Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis.

By differential screening of tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS)- activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a member of the inhibitor of apoptosis (iap) gene family. iap genes function to protect cells from undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap1, hiap2, and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory cytokines TNF-alpha, interleukin 1beta, and LPS, reagents that lead to activation of the nuclear transcription factor kappaB (NF-kappaB). Indeed, overexpression of IkappaBalpha, an inhibitor of NF-kappaB, suppresses the induced expression of iap genes and sensitizes ECs to TNF-alpha-induced apoptosis. Ectopic expression of one member of the human iap genes, human X-chromosome-linked iap (xiap), using recombinant adenovirus overrules the IkappaBalpha effect and protects ECs from TNF-alpha- induced apoptosis. We conclude that xiap represents one of the NF-kappaB-regulated genes that counteracts the apoptotic signals caused by TNF-alpha and thereby prevents ECs from undergoing apoptosis during inflammation.

Hypercoagulability and high lipoprotein(a) levels in patients with type II diabetes mellitus.

Diabetes mellitus is associated with disturbances in hemostasis that could contribute to the development of diabetic vascular disease. We investigated the changes in parameters of blood coagulation and the fibrinolytic system and in plasma levels of lipoprotein(a)(Lp(a)) in 124 patients with type II diabetes mellitus and 44 healthy control subjects matched for age and body mass index (BMI) to determine whether hemostatic disturbances may lead to increased cardiovascular mortality. Median levels of fibrinogen (P < 0.0001), thrombin-antithrombin III complex (TAT) (P < 0.005), and plasminogen activator inhibitor-1 (PAI-1) activity (P < 0.05) in plasma were significantly elevated in diabetic patients compared with controls. The median concentration of Lp(a) was significantly higher in diabetic patients than in normal controls (18.2 vs. 12.6 mg/dl. P < 0.0005). Lp(a) levels tended to be elevated in patients with a prolonged history of diabetes. There was no evidence that Lp(a) levels were affected by metabolic control or by type of treatment. Twenty-two diabetics with coronary heart disease (CHD) had significantly higher levels of fibrinogen (P < 0.05), TAT (P < 0.05), and Lp(a) (24.7 vs. 13.7 mg/dl, P < 0.01) than the 51 patients without diabetic angiopathy. Our data indicate that impaired hemostatic balance in diabetes may cause hypercoagulability and may thus contribute to the increased cardiovascular mortality in diabetes.

Effects of DX-9065a, an orally active, newly synthesized and specific inhibitor of factor Xa, against experimental disseminated intravascular coagulation in rats.

We investigated the protective effects of DX-9065a, an orally active, newly synthesized and specific inhibitor of factor Xa, against two kinds of experimental disseminated intravascular coagulation (DIC) in rats. Endotoxin-induced experimental DIC was induced by a 4-h sustained infusion of endotoxin at a dose of 100 mg/kg. Thromboplastin-induced experimental DIC was induced by a bolus injection of thromboplastin at a dose of 150 mg/kg. The rats were orally administered DX-9065a at 10, 30 or 100 mg/kg 30 min before endotoxin or thromboplastin injection. In both DIC models, DX-9065a showed a protective effect against DIC, at all doses and in all parameters, including fibrin/fibrinogen degradation products (FDP), fibrinogen level, prothrombin time, activated partial thromboplastin time, platelet count and the number of renal glomeruli with fibrin thrombi. When DX-9065a was orally administrated at 100 mg/kg without endotoxin or thromboplastin, no significant changes were seen in hemostatic parameters except PT and APTT, and no fibrin thrombi or abnormal bleeding were seen in renal specimens. These findings suggest that the new oral anti-Xa drug, DX-9065a, has an effect in reducing the severity of DIC. However, further dose-finding and safety studies of this drug have still to be assessed.

Plasma levels of lipoprotein(a) are elevated in patients with the antiphospholipid antibody syndrome.

