Emicizumab, the bispecific antibody to factors IX/IXa and X/Xa, potentiates coagulation function in factor XI-deficient plasma in vitro.

Essentials Emicizumab mimics factor (F)VIIIa cofactor function, augments the intrinsic tenase activity. We assessed the emicizumab-driven hemostatic function in FXI-deficient plasmas. Emicizumab improved the coagulation potentials in severe FXI-deficient plasma. Emicizumab may provide a possibility for clinical application in patients with FXI deficiency. SUMMARY: Background Patients with factor (F)XI deficiency commonly present with markedly prolonged activated partial thromboplastin times (APTT), although bleeding phenotypes are heterogeneous. Emicizumab, a bispecific monoclonal antibody to FIX/FIXa and FX/FXa, mimics FVIIIa cofactor function on phospholipid (PL) surfaces. Antibody reactions were designed, therefore, to augment mechanisms during the propagation phase of blood coagulation. Aim To assess emicizumab-driven hemostatic function in FXI-deficient plasmas. Methods and Results Standard ellagic acid (Elg)/PL-based APTTs of different FXI-deficient plasmas (n = 13; FXI activity, < 1 IU dl-1 ) were markedly shortened dose dependently by the presence of emicizumab. To further analyze the effects of emicizumab, clot waveform analysis (CWA) in FXI-deficient plasmas with emicizumab, triggered by tissue factor (TF)/Elg demonstrated improvements in both clot times, reflecting the initiation phase, and coagulation velocity, which represents the propagation phase. Emicizumab also enhanced the TF/Elg-triggered thrombin generation in FXI-deficient plasmas dose-dependently although the degree of enhancement varied in individual cases. Thrombin generation with either FVII-deficient plasma or FIX-deficient plasma treated with anti-FXI antibody showed little or no increase by the co-presence of emicizumab, suggesting that the accelerated thrombin generation in FXI-deficient plasmas by emicizumab should depend on the FIXa-involved coagulation propagation initially triggered by FVIIa/TF. The ex vivo addition of emicizumab to whole blood from three patients with severe FXI deficiency demonstrated modest, dose-dependent improvements in Ca2+ -triggered thromboelastograms (NATEM mode). Conclusion Emicizumab appeared to improve coagulation function in severe FXI-deficient plasma, and might provide possibilities for clinical application in patients with FXI deficiency.

Clot waveform analysis using CS-2000i™ distinguishes between very low and absent levels of factor VIII activity in patients with severe haemophilia A.

INTRODUCTION: A recently developed method to assess comprehensive coagulation function, clot waveform analysis (CWA), accurately detect low levels (<1 IU/dL) of factor VIII activity (FVIII:C) in haemophilia A patients (HA-pts). Improvements are needed, however, to differentiate patients with very low from absent levels of FVIII:C. AIM: We attempted to optimize CWA using the coagulation analyser CS-2000i™ to distinguish between very low levels and absent FVIII:C in severe HA-pts. METHODS AND RESULTS: Activated partial thrombin time (aPTT)-based clot waveforms were determined in FVIII-deficient plasmas mixed with various amounts of recombinant FVIII. Clot times (CT) were shortened, and maximum coagulation velocity (|min1|) and acceleration (|min2|) were increased in FVIII dose-dependently at levels ranging from 0.25 to 100 IU/dL. The lowest level of FVIII:C detected was 0.25 IU/dL. Plasma samples from modestly severe (MS-HA; 0.5-<1.0 IU/dL), very severe (VS-HA; 0.25-<0.5 IU/dL), extremely severe (ES-HA; <0.25 IU/dL) and inhibitor-positive HA-pts (HA-inh) were examined. The CT was markedly prolonged in all instances but showed significant differences between the different groups insufficiently. The |min1| and |min2| in HA-inh were lower compared to the other groups (P<.05). A new parameter (slope-|min1|) reflecting average coagulation acceleration was derived. This index (median) was lower in HA-inh (0.0042) compared to ES-HA (0.0068) and VS-HA (0.011) with greater significant differences (P<.01), and an index of <.005 reflected the total absence of FVIII in the presence of inhibitor. CONCLUSION: The slope-|min1| parameter could provide a useful index for evaluating very low and absent levels of FVIII and/or the development of FVIII inhibitor in HA-pts.

