Regulation of the procoagulant activity within the bronchoalveolar compartment of normal human lung

The nature of the procoagulant activity of normal bronchoalveolar fluid was examined both qualitatively and quantitatively. Unconcentrated, cell-free lavage freshly obtained from normal volunteers clotted normal plasma in a mean of 84 +/- 20 s. The procoagulant activity was initiated by Factor VII-tissue factor complexes as judged by differential activity in various plasmas genetically deficient in single clotting factors, by neutralization of the procoagulant activity with antibodies to either Factor VII or tissue factor, and by a Factor X activation assay. Preincubation of the lavage with calcium was required to demonstrate Factor VII activity in unconcentrated samples. The cell-free fluid contained about 8,500 thromboplastin units/mg protein, equivalent to a third of the thromboplastin standard and indicating high amounts of cofactor. Quantitation of Factor VII was estimated by functional analysis in coagulation and amidolytic assays with reference to dilutions of normal plasma of known Factor VII concentration. When lavage and diluted plasma were adjusted to yield equivalent amidolytic activities, the average ratio of the Factor VII-clotting activity of the alveolar fluid relative to plasma Factor VII was 19 +/- 7, suggesting the presence of Factor VIIa in lavage. In contrast to previous reports with serum or activated plasma, immunoblots of concentrated lavage revealed only single-chain Factor VII, and 125I-Factor VII added to the fluid was not converted to 125I-Factor VIIa, suggesting a unique control mechanism in the lung compartment which differs from plasma. When equivalent Factor VII amidolytic activities in diluted plasma and cell-free lavage were compared, the rates of Factor Xa formation were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

The number of receptors for factor VII correlates with the ability of cultured cells to initiate coagulation

Previously, we showed that cells derived from nonvascular tissues initiate clotting primarily by markedly increasing the activity of coagulation factor VII. Cells derived from vascular tissue do not normally exhibit this property (tissue factor activity). In this study, we have characterized the relationship between the tissue factor activity of cultured cells derived from normal tissues and the number of receptors they possess for coagulation factor VII. Only cultured nonvascular cells expressed tissue factor activity or possessed receptors for 125I-factor VII. Fetal lung cells, the nonvascular tissue with the largest amount of procoagulant and tissue factor activity, possessed the most receptors for 125I-factor VII (880,000/cell). Bovine corneal endothelial cells, the nonvascular tissue possessing the fewest number of receptors (2,400/cell), had the least amount of procoagulant or tissue factor activity. The affinity of nonvascular cells for 125I- factor VII varied for the cells studied (Kd congruent to 1.3-90 X 10(- 10) M). Vascular cells expressed no tissue factor activity, nor did they bind 125I-factor VII. 125I-factor VII and unlabeled factor VII bound to cells had identical procoagulant activities. These results indicate that the ability of cultured cells to initiate coagulation may be regulated in part by the number of receptors they possess for factor VII.     

Procoagulant (thromboplastin) activity in human bronchoalveolar lavage fluids is derived from alveolar macrophages

Fibrin deposition in the alveolar space and the lung interstitium is a prominent feature of many types of inflammatory pulmonary diseases. Cells of the monocyte/macrophage line are the primary cells supplying procoagulant activity in inflammatory lesions. In the present study we found that both lung alveolar macrophages (LAM) and bronchoalveolar lavage fluids (BALF) from humans contained procoagulant activities. The procoagulant in BALF was associated with membrane vesicles which sedimented at 100,000 g for 1 h. By electron microscopy the BALF ultrasediment was seen to consist almost exclusively of membrane material and this was confirmed by monitoring the content of different marker enzymes for specific subcellular structures. Using macrophage membrane markers, at least part of the BALF-ultrasediment was shown to be derived from LAM. On the basis of phospholipase C sensitivity, antibody neutralization and the site of action of the procoagulant in the sequential activation of coagulation factors, both the LAM-associated and the BALF-associated procoagulant activity was identified as thromboplastin (tissue factor) or thromboplastin-factor VII complexes. This suggests that alveolar macrophages and the LAM-derived thromboplastin-containing microvesicles may contribute to intraalveolar and interstitial fibrin deposition in vivo and probably also have consequences for the development of pulmonary fibrosis.     