The mechanisms underlying clinical abnormalities associated with the antiphospholipid antibody syndrome (APAS) have not been elucidated. We measured plasma levels of lipoprotein(a) [Lp(a)], the active form of plasminogen activator inhibitor (active PAI), thrombin-antithrombin III complex (TAT) and soluble thrombomodulin (TM), to investigate the relationship of these factors to thrombotic events in APAS. Mean plasma levels of Lp(a), TAT, active PAI and TM were all significantly higher in patients with aPL than in a control group of subjects. Plasma levels of Lp(a) and active PAI were significantly higher in patients with aPL and arterial thromboses than in patients with aPL but only venous thromboses. There was a significant correlation between plasma levels of Lp(a) and active PAI in patients with aPL. These findings suggest that patients with aPL are in hypercoagulable state. High levels of Lp(a) in plasma may impair the fibrinolytic system resulting in thromboses, especially in the arterial system.

Effects of aerosol administration of a thromboxane synthetase inhibitor (OKY-046) on bronchial responsiveness to acetylcholine in asthmatic subjects.

Bronchial hyperresponsiveness is one of the major clinical features of bronchial asthma. We previously reported that oral administration of a selective thromboxane synthetase inhibitor, OKY-046, reduced bronchial hyperresponsiveness to acetylcholine in asthmatic subjects. In this study, the effect of aerosol administration of OKY-046 on bronchial hyperresponsiveness was evaluated in ten inpatients with intrinsic asthma. Acetylcholine inhalation tests were performed before and after four days of inhalation of OKY-046 (100 mg/day). The provocative concentration of acetylcholine producing a 20 percent fall in forced expiratory volume in 1 s (PC20-FEV1) and that causing a 35 percent fall in respiratory conductance (PC35-Grs) were measured as indexes of bronchial responsiveness. There was a significant increase in PC20-FEV1 (p less than 0.001) and PC35-Grs (p less than 0.02) after inhalation of OKY-046 from 0.79 (GSEM, 1.41) Mg/ml and 0.96 (GSEM, 1.35) mg/ml to 1.20 (GSEM, 1.41) mg/ml and 1.74 (GSEM, 1.32) mg/ml, respectively. There was no significant difference in forced vital capacity (FVC), FEV1, or respiratory resistance (Rrs) baseline values before and after inhalation of OKY-046. Platelet aggregation was not inhibited by the treatment in other five inpatients. Thus, prophylactic administration of aerosol OKY-046 may be available for treatment of asthma by reduction of bronchial hyperresponsiveness. Further studies are needed to determine the optimum dose.

Transient wheeze. Eosinophilic bronchobronchiolitis in acute eosinophilic pneumonia.

The clinicopathologic features of five patients with acute eosinophilic pneumonia who presented with transient wheeze as well as acute onset of high fever, severe hypoxemia, and diffuse pulmonary infiltrates are described. Eosinophilic pneumonia was diagnosed by bronchoalveolar lavage and transbronchial lung biopsy. The illness resolved rapidly with or without corticosteroid therapy. No relapse occurred. To characterize the transient wheeze, a transbronchoscopic bronchial biopsy and pulmonary function tests were performed. Specimens of bronchial wall revealed eosinophil infiltration into the bronchial mucosa. Pulmonary function tests demonstrated reduced diffusing capacity and small airway dysfunction. These findings suggested that eosinophil infiltration into the bronchial mucosa might temporarily cause transient wheeze, different from bronchial asthma, due to small airway dysfunction in acute eosinophilic pneumonia.

Quantitative estimation of elastase-alpha 1-proteinase inhibitor (E-alpha 1 PI) complex in leukemia: marked elevation in cases of acute promyelocytic leukemia.

The concentrations of elastase-alpha 1-proteinase inhibitor (E-alpha 1 PI) complex were assayed in 43 patients with various types of leukemia. Marked to moderate elevation of E-alpha 1 PI complex levels was observed in patients with acute myelocytic leukemia (AML), acute promyelocytic leukemia (APL), acute myelomonocytic leukemia (AMMoL), or chronic myelocytic leukemia (CML) at diagnosis. The ratio of E-alpha 1 PI complex concentrations in plasma to leukocyte counts markedly elevated in the patients with APL, especially. During the course of remission induction therapy, levels of E-alpha 1 PI complex decreased in parallel with decline of leukocyte counts in the patients with leukemia other than APL, however the E-alpha 1 PI complex was persistently elevated regardless of leukopenia in some patients with APL. In APL, concentrations of fibrin/fibrinogen degradation products (FDP) markedly increased even when levels of plasmin-alpha 2-antiplasmin complex were within normal limits. However, levels of E-alpha 1 PI complex usually increased in these cases. From these results, it is strongly suggested that promyelocytes contain markedly elevated amounts of elastase which participates in degradation of fibrin or fibrinogen in some cases of APL.

