Daryl S. Fair

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.

Monoclonal antibody analysis of purified and cell-associated tissue factor

Tissue factor (TF), one of the cell-surface initiators of blood coagulation, has been implicated as the major molecule of this type and as a critical controlling molecule in hemostasis, thrombosis and inflammation. Analysis of the expression of human TF by cells has been hampered by the lack of suitable molecular probes. We have prepared a library of twenty-four murine hybridomas which stably secrete monoclonal antibodies to human TF. Based on their characteristics, these monoclonals can be categorized into a minimum of five distinct groups. Twenty-three of the hybridoma antibodies strongly inhibited TF activity, which was attributable to blocking of formation of the bimolecular complex of TF and factor VII. We have used these antibodies to demonstrate directly that TF is the sole high affinity factor VII receptor on an intact cell. We have also demonstrated the immunologic relationship between constitutive and induced expression of the protein responsible for TF-like activity by several cells and tissues. Most of the antibodies were found to inhibit TF activity expressed by other primate species, and the potential in vivo therapeutic use of monoclonal antibodies of differing intramolecular specificity is discussed.

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.

Human alveolar macrophages synthesize factor VII in vitro. Possible role in interstitial lung disease.

Both fibrin and tissue macrophages are prominent in the histopathology of chronic inflammatory pulmonary disease. We therefore examined the procoagulant activity of freshly lavaged human alveolar macrophages in vitro. Intact macrophages (5 X 10(5) cells) from 13 healthy volunteers promoted clotting of whole plasma in a mean of 65 s. Macrophage procoagulant activity was at least partially independent of exogenous Factor VII as judged by a mean clotting time of 99 s in Factor VII-deficient plasma and by neutralization of procoagulant activity by an antibody to Factor VII. Immunoprecipitation of extracts of macrophages metabolically labeled with [35S]methionine by Factor VII antibody and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a labeled protein consistent in size with the known molecular weight of blood Factor VII, 48,000. The addition of 50 micrograms of unlabeled, purified Factor VII blocked recovery of the 48,000-mol wt protein. In addition, supernatants of cultured macrophages from six normal volunteers had Factor X-activating activity that was suppressed an average of 71% after culture in the presence of 50 microM coumadin or entirely by the Factor VII antibody indicating that Factor VII synthesized by the cell was biologically active. Endotoxin in vitro induced increases in cellular tissue factor but had no consistent effect on macrophage Factor VII activity. We also examined the tissue factor and Factor VII activities of freshly lavaged alveolar cells from nine subjects with clinical and/or histologic evidence of sarcoidosis. Four of the nine subjects expressed increased tissue factor and seven of nine had increased Factor VII activity over the normal range (P less than 0.01). We estimate the mean Factor VII associated with the cells of sarcoid patients to be 4.7 ng/10(6) cells (range 0.4-20) as compared to a mean of 0.74 ng/10(6) cells (range 0.2-2) for that of normal subjects. Along with previous data showing synthesis of plasminogen activator, these findings indicate that human alveolar macrophages normally synthesize and express measurable amounts of the initial enzymes of proteolytic reactions regulating both fibrin deposition and fibrin resorption. Abnormalities in Factor VII activity in a small group of patients with sarcoidosis raise the possibility that modulation of fibrin turnover by macrophages may contribute to the pathology of this and perhaps other interstitial lung diseases.

Plasma Lipoprotein Induction and Suppression of the Generation of Cellular Procoagulant Activity In Vitro: TWO PROCOAGULANT ACTIVITIES ARE PRODUCED BY PERIPHERAL BLOOD MONONUCLEAR CELLS

In the process of analyzing the effects of lipoproteins on functions of lymphoid cells, it was observed that physiological concentrations of isolated human plasma lipoproteins possess varying capacities to rapidly enhance the expression of procoagulant activity of human peripheral blood mononuclear cells in vitro. In a strict dose-dependent fashion, very low density lipoprotein, intermediate density lipoprotein, and high density lipoprotein enhanced both the surface expression by viable cells and the total cellular content of procoagulant activity during a 6-h incubation. Very low density lipoprotein induced a maximal 6.7-fold increase in the expression of a thromboplastin activity, which was consistent with tissue factor, in that it was dependent on Factors VII, X, and II. Both intermediate density lipoprotein and high density lipoprotein induced approximately a 12-fold increase of a different procoagulant activity which appears to be a direct prothrombin activator. This prothrombinase was calcium dependent and was inhibited by 2.5 mM diisopropylfluorophosphate, but was not neutralized by anti-Factor X antibodies or by inhibitors of Factor Xa. In contrast to the other lipoprotein density classes, low density lipoprotein did not stimulate procoagulant activity, but instead actively suppressed the generation of the two procoagulant activities induced by the stimulatory lipoproteins. Suppression by low density lipoprotein was clearly evident at molar ratios of low density lipoprotein to stimulatory lipoproteins of 1:3 or less. Reconstitution of all lipoproteins to physiological concentrations was not stimulatory as a consequence of the suppressive effects of low density lipoprotein. These data indicate that isolated plasma lipoproteins are capable of regulating the expression of two different procoagulant activities of peripheral blood mononuclear cells in vitro. The possibility that these interactions may be implicated in the association between certain types of hyperlipoproteinemias and thromboembolic disease merits study.

