ABSTRACT
Progressive, irreversible fibrosis is one of the most clinically significant consequences of ionizing radiation on normal tissue. When applied to lungs, it leads to a complication described as idiopathic pneumonia syndrome (IPS) and eventually to organ fibrosis. For its high mortality, the condition precludes treatment with high doses of radiation. There is widespread interest to understand the pathogenetic mechanisms of IPS and to find drugs effective in the prevention of its development. This report summarizes our experience with the protective effects of L 158,809, an angiotensin II (ANG II) receptor blocker, and two angiotensin converting enzyme (ACE) inhibitors in the development of IPS and the role of transforming growth factor beta (TGF-beta) and of alpha-actomyosin (alpha SMA) in pathogenesis of radiation induced pulmonary fibrosis in an experimental model of bone marrow transplant (BMT). Male WAG/Riji/MCV rats received total body irradiation and a regimen of cyclophosphamide (CTX) in preparation for bone marrow transplant. While one group of animals remained untreated, the remainders were subdivided into three groups, each of them receiving either the ANG II receptor blocker or one of the two ACE inhibitors (Captopril or Enalapril). Each of the three drugs was administered orally from 11 days before the transplant up to 56 days post transplant. At sacrifice time the irradiated rats receiving only CTX showed a chronic pneumonitis with septal fibrosis and vasculitis affecting, in particular, small caliber pulmonary arteries and arterioles. Their lung content of hydroxyproline was also markedly elevated in association with the lung concentrations of thromboxane (TXA2) and prostaglandin (PGI(2)), (two markers of pulmonary endothelial damage). A significant increase of alpha actomyosin staining was observed in vessels, septa and macrophages of the same animals which also overexpressed TGF-beta. When L 158,809, Captopril and Enalapril were added to the radiation and cytoxan treatment, a significant amelioration of the histological damage as well as the overexpression of alpha SMA was observed. Lung concentrations of hydroxyproline, PGI(2), TXA2 and TGF-beta were also observed in these animals so that the values of these compounds were closer to those measured in untreated control rats than to their irradiated and cytoxan treated counterparts. Angiotensin II plays an important role in the regulation of TGF-beta and alpha SMA, two proteins involved in the pathogenesis of pulmonary fibrosis. The finding that ACE inhibitors or ANG II receptor blockers protect the lungs from radiation induced pneumonitis and fibrosis reaffirms the role that ANG II plays in this inflammatory process and suggests an additional indication of treatment of this condition, thus opening a new potential pharmacologic use of these drugs.
Subject(s)
Actomyosin/metabolism , Angiotensin Receptor Antagonists , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Lung/radiation effects , Peptidyl-Dipeptidase A/metabolism , Pulmonary Fibrosis/metabolism , Transforming Growth Factor beta/metabolism , Actomyosin/antagonists & inhibitors , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Lung/drug effects , Lung/metabolism , Male , Peptidyl-Dipeptidase A/physiology , Pulmonary Fibrosis/drug therapy , Radiation Injuries, Experimental/drug therapy , Radiation Injuries, Experimental/metabolism , Rats , Receptors, Angiotensin/metabolism , Transforming Growth Factor beta/antagonists & inhibitorsABSTRACT
Thromboelastography (TEG) has been used increasingly as an intraoperative hemostasis monitoring device. Low-molecular-weight heparins are given increasingly to reduce the development of antibodies against the heparin-platelet factor 4 complex, and heparinoids are given to patients who have developed the antibody. We studied the effect of unfractionated heparin, a low-molecular-weight heparin (enoxaparin sodium [Lovenox]), and a heparinoid (danaparoid sodium [Orgaran]) on blood clotting assayed with TEG (TEG clotting) in vitro and the efficacy of protamine sulfate and heparinase for reversing the effect. Heparin, enoxaparin, and danaparoid all caused a dose-dependent inhibition of TEG clotting of normal blood. Concentrations of enoxaparin and danaparoid that totally inhibited TEG clotting only minimally prolonged the activated partial thromboplastin time. While inhibition of TEG clotting by heparin and enoxaparin was reversed by protamine sulfate and heparinase, inhibition by danaparoid was reversed only by heparinase. Abnormal TEG clotting was observed in patients receiving enoxaparin whose plasma level of the drug was more than 0.1 antiXa U/mL. However, the degree of TEG abnormality did not always coincide with plasma levels of the drug.
