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A number of test kits are available for measuring activated partial thromboplastin time (APTT) and are used to screen for intrinsic coagulation reactions. However, results obtained with the same sample by different test kits often vary, causing confusion regarding potential hemostatic activity in the specimen. We investigated the usefulness of 6 different APPT kits, which utilize various phospholipids and activators, to detect prolonged clotting time in plasma from subjects with abnormal coagulopathy, including lupus anticoagulant(LA). In samples from subjects with intrinsic coagulation factor deficiencies and subjects taken heparin, the abnormal APTT detection ratio was high regardless of the kit used, thus any would be acceptable for measuring APTT in such patients. In contrast, that ratio in patients with von Willebrand disease was relatively low regardless of the kit, probably because factor VIII activities in those patients were slightly decreased. The ratio of detected subjects with LA and subjects taking warfarin varied among the APTT kits, however, those that utilized synthetic phospholipids were useful for the detection of LA. Our results suggest that an APTT kit should be selected according to the kind of disorder in the patient. Further, kits that employ synthetic phospholipids are useful for detecting abnormal coagulopathy in patients with intrinsic coagulation factor deficiencies and patients taken heparin, as well as for detection of LA.

Background: Fibrin monomer (FM) and its complex (sFC) exist at high concentrations in hypercoagulable state blood. Two novel immunoassays for sFC (SF and FMC) using specific monoclonal antibodies (IF-43 and F405) were recently developed., Methods: We measured the concentrations of thrombotic markers in 103 patients with DIC and thrombotic disorders., Results: We found that the concentration of FMC was approximately 3.35-fold greater than that of SF. In patients with a high FMC/SF ratio, FDP and D dimer concentrations were increased, suggesting that the discrepancy in sFC concentrations was caused by fibrinolytic activity. Further, plasma samples from those patients were found to contain the X- and Y-fragments of FM in addition to FM and sFC in a Western blotting assay using F405, which binds with those fragments. In an in vitro study, FM formed from pooled plasma containing EDTA was degraded to the X- and Y-fragments of FM by fibrinolytic activity, and we termed those FM degradation products (FMDP)., Conclusion: Determination of FMDP is important for diagnosis of thrombogenic conditions associated with fibrinolysis, such as in patients with DIC, and it may serve as a useful marker for hypercoagulable states with accelerated fibrinolysis.

Antiphospholipid syndrome (APS) is an autoimmune disorder in which vascular thrombosis and recurrent pregnancy loss occur in patients with antiphospholipid antibodies(aPL). Measurements of the beta2-glycoprotein I-dependent anticardiolipin antibody(aCL) and lupus anticoagulant(LAC) are the only laboratory tests available for the diagnosis of APS. Recently, phosphatidylserine-dependent antiprothrombin antibody(aPS/PT) has been detected. aPS/PT was measured by ELISA using the phosphatidylserine-prothrombin complex as an antigen immobilized on ELISA plates in the presence of CaCl2. In our study of 219 patients with APS and autoimmune diseases, the prevalence of aPS/PT-IgG in those with APS was 42.2%, which was significantly higher than that(4.6%) in patients with autoimmune diseases. Furthermore, aPS/PT was closely associated with APS manifestations with an odds ratio (OR) of 2.92 (95% confidence interval (95% CI): 1.33 to approximately 6.40), whereas the OR for aCL was 2.06 (95% CI: 0.91 to approximately 4.66). In addition, aPS/PT-IgG was strongly correlated with the presence of LAC as detected with a diluted Russell viper venom time test (dRVVT) (OR: 38.2, 95% CI: 13.4 to approximately 109.1). The monoclonal antibody (23-1D) of aPS/PT also prolonged the clotting time in LAC tests (aPTT, dRVVT, and kaolin clotting time) in a concentration-dependent manner. In conclusion, aPS/PT is more closely associated with manifestations of APS and LAC, and positive results from an aPS/PT test can mark thrombotic events in APS patients. The determination of aPS/PT in clinical practice, in conjunction with that of other aPL, may improve the likelihood of recognizing APS.

Human T cell leukemia virus type I (HTLV-I) causes adult T cell leukemia (ATL) in 2-5% of carriers after a long latent period. An HTLV-I encoded protein, Tax, induces proliferation and inhibits apoptosis, resulting in clonal proliferation of infected cells. However, tax gene expression in ATL cells is disrupted by several mechanisms, including genetic changes in the tax gene and DNA methylation/deletion of the 5' long terminal repeat (LTR). Because Tax is the major target of cytotoxic T-lymphocytes in vivo, loss of Tax expression should enable ATL cells to escape the host immune system. The 5' LTR of HTLV-I is frequently hypermethylated or deleted in ATL cells, whereas the 3' LTR remains unmethylated and intact, suggesting the involvement of the 3' LTR in leukemogenesis. Here we show that a gene encoded by the minus strand of the HTLV-I proviral genome, HTLV-I basic leucine zipper factor (HBZ), is transcribed from 3'-LTR in all ATL cells. Suppression of HBZ gene transcription by short interfering RNA inhibits proliferation of ATL cells. In addition, HBZ gene expression promotes proliferation of a human T cell line. Analyses of T cell lines transfected with mutated HBZ genes showed that HBZ promotes T cell proliferation in its RNA form, whereas HBZ protein suppresses Tax-mediated viral transcription through the 5' LTR. Thus, the single HBZ gene has bimodal functions in two different molecular forms. The growth-promoting activity of HBZ RNA likely plays an important role in oncogenesis by HTLV-I.