Procoagulant activity of rabbit alveolar macrophages

Alveolar macrophages are thought to participate in the clearance of fibrin from the injured lung, but their ability to facilitate the conversion of fibrinogen to fibrin (procoagulant activity) has not been described. In order to characterize their procoagulant properties, unstimulated alveolar macrophages obtained from normal rabbits were tested for their ability to accelerate the coagulation of plasma in a one-stage clotting assay. Compared with control assays containing no macrophages (coagulation times greater than 500 s), intact cells (10(6)/ml) were shown to display procoagulant activity (coagulation time, 153.6 +/- 11.3 s mean +/- SEM). Cell lysis caused further procoagulant activity to be expressed (125.6 +/- 11.8 s). Alveolar macrophages that were stimulated in vitro with bacterial lipopolysaccharide (LPS) or the purified complement fragments C5a and C5a des Arg caused further significant (p less than 0.002) reductions in coagulation times (intact cells, 71 to 76 s; lysed cells, 27 to 32 s), representing 5- to 6-fold and 30- to 40-fold increases in the procoagulant activity of intact and lysed cells, respectively. The generation of this material was independent of the presence of lymphocytes. The procoagulant material was identified as a cell-associated tissue thromboplastin, acting via the extrinsic coagulation pathway. These findings show that alveolar macrophages have procoagulant activity that is markedly augmented by LPS and complement fragments. This suggests that alveolar macrophages may contribute to intra-alveolar fibrin deposition in vivo.     

Tissue factor in bronchoalveolar lavage fluids. Evidence for an alveolar macrophage source

Local and systemic coagulation and fibrin deposition occur in many types of alveolar injury and inflammation, but clotting factors capable of initiating the coagulation cascade in the alveolus have not been thoroughly identified and characterized. In the present studies, BAL (bronchoalveolar lavage) fluids obtained from rabbits were found to have procoagulant activity detectable in dilutions containing as little as 1.3 ng of protein. The specific activity of the procoagulant in these fluids was within 1 order of magnitude of that found in brain thromboplastin. The BAL procoagulant was shown to be associated with particles having a molecular weight greater than 15 X 10(6) daltons by gel filtration chromatography, and was characterized as tissue factor by showing specific requirements for factors VII, X, and II. Further experiments were performed using membranes purified from alveolar macrophages by sucrose density gradients and characterized by studies of alkaline phosphodiesterase I, a cytoplasmic membrane marker, and electron microscopy. These studies demonstrate that alveolar macrophages, especially low-density subpopulations, generate and release membrane material that is a source of tissue factor in BAL fluids.     

Quantitation of factor VII in the plasma of normal and warfarin-treated individuals by radioimmunoassay

Highly purified single-chain factor VII was isolated from plasma and used to generate monospecific antibodies. A double-antibody equilibrium radioimmunoassay was constructed. The assay was tested for and met all the criteria required for a specific, sensitive, and accurate determination of factor VII in plasma. The range of sensitivity of the assay was between 1 and 500 ng factor VII/ml, and the coefficient of variation was 1%-3% within assay and 12%-16% between assays. Pure factor VII and plasmic factor VII from normal, warfarin-treated, and hereditary deficient individuals inhibited competition assays with parallel slopes, indicating the expression of similar epitopes by these molecules and validating the measurement of this protein in plasma. The concentration of factor VII in normal plasma (n = 41) was 470 +/- 112 ng/ml, and the measurement of factor VII antigen correlated with activity (r = 0.82). Factor VII concentration in the plasma of individuals on warfarin therapy (n = 24) was 238 +/- 73 ng/ml. Factor VII activity was about 38% of normal and correlated less well with factor VII antigen (r = 0.53). The specific activity of these molecules was 78% of normal (p less than 0.01), suggesting the presence of nonfunctional or partially functional molecules in the circulation of individuals undergoing drug therapy. Analysis of two hereditary deficient patients revealed that, while there were significant levels of factor VII protein, the procoagulant activity was less than 2%, indicating a discordant relationship of these parameters in individuals expressing the deficient factor VII phenotype.     

