The production of coagulation factor VII by adipocytes is enhanced by tumor necrosis factor-α or isoproterenol.

BACKGROUND: A relationship has been reported between blood concentrations of coagulation factor VII (FVII) and obesity. In addition to its role in coagulation, FVII has been shown to inhibit insulin signals in adipocytes. However, the production of FVII by adipocytes remains unclear. OBJECTIVE: We herein investigated the production and secretion of FVII by adipocytes, especially in relation to obesity-related conditions including adipose inflammation and sympathetic nerve activation. METHODS: C57Bl/6J mice were fed a low- or high-fat diet and the expression of FVII messenger RNA (mRNA) was then examined in adipose tissue. 3T3-L1 cells were used as an adipocyte model for in vitro experiments in which these cells were treated with tumor necrosis factor-α (TNF-α) or isoproterenol. The expression and secretion of FVII were assessed by quantitative real-time PCR, Western blotting and enzyme-linked immunosorbent assays. RESULTS: The expression of FVII mRNA in the adipose tissue of mice fed with high-fat diet was significantly higher than that in mice fed with low-fat diet. Expression of the FVII gene and protein was induced during adipogenesis and maintained in mature adipocytes. The expression and secretion of FVII mRNA were increased in the culture medium of 3T3-L1 adipocytes treated with TNF-α, and these effects were blocked when these cells were exposed to inhibitors of mitogen-activated kinases or NF-κB activation. The ß-adrenoceptor agonist isoproterenol stimulated the secretion of FVII from mature adipocytes via the cyclic AMP/protein kinase A pathway. Blockade of secreted FVII with the anti-FVII antibody did not affect the phosphorylation of Akt in the isoproterenol-stimulated adipocytes. CONCLUSION: Obese adipose tissue produced FVII. The production and secretion of FVII by adipocytes was enhanced by TNF-α or isoproterenol via different mechanisms. These results indicate that FVII is an adipokine that plays an important role in the pathogenesis of obesity.

Synthetic selective inhibitors of coagulation factor Xa strongly inhibit thrombin generation without affecting initial thrombin forming time necessary for platelet activation in hemostasis.

DX-9065a and JTV-803, synthetic selective inhibitors of activated factor X (FXa), have recently been demonstrated as strongly effective antithrombotic agents in animal thrombosis models, yet with a low risk of bleeding. The aim of the present study was to elucidate these characteristics. Using a chromogenic assay with purified coagulation factors, 73.9% of thrombin generation was suppressed by the addition of DX-9065a (0.20 microm) and 75.7% by JTV-803 (0.18 microm). Inhibition by argatroban (0.19 microm) was less (36.0%) and initial thrombin forming time (T50), the time required to generate 50% thrombin activity in vitro, which is considered important for platelet aggregation in hemostasis, was significantly prolonged by argatroban. In contrast, DX-9065a and JTV-803 had no apparent influence on T50, suggesting that initial thrombin was formed immediately, as in the control. We also investigated platelet aggregation in defibrinated plasma induced by tissue factor, to clarify whether initial thrombin contributes to hemostasis. Aggregation was not affected by the addition of either FXa inhibitor, whereas it was significantly reduced by argatroban. Our results suggest that initial thrombin, which is formed despite the presence of a FXa inhibitor, can activate platelets. We concluded that DX-9065a and JTV-803 are able to inhibit thrombin generation significantly without affecting the formation of initial thrombin for platelet activation, which may contribute to hemostasis through the preservation of normal bleeding time.

Role of physiologic concentrations of stem cell factor in leukemic type growth of myelodysplastic CD34+ cells.