Subject(s)
Anticoagulants/pharmacology , Blood Coagulation/drug effects , Enoxaparin/pharmacology , Heparin/pharmacology , Heparinoids/pharmacology , Thrombelastography , Antibodies/blood , Autoantibodies/blood , Chondroitin Sulfates/pharmacology , Dermatan Sulfate/pharmacology , Drug Combinations , Heparin/immunology , Heparin Lyase/pharmacology , Heparitin Sulfate/pharmacology , Humans , Partial Thromboplastin Time , Platelet Factor 4/immunology , Protamines/pharmacology , Prothrombin TimeABSTRACT
Captopril (D-3-mercapto-2-methylpropanoyl-L-proline) is an angiotensin converting enzyme (ACE) inhibitor, used widely in the treatment of hypertension and congestive heart failure. Captopril also inhibits proliferation of a variety of cell types, including several lacking ACE and renin acitvity. We have previously demonstrated that human mammary ductal carcinoma cells are among the cell types whose mitotic activity is inhibited by captopril. In those cells, captopril also reduces estrogen receptor (ER) and increases progesterone receptor (PR) concentrations. The present study evaluated the mechanism of captopril's antiproliferative action in an ER/PR-negative human mammary ductal carcinoma cell line, Hs578T. Cells grown in a 10% serum medium showed negligible changes in the presence of captopril alone. However, in the presence of subphysiologic concentrations of copper salts or copper-loaded ceruloplasmin, captopril caused a dose-dependent reduction in cell number, thymidine incorporation and mitochondrial dehydrogenase activity. In contrast, iron salts and iron-saturated transferrin had no effect on captopril activity. Catalase and horseradish peroxidase nullified the cytotoxic effects of captopril/Cu++, whereas H2O2 mimicked those effects. These data are consistent with the notion of a copper-catalyzed oxidation of captopril, leading to the generation of H2O2 as the cytotoxin to this clinically important cell type.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/toxicity , Antineoplastic Combined Chemotherapy Protocols/toxicity , Breast Neoplasms/pathology , Captopril/toxicity , Carcinoma, Ductal, Breast/pathology , Copper/toxicity , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/pathology , Copper/physiology , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Drug Synergism , Humans , Hydrogen Peroxide/toxicity , Lisinopril/toxicity , Penicillamine/toxicityABSTRACT
Performance characteristics of a totally synthetic activated partial thromboplastin time (APTT) reagent, recently available commercially, were evaluated and compared with a rabbit-brain extracted reagent. We found that the synthetic reagent, Synthasil, returned significantly higher normal APTT values than the brain-extracted reagent, Thrombosil. APTT ratios (APTT patients/normal mean APTT), yielded by Synthasil were higher in the majority of patients receiving heparin therapy. Synthasil also returned longer APTT values than did Thrombosil on normal plasma spiked with heparin. On patients with lupus anticoagulants, APTTs assayed with Synthasil were generally longer than with Thrombosil. However, the differences disappeared when APTT values were converted to ratios. Factors XII-, XI-, IX- and VIII-deficient plasmas supplemented with normal plasma to yield activities of 2-50%, generally gave longer APTTs with Synthasil than with Thrombosil. However, this was not always the case on plasmas from haemophilias A and B patients. No reduction in Synthasil activity was noted after the reagent had been left at 24 degrees C for 28 days.
Subject(s)
Coagulation Protein Disorders/diagnosis , Heparin/blood , Indicators and Reagents , Partial Thromboplastin Time , Drug Stability , Hemophilia A/diagnosis , Hemophilia B/diagnosis , Humans , Linear Models , Lupus Coagulation Inhibitor/analysis , Sensitivity and SpecificityABSTRACT
We conducted studies to determine at what time point an add-on prothrombin time (PT) or activated partial thromboplastin time (APTT) test can be honored on specimens that have been received in the laboratory hours earlier without yielding results with clinically significant differences from those if the test had been performed on the original unstored plasma. PT and APTT tests were performed on blood samples from 20 healthy subjects, 30 patients receiving warfarin, and 30 patients receiving heparin anticoagulation therapy. The tests were performed on plasma prepared initially after the samples were obtained. The same tests were assayed on plasma that had been left on spun-down blood cells at room temperature for 2, 4, and 8 hours. We found that the PT of the majority of plasma samples from healthy subjects and from patients receiving oral anticoagulant therapy tended to become shorter on storage. However, the difference in PT values was small and had no clinical significance. In most cases, the APTT values for the stored plasma from healthy subjects tended to increase with time. Except in one specimen in which the 8-hour add-on APTT was 1.2 seconds longer than the APTT result for the original sample, all others had APTT results less than 1.2 seconds longer than the original values. In patients receiving heparin, the differences in APTT values between the initial and add-on tests were larger than those observed for healthy subjects. However, those differences are not beyond what we would accept for duplicate checks for heparinized samples with high APTT values. Unlike samples from healthy subjects, there was no obvious trend of time-related prolongation of the APTT in heparinized plasma. These results led us to believe that within an 8-hour period and with plasma on spun-down cells at room temperature, add-on tests for PT and APTT could be performed with results similar to what would be obtained from testing unstored samples.