Assessing the clinical severity of type 1 von Willebrand disease patients with a microchip flow-chamber system.

BACKGROUND: The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. METHODS: Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30 ) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. RESULTS: PL-T10 values correlated with BS (R(2) ~ 0.45) better than VWF:RCo (R(2) ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R(2) ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of < 10 IU dL(-1) than in those with VWF:RCo of 10 IU dL(-1) to < 25 IU dL(-1) and 25-40 IU dL(-1). In patients with VWF:RCo of < 10 IU dL(-1) , BS was significantly higher in those with PL-T10 > 8 min than in those with PL-T10 ≤ 8 min. CONCLUSION: T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

Mild hemophilia A patient with novel Pro1809Leu mutation develops an anti-C2 antibody inhibiting allogeneic but not autologous factor VIII activity.

BACKGROUND: In mild hemophilia A (MHA) patients, the risk of inhibitor development is generally low, but some factor VIII (FVIII) gene missense mutations are associated with a higher inhibitor incidence. OBJECTIVE: To investigate the mechanism(s) of inhibitor development in MHA. METHODS AND RESULTS: A patient, HA78, with MHA with a novel P1809L missense mutation in the A3 domain, exhibited significant residual FVIII activity ( FVIII: C ~10 IU dL(-1) ), despite the development of an inhibitor (5.6 BU mL(-1) ). Purified HA78-IgG significantly depressed FVIII: C from normal plasma but not from patient's plasma without inhibitor, indicating that this IgG inhibited allogeneic but not autologous FVIII. The HA78-IgG blocked thrombin and FXa-catalyzed FVIII cleavage but had little effect on FVIII binding to von Willebrand factor and phospholipid. The IgG recognized a C2 epitope close or overlapping the previously described anti-C2 ESH8 epitope. Similarly, a recombinant FVIII-P1809L mutant was little inactivated by HA78-IgG. This mutant demonstrated ~3-fold lower binding affinities to von Willebrand factor and phospholipid compared with wild-type, while reactions with thrombin or FXa were not impaired. Reaction of FVIII-P1809L with the alternative anti-C2 ESH4 showed only an ~20% inhibition compared with wild-type FVIII but was similar to wild-type after incubation with ESH8. A surface plasmon resonance-based assay demonstrated that anti-C2 ESH4 bound to FVIII-P1809L with ~10(2) -fold lower affinity compared with ESH8. CONCLUSION: These results indicated that the P1809L mutation in A3 induced the conformational change in the FVIII molecule that hampered antigenic determinant(s) located in the C2 domain and might result in the inhibitor development.

Systematic monitoring of hemostatic management in hemophilia A patients with inhibitor in the perioperative period using rotational thromboelastometry.

BACKGROUND: The management of hemophilia A (HA) patients with inhibitors on bypassing therapy remains challenging. In particular, the monitoring of treatment is restricted by the limited reliability and lack of standardization of currently available methods to evaluate the physiological effects of various hemostatic agents. Accurate monitoring of these patients is particularly important in surgical situations. The recently developed comprehensive coagulation assays, including rotational thromboelastometry (ROTEM), may be useful in these circumstances. OBJECTIVE: We have attempted to establish a systematic monitoring protocol using ROTEM (NATEM triggered by CaCl2 ) to evaluate the choice and effectiveness of different bypassing agents in the perioperative period. METHODS AND RESULTS: The hemostatic effects of recombinant factor VIIa (rFVIIa) and activated prothrombin complex concentrates (aPCC) were determined using a three-step procedure (spike, preoperative and perioperative) in eight patients with HA inhibitor admitted for elective surgery and assessed for individually tailored therapy. The ROTEM parameters demonstrated similar improvement to approximately normal levels at each stage after treatment with rFVIIa. Results in the presence of aPCC showed a marked improvement in the spike data, although this appeared to be different from those in the preoperative and perioperative assessments. The information derived from the spike and preoperative findings provided a useful guide for establishing an effective dose of therapeutic material, and facilitated good hemostatic control during and after surgery in all cases. CONCLUSION: The findings suggest that this systematic analysis using ROTEM could provide a promising strategy for the use of bypassing therapy in HA patients with inhibitor.