Tissue factor and factor VII messenger RNAs in human alveolar macrophages: effects of breathing ozone

This study was performed to determine if genes for tissue factor and factor VII proteins are expressed and regulated in vivo in lung macrophages during inflammation. Human alveolar macrophages and alveolar fluids were obtained 18 hours after healthy male adults were exposed, for 2 hours during intermittent exercise, to either air or air with 0.4 ppm ozone, added as a model toxic respiratory agent. Messenger RNA (mRNA) for both tissue factor and factor VII were demonstrated in macrophages isolated after subjects were exposed to unpolluted control air. With the same subjects examined after breathing ozone, the following changes were observed: tissue factor mRNA concentration in macrophages increased 2.6 +/- 0.47-fold. Factor VII mRNA concentration was reduced 0.64 +/- 0.24-fold. Total numbers of macrophages recovered did not change significantly. Ratios of nuclear:cytoplasmic areas of cytocentrifuged macrophages were augmented by 24.8% +/- 3%, giving morphometric evidence that immature cell forms increased in the population. In the lavage, tissue factor activity was increased 2.1 +/- 0.3-fold, while amounts of lipid phosphorous, which estimate total membrane lipids, and estimated volumes of alveolar fluid were not significantly changed. Factor VII activity and fibrinopeptide A levels in lavage were increased approximately twofold. These results using rapidly isolated, noncultured cells indicate that tissue factor and factor VII mRNA are synthesized in the alveolar macrophage population in vivo. In addition, evidence was found that as a result of breathing ozone, a shift in alveolar macrophage maturity occurred in association with tissue factor mRNA, tissue factor activity, and factor VII activity increases, and with formation of fibrinopeptide A in alveolar fluids.     

The distribution of procoagulant and plasminogen activator activities among density fractions of normal rabbit alveolar macrophages

Rabbit alveolar macrophages can directly stimulate either coagulation or fibrinolysis by producing tissue thromboplastin and plasminogen activator activities, respectively. However, it is not known whether these 2 opposed physiologic activities are expressed by the same or different cells within a population of alveolar macrophages. This study was undertaken to determine the distribution of procoagulant and plasminogen activator activities among density-defined populations of rabbit alveolar macrophages. Normal rabbit alveolar macrophages were separated into 4 density fractions on continuous gradients of Percoll, and the distribution of procoagulant and plasminogen activator activities among these fractions was determined. The procoagulant activity of the least dense cells (Fraction 1) was as much as 6 times greater than the activity displayed by denser cells (Fractions 2 to 4). By contrast, both cell-associated and secreted plasminogen activator activities were equally and uniformly distributed among all density fractions. These distributions of activities among density fractions persisted after the cells were incubated in culture medium for 24 h. After culture in vitro with lymphokine, procoagulant activity increased in the denser cells so that the activity became equal among all density fractions. We conclude that procoagulant activity distributes differently from plasminogen activator activity among fractions of normal rabbit alveolar macrophages separated according to cell density. By using density gradient fractionation, alveolar macrophages with predominantly procoagulant or plasminogen activator activities can be enriched from the lavage cell population; the dissimilar distribution of these 2 activities within the alveolar macrophage cell population does not reflect the presence of cells with fixed differences in their functional capacities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hereditary factor VII deficiency: heterogeneity defined by combined functional and immunochemical analysis