Subject(s)
Partial Thromboplastin Time , Plasma , Prothrombin Time , Anticoagulants/therapeutic use , Citrates , Drug Stability , Heparin/therapeutic use , Humans , Reference Values , Sodium Citrate , Time Factors , Warfarin/therapeutic useABSTRACT
Radiation-induced lung fibrosis is a result of collagen accumulation in the interstitium, partly due to increased collagen synthesis by fibroblasts. One feature of active collagen synthesis is increased membrane trafficking in the fibroblasts. A group of proteins called annexins is believed to play a regulatory role in membrane fusion and exocytosis. Therefore, increased annexin activity might be expected in the fibrotic lung. We tested this hypothesis by measuring annexin I levels, hydroxyproline content and ultrastructural changes in radiation-induced pulmonary fibrosis in rat. Three months after a single exposure to 30 Gy of X-rays to the right hemithorax, the right lung of the rat was atrophied and fibrotic with a concomitant increase in size of the shielded left lung. Electron micrographs revealed that the irradiated lung was ladened with interstitial collagen fibrils, with increased number of fibroblasts amongst them. Hydroxyproline concentration in the irradiated lung was nearly twice that in the sham-irradiated lung. Annexin I in the irradiated lung, on the other hand, was markedly reduced, and barely detectable on immunoblots. Since increased annexin I might precede enhanced collagen production, we also measured annexin I levels in rat lungs 3 days after 30 Gy irradiation and correlated that with hydroxyproline concentration. We found no appreciable difference in annexin I levels and hydroxyproline content between sham-irradiated and irradiated lungs at 3 days. To determine whether annexin I levels in cultured fibroblasts were altered by irradiation, we assayed annexin I in cultured rat lung fibroblasts 3 days after 0.10 Gy exposure, with concomitant measurement of 14C-proline incorporation. The annexin I level in fibroblasts irradiated with 10 Gy X-rays was 55% higher than in sham-irradiated fibroblasts. However, incorporation of 14C-proline into collagenase-sensitive macromolecules in the culture medium and extracellular matrix was not different between these two groups of cells. These data demonstrate a radiation-induced increase in immunoreactive annexin I in cultured lung fibroblasts, but fail to support the hypothesis of a positive correlation between annexin I concentration and fibrosis in irradiated rat lung.
Subject(s)
Annexin A1/radiation effects , Lung/radiation effects , Pulmonary Fibrosis/metabolism , Animals , Cells, Cultured , Collagen/metabolism , Fibroblasts/metabolism , Fibroblasts/radiation effects , Hydroxyproline/metabolism , Lung/metabolism , Male , Organ Size/radiation effects , Proline/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
A single point mutation of the factor V (FV) gene, leading to the substitution Arg506Gln in the FV molecule (FV-Leiden) and hence resistance to its breakdown by activated protein C (APC), is the most prevalent risk factor for venous thrombosis in the Caucasians. A ratio determined by activated partial thromboplastin time (APTT) of test plasma in the presence or absence of exogenous APC (the APC ratio), is the method widely used to screen individuals with this risk factor for thrombosis. Because of functional defects of vitamin K-dependent clotting factors in patients on oral anticoagulant therapy, this method cannot be applied to those patients without modification. One modification is to mix test plasma (1:5 or 1:10) with FV-deficient plasma so that 80-90% of functioning vitamin K-dependent factors are supplied by the FV-deficient plasma. Even with 10-20% of FV in the mixture, APC-resistance still can be demonstrated. In this report, we present our results of the modified APC-sensitivity assay using FV-deficient plasma from different commercial sources. APC ratios determined by the original method in which test plasma is not mixed with FV-deficient plasma can be significantly different from those determined by the modified method in which test plasma is diluted 1:5 with FV-deficient plasma. This difference between methods was observed not only in normal individuals, but also in FV-Leiden positive individuals, and in patients on warfarin therapy. Further, APC ratios varied significantly depending on the commercial source of the FV-deficient plasma. The modified method is apparently suitable to identify APC-resistance in patients on warfarin therapy, as well as in individuals not receiving anticoagulant treatment. However, one must be aware that APC-resistance ratios obtained with the modified method are likely to be different from those established with the original method, and the source of FV-deficient plasma can be a factor influencing the ratios in the former cases.