Activated prothrombin complex concentrate (APCC)-mediated activation of factor (F)VIII in mixtures of FVIII and APCC enhances hemostatic effectiveness.

BACKGROUND AND OBJECTIVES: Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their active forms. A recent report has demonstrated that mixtures of APCC and FVIII potentiated thrombin generation, in vitro, in plasma from patients with severe hemophilia A, but the mechanism(s) involved remains unknown. RESULTS: APCC (0.05 U mL(-1) ) increased FVIII activity ~ 4-fold within 1 min in one-stage clotting assays, followed by a return to initial levels within 10 min. This reaction was dependent on the presence of tissue factor and phospholipid. Thrombin generation produced from APCC was ~ 3.5-fold greater in the presence of FVIII than that in its absence. SDS-PAGE analysis revealed that APCC sequentially proteolyzed the heavy chain of FVIII at Arg(372) and Arg(740) , followed by cleavage at Arg(336) . Proteolysis was prevented by FVIIa inhibitor, but not by hirudin, supporting the concept that APCC itself possessed the potential to activate FVIII in early coagulation phases, and that FVIIa in APCC contributed mainly to this reaction. APCC-mediated FVIII activation was unaffected by the addition of anti-FVIII inhibitor antibodies, irrespective of epitope specificity. Anti-C2 type 1 inhibitors, however, diminished the inactivation phase of the APCC reaction by inhibiting cleavage at Arg(336) . CONCLUSION: Small amounts of APCC, relative to the standard concentration used for clinical purposes, could activate FVIII directly, even in the presence of anti-FVIII antibodies. Combination therapy based on mixtures of APCC and FVIII could have significant beneficial implications for the treatment of hemophilia A patients with inhibitors.

Effects of anti-factor VIII inhibitor antibodies on factor VIIa/tissue factor-catalysed activation and inactivation of factor VIII.

Factor (F)VIIa/tissue factor (TF) rapidly activates FVIII activity by proteolysis at Arg372 and Arg740, and subsequently inactivates FVIIIa activity by proteolysis at Arg336, although this activation is weaker than that by thrombin. The effects of anti-FVIII inhibitor antibodies on these reactions remain unknown, however. In this study, 13 of anti-FVIII inhibitor antibodies recognising the A2 or C2 domain were prepared. None of them, irrespective of epitope specificity, significantly affected FVIIa/TF-catalysed FVIII activation in one-stage clotting assays. Anti-A2 and anti-C2 type 2 antibodies had little effect on the inactivation phase. Anti-C2 type 1 antibodies, however, modulated inactivation by 40-60% of that seen with control IgG, suggesting that the activity of FVIIIa generated by FVIIa/TF persisted in the presence of this specific type of inhibitor. SDS-PAGE analysis demonstrated that all antibodies had little effect on FVIIa/TF-catalyzed proteolysis at Arg372 and Arg740. Anti-C2 type 1, however, significantly delayed cleavage at Arg336 in dose- dependent manners. Neither anti-A2 nor anti-C2 type 2 affected this reaction, and the findings were consistent with the results of the functional assays. In addition, anti-C2 monoclonal antibodies with type 1 and 2 demonstrated similar patterns of reaction as the anti-C2 polyclonal antibodies in FVIIa/TF-mediated FVIII mechanisms. We demonstrated that FVIIa/TF activated FVIII even in the presence of anti-FVIII antibodies, but inactivation patterns appeared to depend on inhibitor type. It could be important to determine the characteristic of these inhibitor antibodies for prediction of their effects on FVIIa-related FVIII reactions, and the results could have significant therapeutic implications.