Twenty-six patients with hereditary factor VII deficiency (VII:C less than 10%) were evaluated using a panel of three thromboplastins of varying species and tissue origin in both coagulant and chromogenic assay systems. Normal values for the coagulation and chromogenic assays were 104% +/- 7% and 108% +/- 21%, respectively. Factor VII antigen was measured by a specific radioimmunoassay (normal, 470 +/- 112 ng/mL). The patients were divided into two groups based on the factor VII:Ag assay results. Group 1, 18 patients, had decreased levels of factor VII:Ag and group 2, eight patients, had normal levels of factor VII:Ag. The two groups were further subdivided on the basis of discrepant factor VII:C levels in the chromogenic and coagulant assays. The number of observed patterns of functional factor VII:C activity suggests a high degree of complexity of factor VII and thromboplastin interaction. Whereas no clinical bleeding was reported in any of the nine black patients evaluated, all Caucasians (16) and one Hispanic presented with mild to severe bleeding. Patients with factor VII:C greater than 10% using a human thromboplastin had a negative bleeding history, regardless of the activity measured with other thromboplastins. Factor VII activity measured with a human thromboplastin appears to correlate best with the clinical picture.     

Elevation of factor VII activity and mass in coronary artery disease of varying severity.

The present study was undertaken to determine whether the extent of Factor VII elevation correlated with the severity of coronary artery disease and whether zymogen or activated Factor VII was responsible for this elevation. A group of 69 patients with coronary artery disease with old myocardial infarction was compared with 28 control subjects. The patient groups showed elevated levels of Factor VII procoagulant activity (FVII:C) and more markedly elevated Factor VII antigen (FVII:Ag) levels than the control group; therefore they had a decreased FVII:C to FVII:Ag ratio. The increased Factor VII level in the patient groups was caused by elevated Factor VII zymogen levels, and not by activated Factor VII. Since FVII:C levels strongly correlated with the titer of thrombin-antithrombin III complexes in all patients, the hypercoagulable state accompanying severe coronary atherosclerosis seems to underlie the increase of FVII and TAT in the stable phase of myocardial infarction.     

Elevation of factor VII activity and mass in young adults at risk of ischemic heart disease

The Northwick Park Heart Study found that elevation of factor VII in middle-aged subjects was an independent risk factor for subsequent ischemic heart disease. The present study was designed to determine whether factor VII elevation is present at a younger age and whether zymogen or activated factor VII is responsible for this elevation. A group of 20 asymptomatic first degree relatives (mean age 34.9 years) of patients with premature ischemic heart disease were compared with 15 age-matched normal subjects at low risk of ischemic heart disease and 15 older subjects with established ischemic heart disease (mean age 49.7 years). Factor VII procoagulant, coupled amidolytic and antigenic assays, as well as fasting serum triglyceride and cholesterol assays, were performed on all three groups. Factor VII antigen and coagulant activity was significantly elevated in first degree relatives, as was factor VII antigen in the patients with ischemic heart disease. The increased factor VII level in these subjects was caused by elevated factor VII zymogen, not activated factor VII. The results of this study, combined with those of previous studies, suggest that factor VII may be a useful additional marker of the risk for ischemic heart disease and merits further investigation.     