Fibrinolytic proteins in apoptotic human umbilical vein endothelial cells.

Endothelial cells express fibrinolytic proteins including: urokinase (u-PA) and tissue type (t-PA) plasminogen activators, type-1 (PAI-1) and 2 (PAI-2) plasminogen activator inhibitors, and u-PA receptor (u-PAR). Apoptotic endothelial cells detach, potentially forming both local and circulating microthrombi in vivo. In this article, apoptotic human umbilical vein endothelium was obtained by serum starvation and compared with nonapoptotic cells rescued from death with fresh medium containing serum. Antigen levels for t-PA, PAI-1, PAI-2, and u-PAR were reduced greatly in apoptosis (p< 0.05). In contrast, u-PA levels were similar in apoptotic as compared with rescued cells (p<0.05). Radioactive amino acids were used to determine absolute levels of protein synthesis and degradation in these cells. Reduced antigen levels likely were due to proteolysis as there was 98% total protein degradation and very little protein synthesis in apoptotic endothelial cells. Also, u-PA levels in apoptotic endothelial cells were not affected by the protein synthesis inhibitor cycloheximide. Endothelial cells in inflammatory sites are exposed to cytokines, which increase both apoptosis and u-PA levels. Data from this article support the idea that maintained u-PA levels in apoptotic endothelium may protect from micro-thrombosis in inflammatory sites as well as in the circulation.

Prothrombin Himi; an abnormal prothrombin characterized by a defective thrombin activity.

An abnormal prothrombin has been detected in a 26-year-old female, who had no history of excessive bleeding. Prothrombin activity was approximately 10% when measured using either the classical one-stage assay or the assay with Echis carinatus venom, whereas prothrombin antigen level was normal. In keeping with current nomenclature practices, the abnormal prothrombin was designated "Prothrombin Himi". The electrophoretic behavior and calcium binding properties of Prothrombin Himi did not differ significantly from normal. Prothrombin Himi was isolated by chromatography on Q-Sepharose. Electrophoretic migration of the purified abnormal prothrombin on SDS-PAGE was normal. Upon prothrombin activation by Echis carinatus venom, the clotting activity produced from Prothrombin Himi was only 37% of the normal level after 90 minutes of the activation time, where as the amidolytic activity was almost the same as normal. The cleavage patterns of Prothrombin Himi by factor Xa or Echis carinatus venom investigated by SDS-PAGE, were found to be normal. These results indicate that Prothrombin Himi was characterized by a defective thrombin enzymatic activity.

Prothrombin fragment 1 + 2 measures treatment effect in patients with antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is characterized by recurrent thrombosis. The anticoagulant management of APS thrombosis remains controversial. Few reports on markers of in vivo activation of coagulation have been reported. To determine whether plasma levels of prothrombin fragment 1 + 2 (F1 +2) correlate with thrombotic risk and treatment effect in patients with APS, plasma F1 + 2 levels were followed in 57 patients with this syndrome for more than 2 years. Clinical findings were also observed in these patients. Plasma levels of F1 + 2 in patients with APS were significantly higher when compared with control subjects (p<.05). These results suggest patients with APS are in a hypercoagulable state. Plasma levels of F1 + 2 significantly decreased following treatment with either aspirin, or aspirin plus warfarin (p<.05 and p<.01, respectively). Recurrent thromboses or spontaneous abortions occurred in all eight patients whose plasma levels of F1 + 2 remained higher than 1 nmol/l after treatment with either aspirin alone or no anticoagulants. These patients were subsequently treated with warfarin as well as aspirin, and plasma levels of F1 + 2 decreased to less than 1 nmol/l, with no additional thrombotic events over the remainder of the 2-year follow-up. No fatal bleeding was observed in treated patients. Our results suggest plasma levels of F1 + 2 are useful indicators of successful treatment. It is also suggested that warfarin plus mini-dose aspirin therapy is effective for patients with APS to protect from recurrent thromboses without harmful side effects. Further, prospective cohort studies are needed to substantiate these associations.