Casein hydrolysate formula-induced liver dysfunction in a neonate with non-immunoglobulin E-mediated cow's milk allergy.

A 10-day-old male neonate was admitted with bilious vomiting and gross hematochezia. Peripheral eosinophilia, delayed positive skin prick test to artificial milk, and elevated eosinophil cationic protein levels suggested cow's milk allergy. Fluid infusion with prohibition of oral intake improved the digestive symptoms. Breast-feeding was resumed on hospital day 3 and only casein hydrolysate formula was fed from day 7 onward. Nevertheless, eosinophilia and elevated transaminase levels developed on day 14. Liver dysfunction associated with casein hydrolysate formula was suspected and the infant was transferred to soy formula. Eosinophil counts decreased and transaminase levels were normalized on day 19. A cow's milk protein-specific lymphocyte proliferation test was positive for alpha-casein, beta-lactoglobulin, and bovine serum albumin, indicating sensitization of T cells to cow's milk proteins. These observations suggest that careful attention should be paid to liver dysfunction in non-immunoglobulin E-mediated cow's milk allergy, even when hypoallergenic formula is used.

Insulin levels in low birth weight neonates.

BACKGROUND & OBJECTIVES: Foetal undernutrition may have important role in adult insulin resistance and diabetes but insulin kinetics in very early life has not been well studied. The present study was undertaken to determine insulin levels in low birth weight neonates and compare with the levels in normal weight and high birth weight neonates. METHODS: Ten 7 day old children each of low birth weight (< 2500 g, Group 1), normal birth weight (2500-3500 g, Group 2) and high birth weight (> 3500 g, Group 3) selected successively over a period of one month were studied. All children were normally delivered at full term and were not suffering from any major illness. Detailed anthropometry was performed, a 6 h fasting blood sample was obtained for blood glucose, lipids, insulin and C-peptide estimation; 60 min after an intravenous glucose load a second sample was obtained for glucose and insulin. Insulin resistance was calculated using the homeostasis model assessment (HOMA). RESULTS: Levels of total cholesterol, triglycerides, low density and high density lipoprotein cholesterol and glucose were not significantly different in the three groups. Mean fasting and post-glucose load insulin levels (microU/ml) were 2.78 +/- 2.23 and 3.28 +/- 2.04 in Group 1, 1.67 +/- 1.20 and 2.60 +/- 2.32 in Group 2 and 3.37 +/- 2.08 and 4.40 +/- 3.05 in Group 3 and fasting C-peptide levels (microg/ml) were 0.296 +/- 0.22, 0.208 +/- 0.09 and 0.327 +/- 0.23 respectively. There was no inter-group difference in insulin-glucose ratio, insulin levels adjusted for ponderal index and HOMA indices. A significant inverse quadratic correlation (U-shaped curve) of body weight with insulin (fasting and post-glucose) and C-peptide levels was observed (P < 0.05). INTERPRETATION & CONCLUSION: Both low and high birth weight term neonates have high fasting and post-glucose insulin levels. This U-shaped trend suggests influence of foetal undernutrition (environmental) as well as genetic factors in these children.

Effects of recombinant human macrophage colony-stimulating factor on atherosclerotic lesions established in the aorta of high cholesterol-fed rabbits.

Anti-atherosclerotic effects of human macrophage colony-stimulating factor were investigated using rabbits fed a high cholesterol diet. Rabbits fed a diet containing 2% cholesterol for 59 days developed hyperlipidemia and atheromatous aortic plaques. They were then administered 80 microg/kg/day of either macrophage colony-stimulating factor or human serum albumin, as a control, for the next 12 weeks. Compared with the control group, rabbits treated with macrophage colony-stimulating factor had significantly fewer plaques on the inner surface of the thoracic and abdominal aortae, and half the sectional area of thickened intima in the aortic arch, as well as in the thoracic and abdominal aortae. Macrophage colony-stimulating factor also decreased the cholesterol content of the atherosclerotic lesions. Serobiochemical analyses revealed that macrophage colony-stimulating factor increased the levels of high density lipoprotein-cholesterol significantly, without influencing other lipid parameters such as the level of low density lipoproteins. The effects of macrophage colony-stimulating factor were evident until the fourth week of drug injection, at which time anti-human macrophage colony-stimulating factor antibodies were clearly induced in the serum. These results indicate that exogenously administered macrophage colony-stimulating factor suppresses atherosclerotic lesions induced by a high cholesterol diet by activating lipid metabolism in vivo.