Isolation and antigenic identification of hamster lung interstitial macrophages

Lung interstitial macrophages (IMs) are a large, distinctive population of cells with important proliferative capacities. Characterization of their role in health and disease has been hampered by inadequate methods to separate interstitial from residual alveolar macrophages (AMs) in preparations of individual mononuclear cells from lung tissue. In this study, a specific cell-surface antigen (HAM1) present on more than 90% of hamster AMs, but not expressed by hamster IMs, was used to distinguish these populations. After collagenase digestion of lung tissue slices from exhaustively lavaged and perfused hamster lungs, mononuclear phagocytes were isolated by density gradient centrifugation. The mean yield of lung digest macrophages (3.9 +/- 1.9 (SD) x 10(6] was comparable to the yield of lavaged AMs (4.2 +/- 1.9 x 10(6]. The proliferative capacity of lavaged AMs, blood monocytes, and lung digest macrophages was compared using a soft-agar colony-forming unit (CFU) assay. Both lung digest macrophages and blood monocytes had significantly more CFUs (68.7 +/- 2.6 and 53.5 +/- 8.4 CFU/10(3) cells [mean +/- SEM], respectively) than did AMs (16.5 +/- 1.7) (p less than 0.01). To further define the composition of the lung digest macrophage population, flow cytometric analysis of fixed cells from six experiments was performed using a mouse monoclonal antibody specific for the HAM1 antigen found only on AMs. The lung digest macrophage population consisted of both antigen-negative IMs (78.2% +/- 3.7% [SEM]; n = 6) and antigen-positive, residual AMs (21.8% +/- 3.7%). Morphometric counts confirmed that substantial numbers of AMs are left behind after lavage and contribute to macrophages obtained from lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maintenance of the normal rat alveolar macrophage cell population. The roles of monocyte influx and alveolar macrophage proliferation in situ

We investigated the relative importance of monocyte influx and alveolar macrophage proliferation in maintenance of the alveolar macrophage population in normal rats. Two experimental approaches were used. To detect possible monocyte influx, we radiolabeled blood monocytes by a continuous infusion of tritiated thymidine for 7 days and assayed lavaged alveolar macrophages for radiolabel by autoradiography. Although heavily labeled monocytes occurred in the blood, no heavily labeled cells were detectable among lavaged alveolar macrophages by cohort analysis. To evaluate macrophage proliferation in vivo, we injected normal unlabeled rats with colchicine and examined lavaged alveolar macrophages for arrested mitoses. Mitotic figures were observed among lavaged alveolar macrophages, and calculations at serial times after colchicine injection indicated that 1.83% of alveolar macrophages entered mitosis each day. On the basis of these 2 lines of evidence, we conclude that the alveolar macrophage cell population in normal rats is supported mainly by cellular proliferation in situ rather than direct monocyte influx from the blood compartment.     

Presence of a bradykinin-like substance in pulmonary washings.

Pulmonary washings were studied for the presence of bradykinin-like substances and the capacity for their formation and degradation. Bronchopulmonary lavage with saline was performed in anesthetized, exsanguinated Syrian golden hamsters with a cell yield of 99 per cent alveolar macrophages. In the presence of inhibitors of both kinin formation (hexadimethrine) and destruction (1,10-phenanthroline), immunoreactive kinin was found in bronchopulmonary lavage fluid (12.5 ng per 5 x 10(6) cells lavaged), washings of pooled macrophages (5.62 ng per 5 x 10(6) cells washed), and lysates of alveolar macrophages (3.97 ng per 5 x 10(6) cells lysed). Higher kinin concentrations were obtained by selective inhibition of kinin-destroying enzymes with phenanthroline. Inhibition of kinin formation with hexadimethrine alone rendered nearly all concentrations below the limit of detectability. These findings demonstrated that the capacity for kinin formation and degradation exists in pulmonary washings. The precise origin of this kinin and its forming and destroying enzymes (whether from the pulmonary alveolar macrophage, other sites in the bronchopulmonary tree, or plasma) is not yet known.     

Bronchoalveolar macrophages from patients with idiopathic pulmonary fibrosis are unable to kill facultative intracellular bacteria

The accumulation of inflammatory and immune effector cells in the lungs of patients at early stages of interstitial lung disease has been well documented, but little is known about the functional activity of these cells, particularly alveolar macrophages. The purpose of the experiments described here was to determine whether alveolar macrophages from patients with idiopathic pulmonary fibrosis (IPF) differed from alveolar macrophages of normal subjects using a model system that assesses a component of cell-mediated immunity. Alveolar macrophages were tested for their ability to phagocytose and kill the facultative intracellular bacterium Listeria monocytogenes. Because this system requires the stimulation of macrophages by antigen-specific T-cells, it allows the assessment of effector functions of macrophages found in the lower respiratory tract of patients with IPF. Data showed that alveolar macrophages from normal subjects both phagocytosed and killed those bacteria. However, alveolar macrophages from patients with IPF phagocytosed bacteria normally but expressed little bactericidal or bacteriostatic activity. Further, we found no difference in HLA-DR expression by normal and IPF alveolar macrophages. Therefore, it does not appear that this defect in bactericidal activity in IPF macrophages results from a defect in the antigen-presenting function of these cells. These data suggest that alveolar macrophages from patients with IPF express a functional deficiency in their ability to kill facultative intracellular bacteria.     

