Local abnormalities of coagulation and fibrinolytic pathways that promote alveolar fibrin deposition in the lungs of baboons with diffuse alveolar damage.

Because alveolar fibrin is a prominent histologic feature of diffuse lung injury in baboons, we hypothesized that local abnormalities of pathways of fibrin turnover would favor fibrin deposition in the alveolar space. To test this hypothesis, procoagulant and fibrinolytic activities were characterized in serial bronchoalveolar lavage (BAL) of baboons with evolving diffuse alveolar damage (DAD) induced by exposure to 100% O2. BAL procoagulant activity, characterized mainly as the tissue factor-Factor VII complex, was markedly increased after induction of DAD. Extrinsic pathway inhibitor was likewise increased in BAL during evolving DAD but was insufficient to control coagulation. Urokinase-like fibrinolytic activity was usually detectable in baseline BAL but was undetectable after 7 d of O2. DAD BAL contained significantly increased plasminogen levels, plasmin inhibitor activity sufficient to neutralize all plasmin produced by BAL plasminogen activator found in control BAL and detectable plasminogen activator inhibitor-1. Antiplasmin activity was due, in part, to increased alpha 2-antiplasmin. These changes correlated with quantitatively increased alveolar fibrin deposition demonstrated by histologic and morphometric analyses. Multiple abnormalities of pathways of fibrin turnover occur concurrently in the alveolar compartment of the lungs of baboons with DAD, which collectively predispose to diffuse alveolar fibrin deposition.

Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome.

To determine the possible mechanism(s) promoting alveolar fibrin deposition in the adult respiratory distress syndrome (ARDS), we investigated the initiation and regulation of both fibrinolysis and coagulation from patients with ARDS (n = 14), at risk for ARDS (n = 5), and with interstitial lung diseases (ILD) (n = 8), and normal healthy individuals (n = 13). Bronchoalveolar lavage (BAL) extrinsic pathway inhibitor activity was increased in ARDS BAL compared with patients at risk for ARDS (P = 0.0146) or normal controls (P = 0.0013) but tissue factor-factor VII procoagulant activity was significantly increased in ARDS BAL compared with all other groups (P less than 0.001). Fibrinolytic activity was not detectable in BAL of 10 of the 14 patients with ARDS and low levels of activity were found in BAL of the other four ARDS patients. Depressed fibrinolysis in ARDS BAL was not due to local insufficiency of plasminogen; rather, there was inhibition of both plasmin and plasminogen activator. Plasminogen activator inhibitor 1 was variably detected and low levels of plasminogen activator inhibitor 2 were found in two ARDS BAL samples, but plasminogen activator inhibitor 2 was otherwise undetectable. ARDS BAL antiplasmin activity was, in part, due to alpha 2-antiplasmin. We conclude that abnormalities that result in enhanced coagulation and depressed fibrinolysis, thereby predisposing to alveolar fibrin deposition, occur in the alveolar lining fluids from patients with ARDS.

Thrombin-thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis.

Fibrin deposition is a hallmark of pleural inflammation and loculation but understanding of mechanisms by which mesothelial cells regulate intrapleural fibrinolysins remains incomplete. We speculated that pleural mesothelial cells regulate local fibrinolytic capacity via processing of single chain urokinase type plasminogen activator (scuPA). Pretreatment of human pleural mesothelial (MeT-5A) cells with TGF-beta or thrombin, either alone or in combination, inhibited urokinase (uPA)-mediated fibrinolysis by MeT-5A. Thrombin, unlike TGF-beta, inhibited fibrinolysis without induction of PAI-1, suggesting that thrombin-mediated cleavage of scuPA inhibits the fibrinolytic capacity of MeT-5A cells. Thrombin cleaves both purified scuPA as well as that secreted by MeT-5A cells and cell surface thrombomodulin accelerates thrombin-mediated cleavage of scuPA to inhibit cellular fibrinolytic activity. Molecular dynamics analyses demonstrated that thrombin-cleaved scuPA (uPAt) do not acquire a catalytically active conformation and that secondary plasminogen binding sites of uPA implicated in plasminogen activation are distorted in uPAt, explaining, at least in part, why uPAt is a poor enzyme. uPAt was detectable in transudative and exudative pleural effusions from patients. Intrapleural administration of scuPA generated increased levels of uPAt in PF of rabbits with pleural injury and loculation induced by tetracycline in vivo. This pathway is operative in diverse forms of pleural injury, restricts the urokinase-dependent fibrinolytic capacity of pleural mesothelial cells and contributes to local control of fibrinolytic activity via processing of endogenous or exogenous scuPA within the pleural compartment.

