Guadiz G, Sporn L A, Simpson-Haidaris P J
Department of Medicine, University of Rochester School of Medicine and Dentistry, NY, USA.
Blood. 1997 Oct 1;90(7):2644-53.
Lung 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, beta15-21. 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 (gamma-gamma dimers or alpha-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, beta15-21. The majority (approximately 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 (A alpha/B beta/gamma gamma) 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.
肺上皮细胞（A549）在受到白细胞介素-6和地塞米松刺激时，可在体外合成并分泌纤维蛋白原（FBG）。这种FBG分泌在基底外侧方向呈极化状态，表明FBG是细胞外基质（ECM）的一个组成部分。对A549细胞进行免疫荧光染色显示FBG呈纤维状模式，类似于使用针对基质成分IV型胶原和纤连蛋白（FN）的抗体检测到的染色。使用针对纤维蛋白肽A和B的抗体，以及针对纤维蛋白特异性表位β15 - 21的T2G1单克隆抗体，检测到相同的染色模式。在存在凝血酶抑制剂水蛭素或纤溶酶抑制剂抑肽酶的情况下，基质染色未改变，这与FBG的基质沉积不需要这种酶促作用的解释一致。代谢标记研究证实，从A549细胞分泌或沉积到ECM中的FBG没有凝血酶或纤溶酶蛋白水解加工或转谷氨酰胺酶介导的共价交联（γ - γ二聚体或α - 聚合物）的证据。将A549细胞来源的或纯化的血浆FBG与人包皮成纤维细胞培养物一起孵育，导致FBG沉积在ECM中，与含有内源性产生的FN和层粘连蛋白（LN）的基质纤维共定位。通过抑制凝血酶和纤溶酶的作用，FBG与这种外源性产生的基质的结合未改变，但也显示出纤维蛋白特异性表位β15 - 21的暴露。通过其对胰蛋白酶的敏感性测定，新合成和分泌的FBG大部分（约70%）与细胞表面结合。细胞表面结合的FBG最初可溶于脱氧胆酸盐，随着时间的推移，会以与FN类似的方式整合到不溶于脱氧胆酸盐的ECM中。这些数据表明，基质整合需要分泌的FBG与细胞相关的基质组装位点结合。然而，与FN不同，ECM中的FBG由二聚体原体（Aα/Bβ/γγ）组成，而不是指示纤维蛋白的高分子量聚合物。这项研究提供了证据，表明FBG在内源性和外源性产生的基质中的沉积都会导致构象变化，且这种变化独立于凝血酶裂解而发生。这种与基质结合的FBG可能暴露独特的细胞反应结构域，可增强损伤和炎症期间引发的细胞反应机制。