Role of tissue factor in disseminated intravascular coagulation.

We examined plasma antigen levels of tissue factor (TF) in 95 cases of disseminated intravascular coagulation (DIC), to investigate the role of TF in DIC. A significant elevation of plasma antigen levels of TF was observed in cases of DIC associated with cancer. However, no such significant elevation was observed in cases of DIC associated with acute promyelocytic leukemia (APL), acute leukemia except APL, blastic crisis of chronic myelogenous leukemia, non-Hodgkin lymphoma (NHL), sepsis or fulminant hepatitis. No significant elevation of TF was observed in patients without DIC, except 4 cases of cancer who developed DIC thereafter. Plasma antigen levels of TF were higher in both cases of DIC with renal failure and chronic renal failure without DIC than its levels in those without renal failure. Therefore, plasma antigen levels of TF in DIC patients with renal failure were considered to be carefully estimated. The levels of TF were decreased with the clinical improvement in some cases of DIC but were further increased or remained at high levels in patients who showed no improvement of DIC. Thus, plasma antigen levels of TF is an important marker to predict the development and/or prognosis of DIC, especially in patients with cancer.

Fibrinogen Kanazawa: a congenital dysfibrinogenaemia with delayed polymerization having a replacement of proline-18 by leucine in the A alpha-chain.

Congenital dysfibrinogenaemia was found in a 39-year-old female and her two children. The proposita, apparently heterozygous for this abnormality, had no episode of bleeding or thrombosis. The abnormal fibrinogen showed normal release of fibrinopeptides A and B but impaired polymerization of the fibrin monomer. Amino acid sequence analysis of the whole A alpha-chain isolated from fibrinogen Kanazawa showed a substitution of Leu for Pro at position 18 in the A alpha-chain. This substitution was corroborated by the analysis of the amino acid sequence which demonstrated the lysyl endopeptidase peptides derived from the A alpha-chain of fibrinogen Kanazawa. The minimal genetic exchange responsible for this substitution was a C----T transition in the middle position of the Pro codon. We conclude that Pro-18 in the A alpha-chain is crucial for the polymerization of the fibrin monomer.

The course of disseminated intravascular coagulation is predicted by changes in thrombin-antithrombin III complex levels--is there any difference between treatment with standard heparin or low-molecular-weight heparin?

Changes in thrombin-antithrombin III complex (TAT) over a one week period studied in 42 cases of disseminated intravascular coagulation (DIC); 19 treated with standard (or unfractionated) heparin (UFH) and 23 treated with low-molecular-weight heparin (LMWH). Closer examination of short term changes in TAT (determined 2, 6, 12, 24, 48, and 72 h after starting anticoagulant therapy) was performed in ten cases of DIC; six treated with UFH and four treated with LMWH. In twelve of the 19 cases of DIC treated with UFH and 19 of the 23 cases treated with LMWH, plasma levels of TAT decreased one day after starting anticoagulant therapy, and no exacerbation of DIC was observed for the following week. In the other cases, these levels further increased and most patients had persistently high levels of TAT for the next week. Plasma levels of TAT were significantly lower in patients treated with LMWH than in those treated with UFH, which may suggest that LMWH is more beneficial in DIC. A transient increase in plasma levels of TAT was observed 6 h after the start of anticoagulant therapy in two of the six cases treated with UFH and one of the four cases treated with LMWH. From these results we conclude that fluctuation of TAT was not influenced by the type of heparin (UFH or LMWH), and that the course of DIC for the following week can be predicted by the changes in plasma TAT levels one day after starting anticoagulant therapy.

Study of the balance between coagulation and fibrinolysis in disseminated intravascular coagulation using molecular markers.