Superoxide dismutase in alveolar macrophages from patients with diffuse panbronchiolitis

BACKGROUND: Diffuse panbronchiolitis (DPB) is characterized by chronic neutrophil-mediated inflammation of the airway mediated by oxygen radical production. DPB can be controlled with low-dose and long-term erythromycin therapy based on its anti-inflammatory effect. OBJECTIVE: In this study, the antioxidant levels were analyzed as an anti-inflammatory effect of erythromycin in the patients. METHODS: We investigated the activity and protein level of an antioxidant enzyme, Cu, Zn-superoxide dismutase (SOD) in alveolar macrophages (AMs) of patients with DPB before and after erythromycin therapy. AMs were obtained from bronchoalveolar lavage fluid. RESULTS: There was no significant difference in the activity of Cu, Zn-SOD between normal subjects and untreated patients. Erythromycin therapy (600 mg/day) significantly increased the activity of the enzyme relative to that before therapy and normal subjects [18.2 units/10(6) cells (9.2-26.2) vs. 4.4 units/10(6) cells (1.1-9.3), p < 0.01 and 10.4 units/10(6) cells (2.4-20.6), p < 0.05, respectively]. Furthermore, the protein level of Cu, Zn-SOD in AMs in treated patients was significantly higher than in the other two groups [69.4 ng/10(6) cells (34.2-147.1) vs. 20.1 ng/ 10(6) cells (16.9-39.8) for untreated patients, p < 0.01 and 43.2 ng/10(6) cells (32.6-68.2) for normal subjects, p < 0.01], but the levels in the latter groups were not different. CONCLUSION: Our results suggest that one of the anti-inflammatory effects of erythromycin in DPB may be, in part, mediated by enhancement of antioxidant activity in AMs.     

Heterogeneity of immunologic function among subfractions of normal rat alveolar macrophages. II. Activation as a determinant of functional activity

Previous studies have demonstrated differences in immunologic function among density-fractionated alveolar macrophages. The present study was undertaken to correlate these functional differences among alveolar macrophage density fractions with parameters of macrophage activation. Alveolar macrophages were lavaged from normal rats and separated into 5 density fractions by density gradient centrifugation. Cells from each density fraction were analyzed for parameters of macrophage activation: in vitro cytotoxic function directed against cultured neoplastic cells, ectoenzyme activities, and surface expression of Ia-like determinants. Lavaged alveolar macrophages with in vitro cytotoxic function were concentrated among cells from higher density fractions, and cytotoxic function among the density fractions correlated inversely with ectoenzyme activities. Although the percentage of Ia-positive cells varied among the density fractions, surface expression of Ia-like determinants did not correlate positively with in vitro cytotoxic function. The results show that there is a variable distribution of cytotoxic function among alveolar macrophage density fractions that correlates with enzymatic markers of activation. Thus, we conclude that differential macrophage activation is a potential determinant of the functional heterogeneity observed in rat alveolar macrophages.     

