390-396A limits metastatic potential.