Sarah O. Lawrence

Regulated de novo biosynthesis of fibrinogen in extrahepatic epithelial cells in response to inflammation

Hepatic fibrinogen (FBG) is upregulated during an acute phase response (APR) induced by glucocorticoids and interleukin (IL)-6. Furthermore, intestine and lung epithelium synthesize FBG after exposure to inflammatory mediators, and both plasma and lung cell-derived FBG, along with fibronectin, assemble in detergent-insoluble extracellular matrices (ECM) of pneumocytes and fibroblasts independent of thrombin or plasmin cleavage. An epitope cryptic in soluble FBG (beta(15-21)) but exposed in matrix-FBG and fibrin induces cell proliferation and actin cytoskeleton reorganization during wound repair and angiogenesis. Although fibrin(ogen) is involved in hemostasis and homeostasis, mechanisms regulating extrahepatic FBG expression remain unexplored. Herein we examined FBG production by lung compared to liver epithelial cell lines in response to dexamethasone (DEX)+IL-6. Regulated synthesis of HepG2-FBG follows the pathway shown for constitutive synthesis by liver epithelium. Constitutive A549-FBG expression was not detectable, however, intracellular FBG precursors in DEX+IL-6-treated A549 lung cells were similar to HepG2 cells with two notable exceptions. The relative rate of chain synthesis in HepG2 cells was unequal, whereas nascent synthesis of all three chains occurred at equivalent rates in stimulated A549 cells. Unlike HepG2 cells, which rapidly secreted intact FBG, nascent dimeric FBG accumulated in the A549 cell-associated fraction prior to release into medium. Furthermore, soluble A549-FBG was susceptible to thrombin and plasmin cleavage. Interestingly, many functionally diverse proteins possess FBG-related domains that direct cell-fate determination during development or wound repair, suggesting that extrahepatic FBG biosynthesis evoked only during inflammation plays such a role during localized injury and repair to restore tissue homeostasis.

Calcium modulates plasmin cleavage of the fibrinogen D fragment γ chain N-terminus: mapping of monoclonal antibody J88B to a plasmin sensitive domain of the γ chain

Plasmin sensitive sites are found on the Aα, Bβ and γ chains of fibrinogen at regions joining the two C-terminal D fragments with the central E fragment. We have developed a monoclonal antibody (MoAb) reactive with this plasmin sensitive region on the human fibrinogen y chain and mapped its epitope. MoAb J88B reacts with γ chains of both native as well as with reduced and denatured fibrinogen and fibrin, the CNBr fragment of the fibrinogen central domain, plasmin cleaved fragments D, γ-γ dimers, but not with plasmic fragments E. These data indicate that J88B maps to the plasmin sensitive domain localized to γ63–78. MoAb J88B failed to react with synthetic peptide γ70–78, which suggests that the epitope includes the newly exposed N-terminal residues γ63–70 of the early plasmic fragment DIA. As calcium has a marked influence on plasmin cleavage of C-terminal sites on the γ chain, the effects of calcium on modulating plasmin cleavage of DIA to D1 were assessed in the absence or presence of J88B. The results indicated that calcium delays and J88B (±calcium) protects the γ chain from plasmin cleavage at the N-terminus of DIA, suggesting that this enzymatically labile site is calcium-sensitive. Thus, MoAb J88B should prove useful in studies examining the structure of plasmin cleaved fibrinogen and fibrin.