Cockade-like structures in alveolar macrophages in extrinsic allergic alveolitis

BACKGROUND AND OBJECTIVES: In immunocytochemical preparations of bronchoalveolar lavage (BAL) cells from patients with extrinsic allergic alveolitis (EAA), we observed the presence of alveolar macrophages with cockade-like structures in their cytoplasm (cockade+ alveolar macrophages). These cockade+ alveolar macrophages may reflect a subpopulation of alveolar macrophages which may show a different predominance in various interstitial lung diseases. In this study we aimed to compare the frequency of cockade+ alveolar macrophages in patients with EAA (n = 14) with the results obtained in patients with sarcoidosis (n = 11), idiopathic interstitial pneumonia (IIP; n = 10) and control subjects (n = 8). We also investigated the expression of the transferrin receptor CD71 on cockade+ alveolar macrophages. METHODS: In BAL fluid, the total number of cells and differential counts were determined, and immunocytologic examinations of macrophages and lymphocytes were done using monoclonal antibodies. The percentage of cockade+ alveolar macrophages was determined by counting 300 macrophages in the CD20 field of an immunocytochemical slide. RESULTS: The percentage of cockade+ alveolar macrophages was significantly higher in the EAA group (36 +/- 9%) compared to patients with sarcoidosis (12 +/- 5%) or IIP (11 +/- 10%) and control subjects (3 +/- 1%; p < 0.001). The proportion of CD71+ alveolar macrophages was significantly lower in EAA than in the other groups (p < 0.01), and the CD71 antigen was expressed on a significantly lower proportion of cockade+ alveolar macrophages compared to cockade- alveolar macrophages in EAA (p < 0.001). CONCLUSION: We conclude that cockade+ alveolar macrophages could play a role in the pathogenesis and differential diagnosis EAA.     

Hemophilia B with associated factor VII deficiency: A distinct variant of hemophilia B with low factor VII activity and normal factor VII antigen

Summary Factor VII activity and cross-reacting material was assayed in fresh and deep frozen non-contacted plasma in 43 patients with Hemophilia B belonging to different kindreds.Factor VII activity was found to be slightly decreased (about of 50% normal) in 12 patients, regardless of the thromboplastin used. In an additional patient (hemophilia B_m) factor VII was slightly decreased in 1 10 diluted plasma but was normal in further diluted plasma. In the remaining 30 patients factor VII activity was normal. No significant variation was found between fresh and deep frozen plasmas. Factor VII antigen or cross-reacting material was normal.These patients with associated factor VII defect represent a distinct variant of hemophilia B. The defect seems to be due to a faulty activation of factor VII but the underlying mechanism remains unclear.This study was supported in part by a grant from the M.P.I., Rome (grant 1592-77) and by a grant from the Venetian Region, Venice.     

A fibrinolytic inhibitor of human alveolar macrophages. Induction with endotoxin

Alveolar macrophages are intimately involved with fibrin during the course of acute and chronic inflammatory processes in the lung. In this study, the capability of macrophages to impede fibrinolysis was investigated. Human alveolar macrophages obtained by lavage from normal volunteers released a fibrinolytic inhibitor during the first 24 h of in vitro culture but only inconsistently and in some cases (7 of 15) not at all. Lysates of freshly lavaged cells had no activity. Endotoxin, 5 to 100 ng/ml, consistently induced intracellular accumulation and extracellular release of a fibrinolytic inhibitor by cultured macrophages. Induction required protein synthesis. The intracellular and secreted forms of the inhibitor were true plasminogen activator (PA) inhibitors, as judged by their ability to block urokinase-mediated conversion of 125I-plasminogen. On average, 10(7) endotoxin-stimulated macrophages secreted sufficient PA inhibitor during a 24-h culture in vitro to neutralize 2 picomoles urokinase (16 international units). Analysis of the interaction of the human macrophage PA inhibitor with 125I-urokinase (apparent size, 33 kilodaltons) by SDS-gel electrophoresis showed that the enzyme and inhibitor mostly dissociated in SDS, but a stable complex occurred at 60 to 65 kilodaltons and a broad band of enzyme or enzyme-inhibitor complexes between 33 and 40 kilodaltons. Either heat treatment of the inhibitor or active site inhibition of urokinase with p-nitrophenylguanidinobenzoate blocked both types of interaction. The pattern of interaction was virtually indistinguishable from that of a partially purified human placental urokinase inhibitor but different from that of serum protease inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)