The stem cell factor (SCF: a ligand for c-kit) plays a central role in the growth of myelodysplastic (MDS) progenitor cells with leukemic type growth. In this study, the role of physiologic concentrations of SCF on the proliferation and differentiation on MDS progenitor cells was further analyzed in the presence of combined cytokines. For this purpose, marrow CD34+ cells were purified up to 94% for 12 normal individuals and 90% for 18 MDS patients, using monoclonal antibodies and immunomagnetic microspheres. The purified CD34+ cells were cultured for 14 days with saturating doses of cytokines, including recombinant human macrophage colony stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte/macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3) and rSCF. The clonal growth of MDS CD34+ cells supported by a combination of all the above cytokines was then subdivided into the two patterns of leukemic or non-leukemic. The role of various concentrations of rSCF (0, 0.5, 5, 50 and 500 ng ml(-1)), with or without the above cytokines, in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. The physiologic concentration of SCF at 5 ng ml(-1) significantly increased undifferentiated 'blast cell' colonies or clusters in leukemic type growth of MDS CD34+ cells over that seen in normal CD34+ cells. SCF is present in plasma at a level of ng ml(-1). This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blasts.

Subclinical alterations in coagulation and fibrinolysis in patients undergoing autologous peripheral blood stem cell transplantation.

We monitored 30 laboratory hemostatic parameters in an attempt to better comprehend alterations in coagulation and fibrinolysis in 10 patients with hematological malignancies subjected to autologous peripheral blood stem cell transplantation (APBSCT). These parameters were assessed before and just after high-dose conditioning chemotherapy, on days 1, 7, 14 and 28. Although, clinical manifestations associated with fibrino-coagulation disorders never occurred, including veno-occlusive disease, a statistically significant increase was seen in 7 of 30 parameters, compared to values seen before conditioning chemotherapy. These were subdivided into early and late phase parameters. The early phase parameters, which increased during the first day after the conditioning chemotherapy was given, then returned to baseline values, included protein C, plasma tissue factor and tissue-plasminogen activator. The late phase parameters, which increased over baseline values during days 7 to 28, included free-protein S, fibrinogen, plasmin-alpha2-plasmin inhibitor complex and soluble-thrombomodulin. The increase of early phase parameters, as produced by the liver and by endothelial cells, may reflect tissue damage by conditioning chemotherapy. Late phase parameters increased in parallel with C-reactive protein, which suggests a correlation with the degree of inflammation, such as the presence of infective disease during neutropenia. These subclinical alterations in coagulation and fibrinolysis which take on a biphasic pattern during the course of APBSCT should be kept in mind by the attending physicians during therapy.

In vivo kinetics of 99mTc labeled recombinant tissue plasminogen activator in rabbits.

Our previous studies demonstrated that 99mTc labeled recombinant tissue plasminogen activator (rt-PA) retained high affinity with fibrin in vitro but showed unexpectedly low uptake in fresh thrombi in vivo. The present study was performed to determine the in vivo kinetics of radiolabeled t-PA in the rabbit. Sequential images and blood samples after the intravenous administration of 99mTc labeled rt-PA in thrombus-bearing rabbits were taken. The radioactivity and immunological level of t-PA and PAI-1 in the solution eluted to each fraction by gel permeation chromatography were measured by means of a well scintillation counter and enzyme-linked immunosorbent assay (ELISA). Most of the radioactivity was eluted in the fraction (Fr. 7) of larger molecular weight than that (Fr. 9) of intact t-PA. The level of intact rt-PA was increased with a regimen involving the preadministration of cold rt-PA which was followed by the administration of hot rt-PA. The level of PAI-1 in plasma showed an increased rebound 15 minutes after the intravenous injection. These results suggest two possible reasons why rt-PA retains high affinity with fibrin in vitro, once radiolabeled, but was ineffective in delineating fresh thrombi with a gamma camera: 1) some plasma components such as PAI-1 combine with circulating radiolabeled rt-PA and form a larger molecule immediately and/or 2) radiolabeled rt-PA is modulated as a consequence of the radiolabeling and forms a larger molecule than intact rt-PA.

Tc-99m labeled tissue-type plasminogen activator: preparation, stability and preliminary imaging of thrombus-bearing rats.