Plasma levels of thrombin-antithrombin III complex (TAT), plasmin-alpha 2-plasmin inhibitor complex (PIC) and active plasminogen activator inhibitor (PAI) were assayed in 66 cases of disseminated intravascular coagulation (DIC). Significant elevation of both TAT and PIC was observed in all cases of DIC. Most elevated levels of TAT were seen in DIC with acute promyelocytic leukaemia (APL) and sepsis. The highest levels of PIC were seen in DIC with APL but were much lower in sepsis. A significant elevation in active PAI was observed in DIC due to acute leukaemia (apart from APL), chronic myeloid leukaemia and sepsis, but not in APL, non-Hodgkin lymphoma and cancer. Active PAI was higher in patients with multiple organ failure (MOF) than in those without MOF while PIC was lower in patients with this complication. Thus, the balance of coagulation and fibrinolysis varied according to the underlying cause of DIC; APL had more dominant activation of fibrinolysis, while sepsis had greater activation of coagulation. It is suggested that the inhibition of secondary fibrinolytic activation plays an important role in the progression of MOF by the disturbance of the microcirculation.

Prothrombin fragment F1 + 2 and thrombin-antithrombin III complex are useful markers of the hypercoagulable state in atrial fibrillation.

It is well known that atrial fibrillation (AF) is one of the most important diseases that predispose patients to thrombosis. We have attempted to identify patients with AF in the hypercoagulable state by measuring molecular markers such as thrombin-antithrombin III complex (TAT) and prothrombin fragment 1 + 2 (PTF) and determining the effect of antithrombotic therapy on these markers; 83 patients with AF were studied. Increased levels of plasma TAT and PTF were more frequently observed in patients with AF and associated mitral stenosis than in patients with AF alone. In cases of AF without mitral stenosis, plasma levels of TAT and PTF were significantly lower in those patients receiving antithrombotic agents (aspirin or warfarin) than in those receiving no antithrombotic agents. Furthermore, plasma levels of PTF were significantly lower in patients given warfarin than in those receiving aspirin. These results suggest that (1) patients with AF and mitral stenosis who are not given warfarin are in an extremely hypercoagulable state and (2) some patients with AF without mitral stenosis who are not given antithrombotic agents are also moderately hypercoagulable. In vivo activation of blood coagulation was more effectively controlled in patients receiving warfarin than in those taking aspirin.

Successful treatment of a patient with idiopathic factor VIII inhibitor with double filtration plasmapheresis and steroid administration.

We report a 74-year-old Japanese woman who had bleeding due to a factor VIII inhibitor in the absence of diseases known to be associated with its development. She abruptly developed a large painful purpura extending from the right hip to the thigh followed by an intramuscular haematoma of her left anterior chest. Examinations revealed a marked depression of factor VIII activity (5%) and the presence of 31 Bethesda units/ml factor VIII inhibiting activity. The phenotypes of these inhibitors were of the IgG-kappa and IgG-lambda types. She was treated with oral prednisolone and double filtration plasmapheresis (DFPP). The inhibitors rapidly disappeared after three sessions of plasmapheresis and her plasma factor VIII activity increased to a normal level. During this treatment, no major adverse effects such as thrombosis and infection were observed and transfusion of fresh frozen plasma (FFP) was not necessary. Heterogeneity of idiopathic factor VIII inhibitors in elderly patients is common and spontaneous disappearance or elimination of such inhibitors by treatment is often difficult to achieve. However, the combination of oral prednisolone and double filtration plasmapheresis is effective and safe for idiopathic factor VIII inhibitors and is a worthwhile approach to treatment.

Relationship between elevation in the plasma concentration of elastase-alpha 1 proteinase inhibitor complex (E-alpha 1 PI) and haemostatic parameters during haemodialysis.

