Thrombin Cleavage-Independent Deposition of Fibrinogen in Extracellular Matrices

Abstract

AbstractLung epithelial cells (A549) synthesize and secrete fibrinogen (FBG) in vitro when stimulated with interleukin-6 and dexamethasone. This FBG secretion is polarized in the basolateral direction, suggesting that FBG is a component of the extracellular matrix (ECM). Immunofluorescent staining of A549 cells showed a fibrillar pattern of FBG, similar to the staining detected using antibodies against the matrix constituents, collagen type IV and fibronectin (FN). The same pattern of staining was detected using antibodies against fibrinopeptides A and B, as well as with the T2G1 monoclonal antibody against the fibrin-specific epitope, β15-21. Matrix staining was unaltered in the presence of the thrombin inhibitor, hirudin, or the plasmin inhibitor, aprotinin, consistent with the interpretation that matrix deposition of FBG does not require such enzymatic action. Metabolic labeling studies confirmed that FBG secreted from A549 cells or deposited into the ECM showed no evidence of thrombin or plasmin proteolytic processing or of transglutaminase-mediated covalent cross-linking (γ-γ dimers or α-polymers). Incubation of either A549 cell-derived or purified plasma FBG with cultures of human foreskin fibroblasts resulted in FBG deposition in the ECM that colocalized with matrix fibrils containing endogenously produced FN and laminin (LN). Binding of FBG to this exogenously produced matrix was unaltered by inhibition of thrombin and plasmin action, yet also exhibited exposure of the fibrin-specific epitope, β15-21. The majority (∼70%) of newly synthesized and secreted FBG is bound to the cell surface as determined by its trypsin-sensitivity. Cell surface-bound FBG is initially deoxycholate-soluble, which, over time, becomes incorporated in the deoxycholate-insoluble ECM in a similar fashion to FN. These data suggest that matrix incorporation requires the binding of secreted FBG to cell-associated matrix assembly sites. However, unlike FN, FBG in the ECM is composed of the dimeric protamer (Aα/Bβ/γγ) and not high molecular weight polymers indicative of fibrin. This study provides evidence that deposition of FBG in both endogenous and exogenously produced matrices results in conformational changes that occur independently of thrombin cleavage. This matrix-bound FBG, on which unique cell-reactive domains are likely exposed, could augment cellular response mechanisms evoked during injury and inflammation.