Subject(s)
Biological Assay/methods , Drug Resistance , Factor V Deficiency/blood , Protein C/metabolism , Warfarin/therapeutic use , Anticoagulants/therapeutic use , Factor V/genetics , Factor V/metabolism , Factor V Deficiency/drug therapy , Female , Humans , Male , Point Mutation , Protein C/analysis , Reference Standards , Sensitivity and SpecificityABSTRACT
The purpose of this study was to gather additional evidence in irradiated rat lung on the relationship between annexin I and prostaglandin synthesis. The right hemithorax of the animal was exposed to a single dose of 0 or 30 Gy of X-rays, and the animals were killed 3 months postirradiation. Levels of annexin I and synthesis of prostacyclin (PGI2) were determined in lungs, in cell-free bronchoalveolar lavage (BAL) fluid, and in macrophages lavaged from those lungs. In addition, protein concentration, lactate dehydrogenase (LDH) activity and macrophage count in BAL fluid obtained from irradiated lung were compared with that from sham-irradiated (0 Gy) lung. Levels of annexin I, the putative inhibitor of phospholipase A2, in lung and cell-free BAL fluid were decreased in samples from irradiated animals. By contrast, the level of annexin I in macrophages lavaged from irradiated lung was higher than that in macrophages from sham-irradiated lung. The irradiated lung produced nearly 3.5 times more prostacyclin than did the control lung. However, prostacyclin synthesis by macrophages lavaged from irradiated lung was no different than that of macrophages from sham-irradiated lung. Protein, LDH and macrophage number in BAL fluid from irradiated lungs were significantly higher than in corresponding specimens from sham-irradiated lungs. These data demonstrate that reduced levels of annexin I, as well as increased protein concentration, LDH activity and macrophage numbers in irradiated rat lung are reflected in BAL fluid. Therefore, information obtained from BAL fluid, but not from BAL macrophages, reflects lung status, and may serve as a minimally invasive index of radiation pneumonitis in this model. In irradiated lung, increased PGI2 synthesis coupled with a decreased annexin I level are consistent with the hypothesis of an inhibitory role of annexin I in prostaglandin metabolism. However, this hypothesis is not supported by findings in BAL macrophages, where increased annexin I concentration is not accompanied by a decrease in PGI2 production. In view of the latter findings, and a previous study from our laboratory demonstrating that phospholipase activity in irradiated rat lung is in fact decreased, despite the reduction in annexin I concentration and the hyperproduction of prostanoids, it would seem unlikely that annexin I negatively modulates prostaglandin synthesis via inhibition of phospholipase in this model.
Subject(s)
Annexin A1/metabolism , Epoprostenol/biosynthesis , Lung/radiation effects , Macrophages, Alveolar/radiation effects , 6-Ketoprostaglandin F1 alpha/metabolism , Animals , Arachidonic Acid/metabolism , Blotting, Western , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Cell Count/radiation effects , Gamma Rays , L-Lactate Dehydrogenase/metabolism , Lung/metabolism , Macrophages, Alveolar/metabolism , Male , Rats , Rats, Sprague-Dawley , X-RaysABSTRACT
Positive pressure ventilation with hyperdistention of the lungs (PPVHDL) causes microscopic lung injury in rats and in mice. This study compared lung lavage and serum levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatinine phosphokinase (CPK), lung lavage and plasma endothelin-1 (ET-1) concentration, lung tissue ET-1 mRNA expression, angiotensin converting enzyme (ACE) activity of lung homogenates, and histology of the lung structure in control and PPVHDL rats. Rats were anesthetized with pentobarbital. While control rats were breathing spontaneously, the PPVHDL rats were ventilated with a rodent ventilator delivering 30 percent oxygen, a tidal volume of 18.6 +/- 4.5 ml/kg, and a respiratory rate of 55 to 60 per minute. End-tidal CO2 was maintained at 38-40 mm Hg. After seven hours, rats were killed and the lungs were lavaged. Red blood cells were present in the sediment of lavage fluid in PPVHDL rats and their lung structure showed severe congestion, alveolar septa filled with red cells, and extravasation of red blood cells and inflammatory cells into the alveolar space. Lung lavage fluid AST and LDH were significantly higher in the PPVHDL compared with the control group (P < 0.03 and P < 0.001, respectively). Electrophoresis of the lung lavage LDH showed increased peak-5 in the PPVHDL group. Serum LDH, CPK, AST, and potassium concentrations [K]+ were significantly higher in the PPVHDL rats whereas their serum total protein level was significantly lower than the control group (P < 0.001). Electrophoretic patterns of serum and lung lavage protein were similar in both groups indicating a transmural passage of serum protein from the intravascular to the intra-alveolar space. No significant difference was found in lung tissue ET-1 mRNA expression and lung protein concentration between the two groups. Lung ACE activity, in contrast, was significantly lower in PPVHDL rats. This study demonstrated that moderate alveolar hyperdistention caused significant structural lung damage accompanied by decreased ACE activity after seven hours of mechanical ventilation and that elevated lung lavage and serum LDH and AST levels in lung lavage and in serum might be early markers of ventilator-induced lung injury in this rat model.