Tissue-type plasminogen activator (t-PA) is a thrombolytic agent that directly binds to fibrin formed in clots. In terms of radiolabeling and nuclear imaging, t-PA has several advantages in Tc-99m labeling: it is stable in acidic solution at pH 3, which is suitable for labeling Tc-99m by a method of stannous reduction and blood disappearance after administration is rapid, which is desirable for imaging targets using short-lived radionuclides. Recombinant t-PA was labeled with Tc-99m by a method of stannous reduction without significant degradation of biochemical activity, over 95% of which was retained after the labeling procedure. Labeling efficiency in paper chromatography was over 98%. The moiety of hydrolyzed Tc-99m that was not eluted through the Sephadex column was estimated to be less than 10%. Tc-99m labeled t-PA, however, appeared to become unstable when diluted with normal saline. Nevertheless, in in vitro fibrin binding, Tc-99m labeled t-PA showed high affinity with fibrin: 80% of 100 ng/ml of Tc-99m t-PA bound to 10(-5) mol of the fibrinogen. Preliminary animal studies also showed a concentration of Tc-99m labeled t-PA at fresh thrombi formed in the inferior vena cava. Tc-99m labeled t-PA appears to have potential for thrombus imaging and the preparation of an instant kit.

[Antiphospholipid antibodies and thrombosis: the putative mechanisms of hypercoagulable state in patients with anticardiolipin antibody].

Antiphospholipid antibodies are well recognized as associated with serious clinical complications such as arterial and venous thrombosis and recurrent spontaneous abortion. These complications are collectively called antiphospholipid syndrome(APS). The mechanisms responsible for the thrombosis are unclear. We reported three mechanisms. beta 2-glycoprotein I(beta 2GPI) inhibited activated protein C(APC) activity and, furthermore, APC activity decreased by the addition of monoclonal aCL and beta 2GPI. Monoclonal anticardiolipin antibodies(aCL) seemed to enhance the inhibition of APC procoagulant activity caused by beta 2GPI. Monoclonal aCL in the presence of beta 2GPI also increased the activity of plasminogen activator inhibitor(PAI)-1 in the mixture of tissue-plasminogen activator(t-PA) and PAI-1 by inhibiting the function of beta 2GPI, which increased the remaining t-PA activity in the mixture. The formation of thrombin-antithrombin complexes(TAT) in APS was impaired. The level of TAT in APS did not increase, however the level of prothrombin fragment 1 + 2 (F1 + 2) increased. Therefore, free thrombin present in patients' blood may contribute to thrombosis in APS. These reports indicate that thrombosis in APS may be caused by several thrombogenic factors that stimulate aCL.

[Evaluation of F1 + 2/TAT ratios in Japanese patients with antiphospholipid syndrome].

The clinical manifestations of the antiphospholipid syndrome(APS) include arterial and venous thrombosis and a fetal loss, but the pathogenic mechanisms remain unclear. To clarify the mechanism of thrombogenic state in APS, we investigated the markers for thrombosis including thrombin-antithrombin complex(TAT) in patients with antiphospholipid antibodies(aPL). Prothrombin fragment 1 + 2(F1 + 2) in patients with APS and in autoimmune disease patients with aPL increased significantly compared with those obtained in autoimmune disease patients without aPL or in control subjects. However, there was not a significant difference in the TAT level of each group, suggesting that the formation of TAT was impeded in APS. To investigate which aPL is responsible for the disturbance of the TAT formation, the ratio of F1 + 2/TAT was calculated. The ratio increased in patients with lupus anticoagulant, especially with prolonged kaolin clotting time, and furthermore the ratio strongly increased in patients with IgG type-anticardiolipin antibodies(aCL). Our results suggest that IgG-aCL is associated with thrombogenic state in APS because free thrombin is present in patients' blood by impeding the formation of TAT by mainly IgG-aCL.

[A study on antiprothrombin antibodies in antiphospholipid syndrome].

In this study we investigated the frequency and the type of manifestations of antiprothrombin antibodies (aFII) in patients with antiphospholipid syndrome (APS). In 16 (84.2%) of 19 patients with lupus anticoagulant (LA) and either anticardiolipin antibodies or antiphosphatidylserine antibodies, two types of abnormal patterns were shown on a crossed immuno-electrophoresis technique using anti-human prothrombin murine IgG. The slow-moving peak of prothrombin was detected in 8 patients, while a peak in the other patients had the slow-moving shoulder. These slow-moving materials might represent prothrombin-aFII complexes. In 13 patients who were studied on Western blots, IgGs of 11 patients (84. 6%) bound to human purified prothrombin, and the IgGs of 7 (53.8%) of these patients also bound to human purified alpha-thrombin. Our results indicate that aFII detected in patients with APS may explain part of the mechanism of LA.