Factor Xa (FXa) is a key enzyme that is positioned at the convergence of the intrinsic and extrinsic pathways in the blood coagulation cascade, and inactivation by a specific FXa inhibitor effectively prevents the generation of thrombin. Various types of low molecular weight (LMW) heparin, which function as semi-selective and indirect FXa inhibitors, are replacing unfractionated heparin (UFH) as agents for the prevention and treatment of venous thromboembolism (VTE), as well as in initial treatment for coronary events. Of those, heparinoid has been shown to be safer and more effective for the prevention of postoperative VTE than UFH, especially for treatment of heparin-induced thrombocytopenia (HIT). Further, synthetic pentasaccharide has been found to offer advantages over current thromboprophylactic regimens in a number of patients undergoing major orthopedic surgery. Other studies have shown that pentasaccharide is more effective for overall VTE in comparison with LMW heparin, though it was also associated with an increased rate of major bleeding. Synthetic, selective, and direct inhibitors to FXa, such as DX-9065a, are highly potent and orally bioavailable antithrombotic agents that have demonstrated an improved side effect profile, probably by allowing sufficient thrombin to remain for platelet activation and normal hemostasis, while preventing pathological thrombus formation. For thrombosis therapy, the most desirable type of antithrombotic agent is an orally active drug that has a broad range of effective doses and no hemorrhagic side effects. Presently, many types of direct inhibitors are in various stages of clinical trials and expected to provide significant benefits as compared to currently utilized therapy strategies.

The HFE gene, a member of the class-I transplantation antigen gene family, is responsible for hereditary hemochromatosis, one of the most common inherited diseases in individuals of European descent. Patients exhibit predictable changes in iron homeostasis, including elevations in both transferrin saturation and serum ferritin levels. A subset of patients progress to overt clinical sequelae, resulting from iron overload. A hallmark of the disease is increased absorption of iron by the intestine. Although the HFE protein appears to modulate the function of the transferrin receptor in vitro, its precise role in vivo remains obscure. With multiple cell types involved in iron metabolism, the function of HFE is likely to be complex.

Fragments of fibronectin (FN) corresponding to the N-terminal heparin-binding domain have been observed to promote catabolic chondrocytic gene expression and chondrolysis. We therefore characterized FN species that include sequences from this domain in samples of arthritic synovial fluid using one-and two-dimensional (1D and 2D) Western blot analysis. We detected similar assortments of species, ranging from ~47 to greater than 200 kDa, in samples obtained from patients with osteoarthritis (n = 9) versus rheumatoid arthritis (n = 10). One of the predominant forms, with an apparent molecular weight of ~170 kDa, typically resolved in 2D electrophoresis into a cluster of subspecies. These exhibited reduced binding to gelatin in comparison with a more prevalent species of ~200+ kDa and were also recognized by a monoclonal antibody to the central cell-binding domain (CBD). When considered together with our previous analyses of synovial fluid FN species containing the alternatively spliced EIIIA segment, these observations indicate that the ~170-kDa species includes sequences from four FN domains that have previously, in isolation, been observed to promote catabolic responses by chondrocytes in vitro: the N-terminal heparin-binding domain, the gelatin-binding domain, the central CBD, and the EIIIA segment. The ~170-kDa N-terminal species of FN may therefore be both a participant in joint destructive processes and a biomarker with which to gauge activity of the arthritic process.

The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.

Quantitative genetic analysis of hepatic and splenic iron levels in recombinant inbred mice yielded a quantitative trait locus that was found to coincide with the genomic locale encompassing the tumor necrosis factor receptor 2 gene (Tnfr2). When fed an iron-enriched diet, mice nullizygous with respect to Tnfr2, but not the Tnfr1 gene, showed a significant increase in splenic non-heme iron levels. This result contrasted with mice deficient in the hemochromatosis protein, HFE, which demonstrated a significant increase in normally high hepatic iron levels, but no change in splenic iron, when fed an iron-enriched chow. Both Tnfr2 knockout and HFE knockout mice fed an iron-enriched diet failed to demonstrate intestinal epithelial cell iron following the application of the Perls' stain, as compared to both Tnfr1 knockout and normal control mice. Moreover, intestinal intraepithelial lymphocytes (IELs) isolated from HFE knockout mice did not show an increase in TNF expression following challenge with the iron-enriched diet, in contrast to normal controls. These results suggest that HFE and TNFR2 are both involved in regulating iron deposition in tissues and that the regulation occurs at the level of the intestine through IEL-orchestrated production of TNF following the binding to TNFR2. These data suggest that HFE and TNFR2 may contribute to a common pathway of the iron stores regulator insuring the controlled efflux of gut iron.

A high-resolution crystallographic analysis of the nitrogenase MoFe-protein reveals a previously unrecognized ligand coordinated to six iron atoms in the center of the catalytically essential FeMo-cofactor. The electron density for this ligand is masked in structures with resolutions lower than 1.55 angstroms, owing to Fourier series termination ripples from the surrounding iron and sulfur atoms in the cofactor. The central atom completes an approximate tetrahedral coordination for the six iron atoms, instead of the trigonal coordination proposed on the basis of lower resolution structures. The crystallographic refinement at 1.16 angstrom resolution is consistent with this newly detected component being a light element, most plausibly nitrogen. The presence of a nitrogen atom in the cofactor would have important implications for the mechanism of dinitrogen reduction by nitrogenase.

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits. In this structure, one of the three alpha subunit domains exhibits a substantially changed conformation, whereas the rest of the protein remains essentially unchanged. A predominantly positively charged funnel is revealed; this funnel is of sufficient size to accommodate insertion of the negatively charged cofactor.