Subject(s)
Lung Diseases/etiology , Positive-Pressure Respiration/adverse effects , Animals , Biomarkers , Blood/metabolism , Bronchoalveolar Lavage Fluid/chemistry , Electrophoresis, Polyacrylamide Gel , Lung/metabolism , Lung/pathology , Lung Diseases/metabolism , Lung Diseases/pathology , Male , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
Cytotoxicity indicated by increased release of prelabeled 51chromium (51Cr) and lactate dehydrogenase (LDH) was studied in human prostate cancer and melanoma cells in cell culture following irradiation or exposure to several injurious substances. These changes were compared to those observed in bovine aortic endothelial cells (BAEC) subjected to identical treatments. Further, the effect of irradiation on plasminogen activator (PA) secretion from prostate cancer cells, and the effect of glycine on radiation-induced cytotoxicity in BAEC were also investigated. Radiation, lipopolysaccharide and xanthine/xanthine oxidase stimulated no release of 51Cr or LDH from tumor cells, while these treatments induced a dose- and time-related loss of those cytotoxic indicators from BAEC. Protease, elastase and Triton X-100 incited loss of 51Cr and LDH from all three cell types. Radiation, lipopolysaccharide and xanthine/xanthine oxidase have been shown to cause cell injury via a common pathogenic pathway of oxidant generation. Tumor cells appear quite resistant to oxidant stress. Cell damage precipitated by protease, elastase and Triton probably involves hydrolysis of proteins and phospholipids in the cell membrane, leading to an increased leakage of intracellular proteins such as LDH and those bound with 51Cr. Radiation caused a dose- and time-related reduction in the secretion of PA from prostate cancer cells. PA is alleged to play a role in tumor metastasis; the reduced secretion could be another beneficial effect of radiation, in addition to interruption of cell proliferation, in the impediment of tumor growth and spread. Glycine diminished cytotoxic injury of BAEC inflicted by radiation. This amino acid may prove useful in offering a degree of protection of normal tissue against radiation associated side-effects.
Subject(s)
Endothelium, Vascular/radiation effects , Glycine/pharmacology , Melanoma/radiotherapy , Prostatic Neoplasms/radiotherapy , Radiation-Protective Agents/pharmacology , Animals , Cattle , Cells, Cultured , Chromium Radioisotopes/analysis , Endopeptidases/pharmacology , Endothelium, Vascular/drug effects , Hot Temperature , Humans , L-Lactate Dehydrogenase/analysis , Lipopolysaccharides/pharmacology , Male , Pancreatic Elastase/pharmacology , Plasminogen Activators/metabolism , Polyethylene Glycols/pharmacology , Prostatic Neoplasms/metabolism , Radiation Injuries/prevention & control , Time Factors , Tumor Cells, Cultured , Xanthine Oxidase/pharmacology , Xanthines/pharmacologyABSTRACT
The level of annexin I, a 36 kDa calcium-dependent phospholipid-binding protein (36 kDa PLBP) in the reproductive organs of young, mature, and pregnant rabbits was determined immunologically with antibodies raised against purified rabbit lung annexin I. In the cytosolic fractions of the ovary, fallopian tube, uterus, and placenta, annexin I was the only major immunoreactive protein. The reproductive organs appeared to have higher annexin I levels than most nonreproductive organ tissues, except the lung and the spleen which were also rich in annexin I. A small amount of annexin I and a nearly equal amount of its hydrolytic product, a 33 kDa polypeptide, were detected in the amniotic fluid between 21 and 27 days gestation. Structural similarity of annexin I in the reproductive organs and in the lung was suggested by their identical isoelectric point values. Annexin I in the ovary of adult rabbits was 70% higher than that in the respective organ of immature rabbits. The uterus of pregnant rabbits had about 84% higher annexin I contents than that of the nonpregnant rabbits. The placenta had more annexin I per mg cytosolic protein than either the ovary or the uterus during pregnancy. The high concentration of annexin I in the reproductive organs may reflect specific functions of these organs in the reproductive years and during the reproductive cycle.