To investigate the relationship between changes in plasma concentrations of polymorphonuclear elastase (PMN-E) and haemostatic effects during haemodialysis (HD), changes in the plasma concentrations of elastase-alpha 1 proteinase inhibitor complex (E-alpha 1 PI) and fibrinogen (Fbg), cross-linked fibrin degradation products (XDP), thrombin-antithrombin III complex (TAT), plasmin-alpha 2 plasmin inhibitor complex (PIC) and soluble thrombomodulin (TM) in 49 patients with end-stage chronic glomerulonephritis maintained on chronic HD were measured. Plasma concentrations of TAT, PIC, TM and E-alpha 1 PI significantly increased during a single HD. There was a statistically significant correlation between change in plasma E-alpha 1 PI concentration and changes in plasma concentrations of TAT, PIC and TM during a single HD, as well as between changes in plasma concentrations of TM and TAT during a single HD. These observations suggested that activation of coagulation and fibrinolysis, endothelial cell damage, and activation of polymorphonuclear cells occur during HD. Activation of polymorphonuclear cells may induce activation of coagulation and fibrinolysis, leading to endothelial cell damage, augmented by release of proteases such as elastase.

Recombinant hirudin for the treatment of disseminated intravascular coagulation in patients with haematological malignancy.

The purpose of this pilot study was to determine the effect of recombinant hirudin (r-hirudin) on coagulopathy and the relationship between concentrations of thrombin-antithrombin III (ATIII) complex (TAT) and thrombin-hirudin complex (THC) in patients with disseminated intravascular coagulation (DIC). Five patients with haematological malignancy associated with DIC were studied. r-Hirudin was administered by continuous intravenous infusion at a dose of 0.005 mg/kg/h for 4-9 days to each patient. Fibrin/fibrinogen degradation products (FDP), D-dimer, TAT and plasmin-alpha 2 antiplasmin complex (PAP) concentrations decreased after treatment with r-hirudin in four patients studied. However, in one patient, serum creatinine increased to 1.7 mg/dl and aPTT was prolonged to 74.4s. Statistical analysis disclosed significant positive correlations between plasma concentrations of hirudin and THC, and between concentrations of THC and TAT. The concentrations of THC were much higher than those of TAT. In conclusion, these findings indicate that r-hirudin more strongly inhibited thrombin than did ATIII without heparin, and that administration of r-hirudin to renal insufficiency required individual adjustment of dosage. The present findings also suggest that r-hirudin can be considered a new agent for the treatment of DIC.

Hypercoagulability and high lipoprotein(a) levels in patients with aplastic anemia receiving cyclosporine.

There is some clinical evidence that cyclosporine A (CyA) is associated with thrombotic complications of bone marrow and renal transplantation. We investigated plasma concentrations of lipoprotein(a) [Lp(a)], a potentially atherothrombotic lipoprotein, and hemostatic and vascular status in ten patients with aplastic anemia receiving CyA, eleven patients not taking it, and 38 age-matched healthy controls. Patients receiving CyA had significantly higher concentrations of plasma fibrinogen (P < 0.05), prothrombin fragment 1 + 2 (F1 + 2; P < 0.05), plasminogen activator inhibitor-1 (PAI-1; P < 0.05), and von Willebrand factor antigen (P < 0.05) than did patients not taking CyA. Plasma concentrations of Lp(a) were higher in CyA-treated patients than those not receiving it (P < 0.05) or healthy controls (P < 0.05). The difference in the Lp(a) concentration between controls and patients who did not receive CyA-treatment was not significant. Our results suggest that hypercoagulability is likely to occur during CyA therapy. Further, the presence of high concentrations of Lp(a) may accelerate the process of atherosclerosis and increase thrombotic events in patients receiving long-term CyA.

[Plasma exchange failures in two patients with thrombotic thrombocytopenic purpura].

We treated two patients with thrombotic thrombocytopenic purpura (TTP) with multiple plasma exchanges for a short period. Following 6 plasma exchanges during 11 days in patient 1 and the 8 exchanges during 13 days in patient 2, consciousness disturbance and thrombocytopenia improved with a decrease in serum lactate dehydrogenase (LDH) levels in both patients. However, thrombocytopenia rapidly progressed with a rise of serum LDH levels after a few days from cessation of the treatment. They received a few rounds of plasma exchange without beneficial effects and eventually died of massive hemorrhage. The clinical course of these patients indicates that some patients with TTP are refractory to repeated plasma exchanges for a short period. It seems important to determine the target point in blood chemistry values such as the LDH level for plasma exchange at which the treatment could be discontinued without causing relapse of TTP.