Posttranscriptional regulation of urokinase receptor mRNA: identification of a novel urokinase receptor mRNA binding protein in human mesothelioma cells.

Treatment of human pleural mesothelioma (MS-1) cells with phorbol myristate acetate (PMA) and cycloheximide results in 17- and 10-fold, respectively, increases in steady-state expression of urokinase-type plasminogen activator receptor (uPAR) mRNA. Studies of transcriptional inhibition by actinomycin D showed four- and sixfold extensions of uPAR mRNA half-life in MS-1 cells treated with PMA and cycloheximide, respectively, suggesting that uPAR gene expression involves a posttranscriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 50-kDa uPAR mRNA binding protein (uPAR mRNABp) that selectively bound to a 51-nucleotide (nt) fragment of mRNA corresponding to the uPAR coding region. We investigated the possibility that this 51-nt protein binding fragment of uPAR mRNA contains regulatory information for message stability. Chimeric beta-globin/uPAR/beta-globin mRNA containing the 51-nt protein binding fragment was able to destabilize otherwise stable beta-globin mRNA. Conversely, a control chimeric beta-globin/uPAR/beta-globin mRNA containing a 51-nt fragment of the uPAR coding region that does not bind uPAR mRNABp was stable under identical conditions. Binding of uPAR mRNABp to uPAR mRNA was abolished after treatment with cycloheximide and rapidly down-regulated by PMA. These data suggest that the 51-nt protein binding fragment of uPAR mRNA may be involved in mRNA turnover as well as in cycloheximide-induced uPAR message stabilization. Our results indicate a novel mechanism of uPAR gene regulation in which cis elements within a 51-nt coding region interact with a uPAR mRNABp to regulate uPAR message stability.

The fibrinolytic system: A new target for treatment of depression with psychedelics.

Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists, such as psilocybin, can improve mood through anti- inflammatory and pro-fibrinolytic effects that include blockade of TNF-α activity leading to decreased PAI-1 activity and increased clearance. The process involves disinhibition of tPA and uPA with subsequent increased cleavage of proBDNF which promotes neurogenesis, decreased neuroinflammation, decreased fibrin deposition, normalized glial-neuronal cross-talk, and optimally functioning neuro-circuits involved in mood. We propose that psilocybin can alleviate deleterious changes in the brain caused by chronic stress leading to restoration of homeostatic brain fibrinolytic capacity leading to euthymia.

Tissue factor in experimental acute lung injury.

Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces. Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung. These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines. Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway. Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear. Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS). Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues. The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema. Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function. These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis.

Redistribution on the thallium scan in myocardial sarcoidosis: concise communication.

Resting and redistribution thallium studies were performed in four young patients with sarcoidosis to evaluate the possibility of myocardial involvement. In each case the resting scan showed marked defects that resolved on the redistribution studies. In a different patient population, these results would have implied significant coronary artery disease.

Expression and assembly of procoagulant complexes by human pleural mesothelial cells.

Many pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor VIIa to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

Tissue factor pathway inhibitor in tetracycline-induced pleuritis in rabbits.

Pleural fibrin deposition that promotes loculation and fibrosis after pleural injury is initiated by tissue factor (TF). In this study, we sought to determine if tissue factor pathway inhibitor (TFPI), an inhibitor of the TF-factor VIIa complex, was likewise expressed in tetracycline (TCN)-induced pleural injury and, if so, whether TFPI was locally elaborated. Pleural fluid TFPI activity approximated that of plasma by 24 h and doubled by 3 days after intrapleural TCN. By contrast, pleural fluid coagulation factors VII and V remained below plasma concentrations at these intervals. Immunohistochemical studies demonstrated TF, TFPI and fibrin localized in pleural and subpleural tissues and within intrapleural adhesions. TFPI activity and mRNA were also elaborated by rabbit pleural mesothelial cells and lung fibroblasts. TFPI is locally expressed and pleural fluid TFPI exceeds plasma levels during TCN-induced pleural injury. Resident cells as well as extravasation likely contribute to intrapleural TFPI. TFPI expression temporally and anatomically approximates that of TF and may limit TF-induced fibrin deposition in evolving TCN-induced pleuritis.

Fibrinogen promotes adhesion of monocytic to human mesothelioma cells.