Resolution of monomeric and heterodimeric forms of tissue factor, the high-affinity cellular receptor for factor VII

Tissue factor (TF) is the high affinity cell surface receptor and obligatory cofactor for plasma coagulation factor VII. Purification of TF from human brain and placenta has recently been reported to yield both a monomeric 47 kDa protein as well as a 58 kDa heterodimeric form. In this study, the TF glycoprotein was isolated from human brain by immunoaffinity chromatography using a newly developed monoclonal antibody, TF8-5G9, and was compared to TF isolated by factor VII-affinity chromatography. Except for the greater efficiency of the immunoaffinity method, both methods yielded TF with similar specific activities, and both preparations contained the monomeric and heterodimeric forms of TF. The 58 kDa form was established to be a disulfide-linked heterodimer composed of TF and the alpha chain of hemoglobin. From these results and from studies of immunoprecipitation of TF from cultured fibroblast cells, we conclude that the 47 kDa monomeric form of TF is the naturally occurring cellular form of TF, and that heterodimer formation is a secondary event. The potential significance of the proclivity of TF to form a heterodimer with hemoglobin is discussed.

Acquired factor VII deficiency associated with aplastic anaemia: correction with bone marrow transplantation

Summary. We report a patient with severe aiplastic anaemia found to have a prolonged prothrombin time due to acquired factor VII deficiency. No evidence for a factor VII inhibitor or inactivator was demonstrable. Laboratory studies identified deficiency both of factor VII activity and factor VII antigen. The factor VII deficiency persisted from clinical presentation until approximately 50 d after allogeneic marrow transplantation when restoration of factor VII activity;and antigen was noted. The patients serum could be depleted of factor VII activity by in vitro incubation with Protein A bound to Sepharose, suggesting the presence of an IgCl or IgG containing complex able to bind factor VII, but not neutralize its procoagulant activity. A dual specificity solid phase immunoassay identified a factor VII binding immunoglobulin which was detectable throughout the course of factor VII deficiency. The concordant appearance of this factor VII reactive immunoglobulin and the factor VII deficiency suggested the pathologic role of this immunoglobulin in the aetiology of the factor VII deficiency. This factor VII binding immunoglobulin may have induced rapid plasma clearance of the factor VII molecule or, alternatively, may have modified factor VII synthesis. The immunosuppressive therapy and subsequent lymphohaematopoietic engraftment following allogeneic marrow transplant was accompanied by complete resolution of the factor VII deficiency.

The dysfunction of coagulation factor VIIPadua results from substitution of arginine-304 by glutamine

This study addresses whether a mutation in the factor VIIPadua gene could explain the reduced activity of the inherited variant protein. All nine exons of the normal and Padua factor VII gene were amplified using the polymerase chain reaction, cloned into pUC19 and sequenced. A point mutation (G to A at nucleotide position 10828) was found which results in the substitution of a glutamine (CAG) for arginine (CGG) at amino acid position 304. This substitution creates a PvuII restriction site useful in screening for the defect and in demonstrating homozygosity. This substitution involves an arginine residue in the catalytic domain within a Leu*****Pro******Cys motif which occurs in conserved region 5 in up to 16 coagulation and other serine proteinases. On the basis of conformational homology among serine proteinases, it is suggested that the observed amino acid substitution in factor VIIPadua could cause structural changes affecting its activation and/or catalytic activity.

Inhibition of factor Xa-mediated procoagulant activity of human lung fibroblasts and pleural mesothelial cells.

Extravascular fibrin deposition characterizes diverse forms of lung and pleural injury. Fibrin formation in these compartments is locally potentiated by the assembly and expression of the prothrombinase procoagulant complex (factors Xa, Va and II) at the surface of human lung fibroblasts and pleural mesothelial cells. We sought to identify structural domains on factor Xa that mediate expression of prothrombinase activity by these cells. In order to accomplish this objective, we used panels of monoclonal antibodies (MoAbs) to factor X to block prothrombinase assembly and function on the surface of cultured human lung fibroblasts and pleural mesothelial cells. Of 30 factor X MoAbs that recognized native factors X and Xa, 10 completely inhibited factor Xa function (prothrombin activation), and five others neutralized Xa function without affecting cell-binding, presumably by blocking the prothrombin binding site. Western blots showed that these inhibitory MoAbs reacted with the Xa heavy-chain. One MoAb that recognized the factor Xa light-chain blocked prothrombin activation at the factor Va binding site. Our results indicate that prothrombinase activity at the surface of lung parachymal or pleural cells can be blocked by MoAbs that interact with either the heavy- or light-chain of factors X. Antibodies that neutralize cell surface-expressed prothrombin activation offer a potential means to arrest pericellular fibrin formation in the lung and pleural space.