Leptomeningeal melanomatosis with multiple cutaneous pigmented nevi: tumor cell proliferation and malignant transformation in an autopsy case.

We experienced a rare case of leptomeningeal melanomatosis. The proliferative activity and nuclear accumulation of p53 in this tumor were examined, since the relationship between this tumor type and growth has not yet been elucidated. A 33-year-old Japanese man was shown to have leptomeningeal melanomatosis with multiple cutaneous pigmented nevi. The autopsy findings showed the presence not only of benign diffuse melanosis of the leptomeninges but also of leptomeningeal melanomatosis in the subarachnoid space and brain parenchyma. In the brain parenchyma, the direct invasion of tumor cells from the subarachnoid space and Virchow-Robin spaces filled with melanoma cells were observed. Multiple hemorrhagic areas invaded by melanoma cells were also present. Immunohistochemical staining with a monoclonal antibody to melanoma cells showed positivity in the tumor cells. Proliferation analysis using the MIB-1 antibody demonstrated that the labeling index of tumor cells invading brain parenchyma (2.54%) was higher than that in other lesions of the inner (0.89%) and outer layer (0.76%) of the subarachnoid space. Nuclear accumulation of p53 protein was rarely seen in the tumor cells. We reported a case of leptomeningeal melanomatosis. Higher proliferative activity was found in invading cells of the brain parenchyma. Malignant transformation of the tumor did not appear to be associated with p53 gene mutation.

The antiproliferative effect of HGF on hepatoma cells involves induction of apoptosis with increase in intracellular polyamine concentration levels.

Hepatocyte growth factor (HGF) induced apoptosis and decreased the DNA synthesis in Hep G2 cells. In the HGF group interleukin-1 converting enzyme, ornithine decarboxylase (ODC) activity and intracellular polyamine concentrations were increased compared to those of the control group. Administration of the ODC inhibitor decreased polyamine concentration, and inhibited apoptotic changes in the cells. These changes were reversed by exogenous addition of polyamine. These findings suggest that one of the mechanisms by which HGF exerts its antiproliferative effect is induction of apoptosis and that increase in intracellular polyamine concentration may be one of the triggers of cell death.

[Identification of a sulfhydryl-reducing agent-inducible protein highly homologous to protein synthesis elongation factor Tu in Bacillus subtilis].

It was examined that the effect of beta-mercaptoethanol and dithiothreitol treatments, which should affect disulfide bond formation of proteins, on cellular protein components of Bacillus subtilis. In LB medium, the treatments induced the synthesis of a 50 kD protein (P50), which is synthesized constitutively under normal growth condition and is a major cytoplasmic protein. P50 was also induced by heat shock, but not by sporulation. In Schaeffer's sporulation medium, however, P50 was not induced by the sulfhydryl-reducing agents. This suggests that the sulfhydryl-reducing agent-inducibility of P50 might depent on specific physiological condition(s). The amino terminal sequences of two of the four main V8 protease fragments of P50 were determined. A search in databases revealed that P50 was highly homologous to protein synthesis elongation factor Tu of B. subtilis.

X-ray contact microscopy system for spectromicroscopy of biological specimens.

An X-ray contact microscopy system has been developed for the study of molecular and elemental distributions in biological specimens based on X-ray absorption characteristics. The system consists of a chamber for measuring XANES (X-ray absorption near-edge structure) of biomolecules, and a contact microscopy system for dried specimens with an electronic zooming tube. With this system the elemental distribution of carbon, nitrogen, oxygen, Ca and Fe in an HeLa cell has been studied, and the DNA-related image using a peak from the XANES profile of the DNA at the K-absorption edge of phosphorus has been obtained.