Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central ␤-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1 or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydrylreactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1 bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an argininespecific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin.The adhesive glycoprotein, vitronectin, circulates in human plasma at concentrations of 200 -400 g⅐ml Ϫ1 and serves as a regulatory protein in humoral defense mechanisms by interacting with macromolecules in the reaction cascades of coagulation and fibrinolysis (reviewed in Refs. 1-3). The circulating form of vitronectin is a monomer of 72 kDa, and vitronectin is also found in a multimeric form in platelet releasates and in the extracellular matrix (4 -6). The anti-fibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1), 1 is the major inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (7-11, reviewed in Refs. 12, 13). Like other serpins, PAI-1 has a reactive center loop that mimics the substrate of its target proteases (14, 15). The active conformation of PAI-1 is relatively unstable, so that the protein undergoes rapid conversion to a latent conformation which is characterized by the insertion of the reactive center loop into a central ␤-sheet within the molecule (16). Interactions between strands of the ␤-sheet and the reactive loop stabilize this conformation relative to the active conformation, in which the loop is thought to protrude from the surface of the molecule (7, 16).Binding to vitronectin results in a 2-3-fold increase in the half-life of active PAI-1 (17-19). In addition to stabilizing the active conformation of PAI-1, vitronectin also alters the protease specificity of the serpin so that the vitronectin⅐PAI-1 complex is endowed with the additional ability to inhibit thrombin (20,21). A vitronectin-binding site has been localized on the surface of PAI-1 using site-di...

Summary Acute respiratory distress syndrome (ARDS) is characterized by a rapid‐onset respiratory failure with a mortality rate of approximately 40%. This physiologic inflammatory process is mediated by disruption of the alveolar‐vascular interface, leading to pulmonary oedema and impaired oxygen exchange, which often warrants mechanical ventilation to increase survival in the acute setting. One of the least understood aspects of ARDS is the role of the platelets in this process. Platelets, which protect vascular integrity, play a pivotal role in the progression and resolution of ARDS. The recent substantiation of the age‐old theory that megakaryocytes are found in the lungs has rejuvenated interest in and raised new questions about the importance of platelets for pulmonary function. In addition to primary haemostasis, platelets provide a myriad of inflammatory functions that are poised to aid the innate immune system. This review focuses on the evidence for regulatory roles of platelets in pulmonary inflammation, with an emphasis on two receptors, CLEC‐2 and TLT‐1. Studies of these receptors identify novel pathways through which platelets may regulate vascular integrity and inflammation in the lungs, thereby influencing the development of ARDS.

Abstract Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) affect >200 000 individuals yearly with a 40% mortality rate. Although platelets are implicated in the progression of ALI/ARDS, their exact role remains undefined. Triggering receptor expressed in myeloid cells (TREM)–like transcript 1 (TLT-1) is found on platelets, binds fibrinogen, and mediates clot formation. We hypothesized that platelets use TLT-1 to manage the progression of ALI/ARDS. Here we retrospectively measure plasma levels of soluble TLT-1 (sTLT-1) from the ARDS Network clinical trial and show that patients whose sTLT-1 levels were >1200 pg/mL had nearly twice the mortality risk as those with <1200 pg/mL (P < .001). After correcting for confounding factors such as creatinine levels, Acute Physiology And Chronic Health Evaluation III scores, age, platelet counts, and ventilation volume, sTLT-1 remains significant, suggesting that sTLT-1 is an independent prognostic factor (P < .0001). These data point to a role for TLT-1 during the progression of ALI/ARDS. We use a murine lipopolysaccharide-induced ALI model and demonstrate increased alveolar bleeding, aberrant neutrophil transmigration and accumulation associated with decreased fibrinogen deposition, and increased pulmonary tissue damage in the absence of TLT-1. The loss of TLT-1 resulted in an increased proportion of platelet-neutrophil conjugates (43.73 ± 24.75% vs 8.92 ± 2.4% in wild-type mice), which correlated with increased neutrophil death. Infusion of sTLT-1 restores normal fibrinogen deposition and reduces pulmonary hemorrhage by 40% (P ≤ .001) and tissue damage by 25% (P ≤ .001) in vivo. Our findings suggest that TLT-1 uses fibrinogen to govern the transition between inflammation and hemostasis and facilitate controlled leukocyte transmigration during the progression of ARDS.

Mammals can discriminate among a large number (> 10,000) of unique odorants. The most highly supported explanation for this ability is that olfactory neurons express a large number of seven transmembrane receptors that are not spatially organized at the level of the olfactory epithelium, but whose axonal projections form a distinct pattern within the olfactory bulb. The odor-induced signaling pathway in olfactory neurons includes a Gs-like protein (G(olf)) that activates a specific adenylyl cyclase (type III) isoform, resulting in elevations of cyclic AMP and subsequent activation of a cyclic nucleotide-gated channel. The channel also can be regulated by cyclic GMP. Recently, an olfactory neuron-specific guanylyl cyclase was discovered in rodents, and subsequently a large family of sensory neuronal guanylyl cyclases was identified in nematodes. These guanylyl cyclases are concentrated in the plasma membrane of the dendritic cilia and contain extracellular domains that retain many of the primary sequence characteristics of guanylyl cyclases known to be receptors for various peptides. Thus, the guanylyl cyclases appear to represent a second family of odorant/pheromone receptors.

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A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.

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