Subject(s)
Annexin A1/metabolism , Fallopian Tubes/metabolism , Ovary/metabolism , Placenta/metabolism , Pregnancy, Animal/metabolism , Uterus/metabolism , Amniotic Fluid/metabolism , Animals , Female , Isoelectric Focusing , Lung/metabolism , Pregnancy , Rabbits , Spleen/metabolismABSTRACT
Annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins (PLBP), has been suggested as a regulator of prostaglandin metabolism as a result of its inhibitory effect on phospholipase A2. Synthesis of prostaglandin is increased in irradiated tissue, but the mechanism underlying this increase has not been delineated. It is conceivable that a decrease in the level of annexin I resulting in increased phospholipase activity may be responsible for the enhanced synthesis of prostaglandin. Accordingly, we measured the level of a lung 36 kDa PLBP, which possesses characteristics of annexin I, as well as the activity of phospholipase and the synthesis of thromboxane A2 (TXA2) in irradiated rat lung. The right lung of rats was irradiated with 0, 15 or 30 Gy of X rays and the animals were sacrificed after 3 months. Phospholipid binding protein was assayed by its ability to transfer unilamellar liposomes to multilamellar liposomes and by immunoblotting against anti-36 kDa rabbit PLBP antisera. Production of TXA2 by minced lung tissue was determined by radioimmunoassay of its stable metabolite TXB2. Phospholipase activity was assayed by hydrolysis of [14C]dioleoylphosphatidylcholine. Our results showed that PLBP activity in the lungs irradiated with 30 Gy was lower than that in the lungs irradiated with 0 and 15 Gy (8.82 +/- 0.47 compared to 9.73 +/- 0.49 and 9.95 +/- 0.78 nmol phospholipid transferred/mg protein, respectively). Western blotting demonstrated a near total depletion of annexin I in the lungs irradiated with 30 Gy. Phospholipase activity was also lower in the lungs irradiated with 30 Gy compared to that in the lungs irradiated with 0 Gy (0.23 +/- 0.01 vs 0.32 +/- 0.01 nmol phosphatidylcholine liberated/mg protein/min, P < 0.001). Reduced phospholipase activity was observed not only in the cytosolic or soluble fraction of lung homogenate, but also in precipitates obtained after 21,000g and 100,000g centrifugation. Despite this decline in phospholipase activity, there was a 2.8-fold increase in the synthesis of thromboxane (367 +/- 65 compared to 1076 +/- 143 pg TXB2/mg tissue/10 min for lungs irradiated with 0 and 30 Gy, respectively). These results are not consistent with the hypothesis that increased synthesis of thromboxane A2 in irradiated rat lung is a direct result of elevated phospholipase activity. In fact, phospholipase activity is decreased in the irradiated lung, despite a decline in the concentration of annexin I, its putative inhibitor.
Subject(s)
Annexin A1/analysis , Lung/radiation effects , Phospholipases/metabolism , Thromboxanes/biosynthesis , Animals , Arachidonic Acid/metabolism , Carrier Proteins/analysis , Dose-Response Relationship, Radiation , Lung/metabolism , Male , Phospholipids/analysis , Rats , Rats, Sprague-DawleyABSTRACT
The recent introduction of thromboplastin reagents with low international sensitivity index (ISI) values into the US market for the purpose of generating a more precise international normalized ratio than high ISI thromboplastins could has necessitated an evaluation of the impact of the low ISI reagents on prothrombin time (PT) testing in general. In this study, PT testing with three thromboplastin reagents, one of which (presently used in our laboratory) has an ISI of 2.10 and the other two ISI values of 0.92 and 1.06, respectively, was performed on normal individuals, on quality control reference plasma specimens and single-factor-deficient plasma specimens, and on patients with liver disease, intravascular coagulation, and receiving oral anticoagulant therapy. We found that PTs of normal individuals determined by all three thromboplastins were virtually identical. The thromboplastins with a low ISI generated much longer PTs on abnormal reference plasma specimens than did the high ISI product. Low ISI reagents also produced longer PTs in all three groups of patients. However, the degree of prolongation was far greater for patients receiving warfarin than for the other two groups of patients. Conversion of the PT to an international normalized ratio minimized the discrepancy seen in the PT ratio in patients receiving oral anticoagulants. The two low ISI thromboplastins did not produce near-identical values of PT, PT ratio, or international normalized ratio on plasma specimens obtained from patients who received warfarin therapy. The critical value set for PT with a high ISI thromboplastin would not be adequate if the reagent is to be replaced with a low ISI product.