[Gamma-heavy chain disease associated with MALT lymphoma of the duodenum].

We report a case of a 63-year-old woman with gamma heavy chain disease (HCD) associated with mucosa-associated lymphoid tissue (MALT) lymphoma of the duodenum. She was suffering from drug-resistant tonsillitis with high fever. Examination on admission showed leukocytopenia and thrombocytopenia. Bone marrow aspirate revealed granulocytosis and a hypocellular marrow with no increase in plasma cells or atypical lymphocytes. Serum electrophoresis disclosed, in addition to hypogamma-globulinemia, an abnormal band due to the presence of gamma HCD protein. This abnormal protein was a molecular weight of approximately 40 kd as determined by Western blots technique, and belonged to the IgG1 subclass as determined by ELISA with monoclonal antibodies against IgG. An endoscopic examination of the patient's duodenum found a small tumorous lesion, which was confirmed pathologically to be MALT lymphoma. HCD is known to be associated with lymphoproliferative diseases. In this case, gamma HCD had developed as a secondary complication of MALT lymphoma. gamma HCD associated with MALT lymphoma of the duodenum is rare in the literature.

[A latent form of essential thrombocythemia presented as portal hypertension and associated with acquired von Willebrand syndrome].

A 32 year-old male patient was admitted to our hospital because of abdominal tumor. The examination on admission showed massive splenomegaly and esophageal varices although peripheral blood cell counts were within normal limits. Exploratory laparotomy was performed with the diagnosis of portal hypertension and revealed the multiple thrombus formations in the splenic vein and the extramedullary hematopoietic findings in the spleen by the microscopic examination. In vitro colony forming assay showed the formation of spontaneous erythroid colonies in cultures of progenitor cells (from peripheral blood mononuclear cells) in erythropoietin-poor medium. Increasing thrombocytosis was observed immediately after splenectomy, and hemorrhagic diathesis of nasal bleeding and gastrointestinal bleeding were also detected. The analysis of plasma von Willebrand factor (vWF) revealed the decrease of ristocetin cofactor activity and the lack of large multimeric components of vWF. These abnormal findings observed after splenectomy led to recovery through the administration of busulfan with the improvement of thrombocytosis. Accordingly, the course of the disease clearly indicated it to be the essential thrombocythemia represented as portal vein thrombosis and in latent form with normal cell counts in peripheral blood at the time of diagnosis, and subsequently, to develop into a full-blown form associated with acquired von Willebrand syndrome following splenectomy.

[Human parvovirus-induced transient anemia and leukopenia after delivery].

A 30-year-old female at 27 weeks' gestation, was hospitalized on September 24 1990 because of the premature rupture of the amniotic sac. She underwent Caesarean section on the same day with 700 ml blood loss, but no blood transfusion was required. For several days after the operation, her hemoglobin level remained 7.8 g/dl and did not increase significantly in spite of parenteral iron therapy. On the 9th postoperative day, chills and pyrexia developed with leukopenia. Bone-marrow aspiration revealed severe erythroblastopenia with giant proerythroblasts, suggesting recent HPV infection, which was confirmed by the presence of anti-HPV IgM and HPV antigen by ELISA. The hemoglobin level gradually decreased to 6.0 g/dl by the 21st day, then began to increase rapidly. The serum of acute-phase containing HPV antigens inhibited BFU-E and CFU-E but not CFU-GM. The serum of convalescent-phase inhibited neither erythroid colony growth nor myeloid colony growth. These results indicate that the inhibitory effect of HPV in colony assay is highly specific for erythropoiesis and that HPV play a role in transient cessation of erythropoiesis. The reason, however, for leukopenia in HPV infection remained unclear. This case shows that HPV infection may induce severe hematological disorders even in normal person under erythropoietic stress.

[A case of relapsing polychondritis with IgA nephropathy].

