Bleomycin lung injury in mice leads to an acute alveolitis followed by a fibroproliferative response characterized by the accumulation of extracellular matrix. Because distinct regions of the fibrin(ogen) molecule have unique in vitro biological effects on cells, we quantified, localized, and biochemically characterized the molecular form of extravascular fibrin(ogen) in methoxyflurane anesthetized, bleomycin-injured mice. Bleomycin or saline (controls) was administered intratracheally, and lung tissue was harvested and analyzed at several times thereafter. Immunoreactive fibrin tissue content increased to a maximal 50-fold over controls in a temporal and spatial pattern paralleling that of alveolitis and maximal fibroproliferation. The generation of gamma-gamma-chain dimers and alpha-chain polymers, together with the loss of free alpha- and gamma-chains, indicates that the fibrin is predominantly covalently cross-linked. In fibroproliferative-phase lungs, the fibrin fibrils are branched and colocalize with those of collagen at the electron microscopic level. These observations strongly suggest that fibrin is a significant molecular effector of the in vivo fibroproliferative response after lung injury.

As several forms of lung injury are associated with alveolar fibrin deposition, and fibrin has been pathogenically implicated in the lung fibrotic response, we sought to develop an in vivo gene transfer model of fibrinolytic protease overexpression. To this end, human tissue-type plasminogen activator (t-PA) possesses a high degree of specificity for proteolytic activation of fibrin-bound plasminogen to its active form, plasmin. To construct an effective vector, the cDNA for human t-PA was inserted downstream of a cytomegalovirus early enhancer-promoter into the E1 position of a replication-deficient adenovirus. The adenovirally expressed t-PA was found to be of the expected size and appropriate functional activity both in vitro and in vivo. A single intratracheal instillation of the adenoviral-t-PA construct resulted in a dose- dependent, tissue-specific expression of increased levels of t-PA antigen (100-fold) and t-PA protease activity (4-fold) for at least 2 wk in whole lung lysates. The expressed protein localized to the bronchiolar epithelium and peribronchiolar alveolar cells and did not result in increases in total lung protein or alveolar cell counts at 3 d after instillation. In conclusion, a single intratracheal instillation of adenoviral-cytomegalovirus-t-PA construct will generate dramatic bronchoalveolar compartment overexpression of functional recombinant human t-PA for at least 2 wk. This vector can now be utilized for the determination of the therapeutic potential of t-PA in a number of in vivo model systems.

The crystal structure of the hemagglutinin (HA) of influenza virus A/Aichi/68 (H3N2) from the X-31 reassortant virus was reported in 1981, but as yet there are no X-ray diffraction structures for hemagglutinins of other types or even subtypes of influenza virus. We have used site-directed mutagenesis to probe the structure of the hemagglutinin of influenza B/Hong Kong/8/73. We investigated a region in the globular head domain that is helical in the influenza A HA structure, targeting sidechains that in the H3 HA point toward solvent (Thr196) or into the receptor-binding pocket (Gln197). None of the mutations affected hemagglutination activity, but mutations T196P or Q1971 eliminated binding of a monoclonal antibody. The data suggest that this region of the influenza B HA forms a surface structure different from the alpha-helix of the influenza A HA structure and that it accounts for much of the antigenic activity of influenza B HA.

Persistent fibrin deposition in tissues characterizes the early pathology of many types of injury. In an animal model of bleomycin- induced lung fibrosis, increased expression of type 1 plasminogen activator inhibitor (PAI-1) is associated with accumulation of fibrin in fibroproliferative lesions. Plasmin proteolysis of cross-linked fibrin generates fibrin degradation products (FDPs) with multiple biological activities in several cell types. We reasoned that fibrin fragments may also regulate fibroblast-mediated fibrinolysis. In this study, we describe induction of PAI-1 mRNA, protein, and activity by soluble FDPs and fibrinogen in rat lung fibroblast monolayers. FDPs are more potent than fibrinogen, inducing a concentration-dependent, maximal 3.7 (+/- 0.9)-fold increase in PAI-1 mRNA as measured by northern blotting and a 9.0 (+/- 1.3)-fold induction of PAI-1 antigen levels. Active PAI-1 is demonstrated in fibrinogen- and FDP-stimulated conditioned media. Further characterization of this response shows that PAI-1 expression is induced by the DD/D fragments, but not by immunopurified fragment E. Experiments using Actinomycin D and puromycin indicate that the induction appears to be transcriptionally regulated and is not dependent on new protein synthesis. FDP induction of PAI-1 suggests a matrix-cell feedback process in which a fibrin fragment modulates expression of an important regulator of fibrinolysis.

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