Adhesion between monocytic and mesothelioma or pleural mesothelial cells influences stromal remodeling in pleural neoplasia. We found that cultured monocytic cells (U937) adhere to either human pleural mesothelioma (MS-1) or mesothelial (MeT5A) cells in vitro. 125I-fibrinogen bound specifically and saturably to either cell line, and specific fibrinogen binding increased upon stimulation of these cells with proinflammatory agents such as phorbol myristate (PMA), lipopolysaccharide (LPS) or tumor necrosis factor (TNF-alpha). We purified the fibrinogen receptor protein from a membrane fraction of MS-1 cells and identified it by immunoprecipitation as intercellular adhesion molecule (ICAM-1). Anti-ICAM-1 antibody or antisense oligonucleotides inhibited fibrinogen-mediated cell adhesion and binding of 125I-fibrinogen to mesothelioma or mesothelial cells. Cultured monocytic cells adhere to either mesothelioma or mesothelial cells, and the interaction is promoted by fibrinogen binding ICAM-1 at the cell surface. ICAM-1 is expressed by mesothelioma cells and CD 11b by macrophages in the fibrinous mesothelioma tumor stroma. The data suggest a common mechanism by which monocytic cells could adhere to either malignant mesothelioma cells or the mesothelial surface in pleural neoplasia.

Angiotensin converting enzyme in bronchoalveolar lavage in ARDS.

Angiotensin converting enzyme (ACE) is present in the endothelial cells of the normal lung where it converts angiotensin I to angiotensin II and inactivates bradykinin. It has been suggested that during endothelial injury ACE is sloughed into the blood, and that if the alveolar capillary membrane is injured, also into the alveolar lining fluid. Seven patients with adult respiratory distress syndrome (ARDS), were compared to 11 normal control subjects, nine patients with sarcoidosis, and six with idiopathic pulmonary fibrosis. Total, differential cell counts and ACE determinations were performed on bronchoalveolar lavage fluid in the ARDS group. ACE was detectable in the BAL of all but one ARDS patient. It was concluded that BAL ACE is elevated in some ARDS patients, especially those with infectious causes of lung injury. Increased ACE may reflect endothelial damage or local increase in ACE production in response to sepsis.

Platelet-specific alpha-granule proteins and thrombospondin in bronchoalveolar lavage in the adult respiratory distress syndrome.

Levels of platelet-specific alpha-granule proteins, PF, BTG, and TSP were measured in BAL fluids of patients with the ARDS, ILD, and normal healthy subjects, comprising two separate cohorts. In both groups BAL showed elevated levels of BTG and thrombospondin in ARDS patients. Low levels of PF4 were found in BAL and did not differ between ARDS and control patients. The BTG:PF4 ratio was 2:1 or greater in BAL of ARDS patients and of control subjects with other lung diseases, suggesting in vivo release. In ARDS patients, the ratio of TSP to BTG exceeded that usually found in plasma. In ARDS patients in group 2, BAL levels of TSP, BTG, and total protein correlated strongly with the composite injury scores that were used to quantitate their degree of lung injury. Elevated levels of platelet-derived proteins, which modulate chemotaxis of inflammatory cells and promote connective tissue reorganization, occur in the alveolar compartment of ARDS and ILD patients but are usually undetectable in BAL of healthy control subjects. Levels in these patients in BAL fluid are nonspecific indices of the severity of lung injury in patients with ARDS.

Growth factors for human pleural mesothelial cells in soluble products from formed clots.

Fibrin deposition within the pleural space may influence repair following pleural injury. Although the mesothelial surface can organize fibrin, the contribution of pleural mesothelial cells to pleural repair is unknown. During coagulation thrombin cleaves Fibrinopeptide A (FPA, A alpha 1-16) and fibrinopeptide B (FPB) from the A alpha and B beta chains of fibrinogen to generate fibrin monomer. Since these peptides are mitogenic for human fibroblasts, we considered that they might stimulate replication of human pleural mesothelial cells (HPMC). Application of fluid expressed from fibrin clots significantly increased cell number and stimulated uptake of 3H-thymidine by HPMC compared with untreated cells. The mitogenic response of subconfluent HPMC to dilutions of clot fluid (30-150 micrograms/ml protein) was comparable to that of 0.1 nM TGF-beta. Fibrinopeptide A (7.5-30 microM) stimulated 3H-thymidine uptake in HPMC, but FPB had only a slight effect at 30 microM. Antibody to FPA antibody significantly attenuated the mitogenic effect of clot fluid, indicating that a major component is FPA. Our study suggests that fibrinopeptides released during fibrin formation in vivo may stimulate local mesothelial regeneration following pleural injury.

Approach to adult respiratory distress syndrome and respiratory failure in elderly patients.

Because of physiologic changes associated with aging and the development of chronic illness, the elderly will continue to be at risk for catastrophic illnesses including adult respiratory distress syndrome (ARDS). Current management of elderly patients with ARDS is only supportive and focused on avoiding complications. Truly successful management of these patients requires therapies directed at pathophysiologic initiators and mediators of ARDS.