Subject(s)
Prothrombin Time , Thromboplastin/standards , Administration, Oral , Disseminated Intravascular Coagulation/blood , Humans , Liver Diseases/blood , Quality Control , Reference Standards , Regression Analysis , Sensitivity and Specificity , Warfarin/therapeutic useABSTRACT
This study was undertaken to answer some practical questions physicians and medical directors of clinical laboratories face when they contemplate replacing their high international sensitivity index (ISI) thromboplastins with low ISI ones and using international normalized ratio (INR) in place of prothrombin time ratio (PTR) for monitoring warfarin therapy. To the question of whether low-ISI thromboplastins would produce a prolonged PT on normal patients, the answer is probably no. To the question of the extent of normalization of disparate PTs, determined by high and low ISI thromboplastins, of patients on oral anticoagulants upon the conversion of PTR to INR, the answer is a mixed one. For those whose PTs were 14-20 sec, conversion of PTR to INR would markedly, but not completely, normalize the PTR values. In other words, there would be a lessening of disparity of the PTR after the conversion. For patients whose PTs were > 20 sec, conversion of PTR to INR could even widen the disparity seen with the PTR. Finally, when PTs were assayed on different coagulation devices with the same reagent, highly congenial results were obtained.
Subject(s)
Anticoagulants/therapeutic use , Drug Monitoring/statistics & numerical data , Drug Monitoring/standards , Prothrombin Time , Humans , Reference Values , Time FactorsABSTRACT
The angiotensin converting enzyme (ACE) inhibitor captopril, a free-thiol compound used widely as an antihypertensive agent, also inhibits radiation-induced pulmonary fibrosis in rats (Ward et al., Int J Radiat Oncol Biol Phys 19:1405, 1990). In an attempt to clarify the antifibrotic mechanism of captopril in vivo, the present study examined the effect of the drug on proliferation of human lung fibroblasts in culture. Captopril produced a drug dose-dependent reduction in fibroblast proliferation and 3H-thymidine incorporation during a 24-72-hr incubation. This cytostatic action of captopril was not the result of cytotoxicity as assessed by trypan blue exclusion, or by 51Cr or lactate dehydrogenase (LDH) release. Fibroblasts stimulated to proliferate by basic FGF were more sensitive to the antimitotic effect of captopril than were unstimulated cells. The ability of captopril to inhibit 3H-thymidine incorporation was not reversed by exogenous angiotensin 2, and was not mimicked by the nonthiol ACE inhibitor lisinopril. These data indicate that the cytostatic effect of captopril was not attributable to ACE inhibition. Penicillamine, a thiol compound with virtually no ACE inhibitory activity, also reduced fibroblast 3H-thymidine incorporation, indicating that the antimitotic action of captopril may represent a nonspecific sulfhydryl effect. This study suggests that the antifibrotic activity of captopril in irradiated lung may result in part from a direct inhibition of fibroblast proliferation, particularly in fibroblasts responding to mitogenic stimuli.
Subject(s)
Captopril/pharmacology , Cell Division/drug effects , Lung/cytology , Angiotensin II/pharmacology , Animals , Cell Line , DNA/biosynthesis , Dose-Response Relationship, Drug , Fibroblast Growth Factor 2/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Kinetics , L-Lactate Dehydrogenase/analysis , Mitosis/drug effects , Penicillamine/pharmacology , Pulmonary Fibrosis/prevention & control , Rats , Thymidine/metabolismABSTRACT
The present study determined whether inhibitors of angiotensin converting enzyme (ACE) can ameliorate radiation-induced pulmonary endothelial dysfunction and pulmonary fibrosis in rats sacrificed 2 months after a range of single doses of 60Co gamma rays to the right hemithorax. Four indices of pulmonary endothelial function were monitored: right lung ACE and plasminogen activator (PLA) activity, and prostacyclin (PGI2) and thromboxane (TXA2) production. Hydroxyproline (HP) content served as an index of pulmonary fibrosis. Rats consumed either control powdered chow or feed containing one of five modifying agents continuously after irradiation. The modifiers included three ACE inhibitors: Captopril, CL242817, and CGS13945, respectively, a thiol, a thioacetate, and a nonthiol compound. All of the ACE inhibitors are analogues of proline. Two additional modifiers were tested: penicillamine, a thiol with no ACE inhibitory activity; and pentoxifylline, a vasodilator that is neither a thiol nor an ACE inhibitor. Radiation produced a dose-dependent decrease in lung ACE and PLA activity, and an increase in PGI2 and TXA2 production and in HP content. All ACE inhibitors attenuated the radiation-induced suppression in lung ACE and PLA activity. All thiol or thioacetate compounds ameliorated the radiation-induced increase in PGI2, TXA2, and HP. The two agents that were both thiols and ACE inhibitors (Captopril and CL242817) spared all of the radiation reactions, while the compound that was neither a thiol nor an ACE inhibitor (pentoxifylline) spared none of the reactions. These data suggest a novel application for ACE inhibitors in general, and for Captopril in particular, as modifiers of radiation pneumotoxicity.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Lung/radiation effects , Pulmonary Fibrosis/drug therapy , Animals , Captopril/therapeutic use , Cobalt Radioisotopes , Indoles/therapeutic use , Male , Proline/analogs & derivatives , Proline/therapeutic use , Pulmonary Fibrosis/etiology , Rats , Rats, Inbred Strains , Structure-Activity RelationshipABSTRACT