We report a case of a 37-year-old man with relapsing polychondritis and IgA nephropathy. He visited our hospital with high fever and the swelling of his ears and eyelids. His symptoms and the results of the biopsy of his right auricle fulfilled the Damiani's criteria. Laboratory examination on admission showed an increase of serum IgA level, a presence of immune complex, remarkable hematuria (grade III) and proteinuria (grade II). Most of his symptoms were improved by the administration of antibiotic and NSAIDs, however, urinary findings still remained unchanged. The biopsy of his right kidney led to a diagnosis of IgA nephropathy (group II). Although relapsing polychondritis is known to associate rarely with renal involvement, it is very rare to associate with IgA nephropathy. This case indicates that immune disorders including IgA nephropathy should be investigated in patients with relapsing polychondritis.

[Analysis of plasminogen activator in human plasma using the method of fibrin/celite column chromatography--with special reference to the determination of fibrin adsorbable tissue-plasminogen activator by ELISA].

To clarify the clinical importance of tissue-plasminogen activator (t-PA) on the thrombosis, it is essential to be measured t-PA level in human plasma. Taking advantage of strong affinity of t-PA to fibrin, t-PA was isolated with Fibrin/Celite column chromatography. Immunological activities of t-PA of plasma samples determined by ELISA and the elution pattern of it on Fibrin/Celite column chromatography were studied. The results are as follows: The t-PA in human plasma was consisted of fibrin adsorbable t-PA and fibrin nonadsorbable t-PA. Both types of t-PA could be measured separately by using of Fibrin/Celite column chromatography. The fibrin adsorbable t-PA fraction showed two types of molecular size (major peak was Mr congruent to 70,000, minor peak was Mr congruent to 100,000) on Sephadex G-200 chromatography. The level of t-PA in plasma samples taken from 20 healthy subjects at rest was 2.4 +/- 1. 9 ng/ml, and concentration of t-PA increased to 10.4 +/- 3.2 ng/ml after venous stasis. But the t-PA level of plasma samples from patients with cerebral thrombosis did not increase after venous stasis. The changes of immunologically determined t-PA in plasma samples induced by venous stasis were not reflected in the fibrinolytic activities of the euglobulin fractions, but well reflected in the fibrinolytic activities of the elution fractions on Fibrin/Celite column chromatography. The level of fibrin nonadsorbable t-PA did not change by venous stasis, but the level of fibrin adsorbable t-PA increased from 5.1 +/- 1.9 ng/ml to 9.7 +/- 2.7 ng/ml in the case of 6 healthy subjects. It is concluded that, the plasma t-PA increased by venous stasis was the fibrin adsorbable t-PA, and it may play a very important role in physiological fibrinolysis.

APTT reagent with ellagic acid as activator shows adequate lupus anticoagulant sensitivity in comparison to silica-based reagent.

BACKGROUND: Lupus anticoagulant (LA) is an antibody that interferes with phospholipid-dependent coagulation reactions. Activated partial thromboplastin time (APTT) is widely used as a test for LA screening. APTT reagents are composed of activators, such as silica or ellagic acid, and phospholipids, and APTT reagents with silica are recommended for LA screening because of greater sensitivity. However, the effects of activators on LA activity have not been adequately investigated. OBJECTIVES: In this study, we examined whether an ellagic acid-based reagent was highly sensitive to LA in a low phospholipid condition and useful for LA screening. METHODS: Silica-based (SL) and ellagic acid-based (EA) reagents were prepared in-house with the same composition and concentration of phospholipids, while the commercial APTT reagents APTT-SLA (SLA), Actin FSL (FSL), APTT-SP (SP) and PTT-LA (PTT) were also included in the study. RESULTS: The normal reference ranges for SL and EA were 30.1-47.0 and 28.0-40.2 s, respectively, while the cut-off index values for circulating anticoagulant activity (ICA) calculated from the results obtained with SL, EA, SLA, FSL, SP and PTT were 12.9, 11.5, 13.2, 15.6, 14.3 and 14.0, respectively. The sensitivity of those reagents based on those cut-off values was 91%, 96%, 68%, 46%, 91% and 86%, respectively. CONCLUSIONS: Our results showed that the ellagic acid-based reagent was more sensitive to LA than silica-based reagents in a low phospholipid condition and had adequate sensitivity to detect LA. We concluded that the sensitivity of APTT reagents for LA is dependent on phospholipid concentration and not the activator.