Management of undiagnosed persistent pleural effusions.

Difficult to diagnose pleural effusions are not uncommonly encountered in clinical practice. We define these effusions as those that remain undiagnosed after initial thoracentesis and repeat thoracentesis with pleural biopsy. Tuberculosis and malignancy are often found to be the underlying causes when a diagnosis is ultimately made, but other causes, including pulmonary embolism and intra-abdominal conditions, need to be considered as potential causes of the difficult to diagnose effusions. In selected cases, presumptive treatment of tuberculosis is indicated whereas in others, a decision must be made either to obtain a definitive diagnosis by invasive surgical procedures or to follow the patient with careful observation and watchful waiting.

Bronchoalveolar lavage in patients with the adult respiratory distress syndrome.

BAL in patients with ARDS provides material containing the soluble and cellular constituents of the alveolar compartment, and hence is a useful tool for the study of the pathogenesis of ARDS. The technique is imperfect as it is prone to problems of data acquisition and interpretation. However, it is lung-specific and may be used in serial studies of patients over the course of their disease. A large amount of evidence is rapidly being accumulated which documents the presence of effectors of inflammation in the BAL fluids of patients with ARDS. Confirmation of the importance of such mediators, pathways, or cellular constituents of BAL fluid in establishing the pathogenesis of ARDS ultimately depends upon proof of the efficacy of specific clinical interventions which both arrest the activity of the effector and predictably alter the course of the disease.

Primary systemic amyloidosis with a retroperitoneal mass.

A 73-year-old black woman presented with congestive heart failure, abdominal distension and ascites. A large retroperitoneal mass was demonstrated by gray-scale abdominal ultrasonography and confirmed by laparatomy and postmortem examination. This retroperitoneal mass consisted mainly of amyloid, as demonstrated by green birifringence with alkaline Congo red staining. Resistance of this staining pattern to permanganate treatment and the absence of inflammatory disease or malignancy at autopsy suggest the diagnosis of primary systemic amyloidosis. This is the first reported case of this disease manifesting as a retroperitoneal mass.

Exercise training in patients with chronic obstructive pulmonary disease.

Most patients with chronic obstructive pulmonary disease (COPD) demonstrate positive responses to exercise conditioning. Dyspnea is reduced and work tolerance is extended with little or no change in pulmonary function noted. Possible explanations for the increased ability to better tolerate exercise and activities of daily living (ADL) after training include: 1) psychological encouragement, 2) improvements in mechanical efficiency, 3) improved cardiovascular conditioning, 4) improved muscle function, 5) biochemical adaptations responsible for reducing glucose utilization, 6) desensitization to dyspnea, and 7) contributions from better self-care. However, not all patients respond positively to exercise conditioning. This may represent differences in patient selection, training approaches, and/or comorbidity issues commonly seen in patients with COPD. Alternatively, the answer may reside in devising an optimal training intensity, duration, and frequency combination for patients with COPD. This is not an easy matter because of the diversity of patients categorized as COPD. We have reviewed these issues from the available data and presented areas where additional research is warranted. What is needed at present is a series of well-controlled studies that focus on identifying and improving training responses in patients with COPD. Secondary to this issue is the long term epidemiologic surveillance of trained patients to document sustained effects.

Plasminogen activator inhibitor-1 inhibits factor VIIa bound to tissue factor.

BACKGROUND AND OBJECTIVE: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI-1). The present study was designed to investigate whether PAI-1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well-known procoagulant risk factor. METHODS AND RESULTS: The ability of PAI-1 to inhibit FVIIa-TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI-1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa-TF (PAI-1(IC50) , 817 and 125 nm, respectively); (ii) FVIIa-TF-mediated FX activation (PAI-1(IC50) , 260 and 50 nm, respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI-1(IC50) , 260 and 120 nm, respectively). The association rate constant (k(a)) for PAI-1 inhibition of FVIIa-TF was determined using a chromogenic assay. K(a) for PAI-1 inhibition of FVIIa bound to relipidated TF is 3.3-fold higher than that for FVIIa bound to soluble TF (k(a) = 0.09 ± 0.01 and 0.027 ± 0.03 µm(-1) min(-1), respectively). Vitronectin increases k(a) for both soluble and relipidated TF by 3.5- and 30-fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 µm(-1) min(-1)). However, only a 3.5- to 5.0-fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI-1/vitronectin/FVIIa/relipidated TF non-covalent complex. CONCLUSIONS: Our results demonstrate potential anticoagulant activity of PAI-1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI-1 and TF are overexpressed.