Male rats were exposed to single doses (0-30 Gy) of 60Co gamma rays to the right hemithorax. Half of each dose group consumed only control powdered chow after irradiation, and half consumed feed containing 0.10% (w/w) pentoxifylline (50 mg/kg/day). The severity of epilation and desquamation in the field of the radiation port was scored weekly. Two months after irradiation the animals were killed, and pulmonary endothelial function was monitored by the activity of lung angiotensin converting enzyme (ACE) and plasminogen activator (PLA), and by production of prostacyclin (PGI2) and thromboxane (TXA2). The amount of hydroxyproline (HP) in the lung served as an index of pulmonary fibrosis. Radiation produced a dose-dependent decrease in ACE and PLA activity in the right lung and an increase in the production of PGI2 and TXA2. This endothelial dysfunction was accompanied by an increase in wet weight and in protein and HP content in the irradiated lung. Pentoxifylline spared only the increase in lung wet weight and protein content, and actually elevated the radiation-induced hyperproduction of PGI2 and TXA2. The severity of the epilation and desquamation reactions increased with increasing radiation dose and time but was independent of diet. These data indicate that pentoxifylline, despite some promising pharmacological actions, has no beneficial effect on acute radiation reactions in rat lung and skin.
Subject(s)
Lung/radiation effects , Pentoxifylline/therapeutic use , Radiation Injuries, Experimental/prevention & control , Skin/radiation effects , Administration, Oral , Animals , Lung/drug effects , Male , Pentoxifylline/administration & dosage , Rats , Rats, Inbred Strains , Skin/drug effectsSubject(s)
Bucladesine/pharmacology , Endothelium, Vascular/radiation effects , Animals , Aorta , Cattle , Dose-Response Relationship, Radiation , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , In Vitro Techniques , L-Lactate Dehydrogenase/metabolism , Plasminogen Activators/metabolism , Radiation Tolerance/drug effectsABSTRACT
May-Hegglin anomaly (MHA) is a rare hereditary condition that is characterized by cytoplasmic inclusions in leukocytes and giant platelets. Many patients have some degree of thrombocytopenia. Most individuals with MHA are asymptomatic, but 25-43% of patients previously reported have had a hemorrhagic tendency. The authors describe a patient with MHA who had no history of hemorrhage but who developed complete coronary thrombosis after attempted angioplasty despite an apparent platelet count of 24,000 per mm3. Laboratory investigations revealed a normal bleeding time, normal platelet aggregation, and an increase in the size of approximately two-thirds of the platelets. The calculated platelet mass was near normal, which probably explains the thrombosis despite a decrease in platelet numbers. The authors conclude that in some patients with MHA platelets are functionally active both in vivo and in vitro.
Subject(s)
Blood Platelets/pathology , Coronary Thrombosis/etiology , Hematologic Diseases/complications , Inclusion Bodies/ultrastructure , Neutrophils/pathology , Blood Platelets/physiology , Blood Volume/physiology , Coronary Thrombosis/pathology , Coronary Thrombosis/physiopathology , Female , Hematologic Diseases/pathology , Hematologic Diseases/physiopathology , Humans , Microscopy, Electron , Middle Aged , Neutrophils/ultrastructure , Platelet Aggregation/physiology , Platelet CountABSTRACT
Tumor plasminogen activator (PA) has been alleged to play a role in the growth and metastasis of tumors. Before such a role can be realized, PA first must be released from tumor cells. Having determined intra- and extracellular PA and PA-inhibitor activities in an experimental pancreatic ascites tumor grown in hamsters, the release of PA from these cells was investigated. No PA activity was detected in the suspension medium of freshly isolated tumor cells; inclusion of plasminogen, fibrinogen, or collagen in the medium yielded similar negative results. On the other hand, PA activity was demonstrated to be released in a time-dependent manner from these tumor cells embedded in fibrin clots. Plasminogen activator activity also was not found in the suspension medium of frozen-thawed tumor cells, despite the fact that most of them had breaks on their cell membrane. Unlike freshly isolated tumor cells, PA was not released from frozen-thawed cells embedded in fibrin clots. Full PA activity was demonstrated in frozen-thawed cells treated with Triton X-100, however. Frozen-thawed cells exhibited signs of severe damage, and more than 80% of them failed to exclude trypan blue. Obviously PA is released from viable tumor cells embedded in fibrin clots but not suspended in artificial medium. The PA-release mechanism, not PA itself, is destroyed in cells rendered nonviable by freeze thawing.