Hiroyuki Nishikado

Measurement of membrane fluidity of polymorphonuclear leukocytes by flow cytometry

A method was established for measuring membrane fluidity of polymorphonuclear leukocytes (PMN) by the excimer-forming lipid technique with pyrenedecanoic acid in flow cytometry. When cells were labeled, the use of 2-25 microM of pyrenedecanoic acid provided similar results. Neither the removal of the unincorporated pyrenedecanoic acid nor adjustment of PMN counts exhibited any effect. By the gate analysis method, membrane fluidity of PMN could be measured with 100 microliters of heparinized whole blood in a short time and results with PMN in whole blood was similar to those with purified PMN. Therefore, purification and count adjustment of PMN could be omitted. By this method, membrane fluidity of PMN, which were treated with membrane fluidizer, was measured successfully. This method could be applied to the study of PMN function in various diseases.

Murine monoclonal antibodies to human factor XI

Murine monoclonal antibodies to human factor XI (F.XI) are described. The monoclonal antibodies (2-1, 4-1, 7-1 and 10-1) consisted of IgG1. 4-1 inhibited the activation of F.XI completely in the presence of high molecular weight kininogen and kaolin and the others did so partially, whereas these antibodies had no effect on the activation of F.XI with activated factor XII (beta-XIIa). Four antibodies had no effect directly on the amidolytic activity of activated F.XI (F.XIa). 10-1 inhibited the activation of factor IX in coagulant assay for F.XIa by Mannhalter. And 4-1 and 7-1 did so partially, whereas 2-1 did not. In immunoblotting analysis, all antibodies bound to F.XI, its reduced form and F.XIa. All were directed against the heavy chain of F.XI. All antibodies recognized F.XIa-alpha 1 antitrypsin complex.

Determination of main inhibitor of activated factor XI

To study the contribution of alpha 1 antitrypsin (alpha 1AT) and antithrombin III (AT III) to the inactivation of F.XIa in plasma, we developed the assay system of F.XIa-AT III complex according to our method of F.XIa-alpha 1AT complex, and measured the levels of both complexes in the patients with disseminated intravascular coagulation (DIC) derived from various triggers. F.XIa-alpha 1AT complex level was always higher than F.XIa-AT III complex level in each patient, independently on the trigger of DIC, the administration of heparin and the levels of F.XI, alpha 1AT and AT III. These results indicate that alpha 1AT is the main inhibitor of F.XIa in plasma.

Factor XIa-α1antitrypsin complex — Elevation in the patients with DIC —

We developed an assay for the factor XIa-α1antitrypsin complex (F.XIa-α1AT complex) in plasma. The purified factor XI (F.XI) activated with β-XIIa and treated with α1antitrypsin (α1AT) served as the standard complex. The assay is an enzyme-linked differential antibody immunosorbent assay. The complex level of tested plasma was measured with peroxidase-labeled anti-α1AT Fab′ after the addition of 10-fold diluted test plasma (200 μl) to the anti-F.XI monoclonal antibody beads. To eliminate the effects of plasma, the standard F.XIa-α1AT complex was diluted with F.XI-deficient plasma (10-fold diluted) which did not contain the complex. Purified F.XI (0.08 μg/assay, i.e. 100%) was added to the standard F.XIa-α1AT complex, because the absorbance of the standard complex containing F.XI (0.016-0.12 μg/assay, i.e. 20–150%) was a little lower than that of the complex alone. The recovery of the F.XIa-α1AT complex added was over 90%. Neither F.XI nor α1AT alone had the color development. The complex level of 25 normal individuals was below the detectable limit (<0.18 ng/assay), whereas the 30 patients with disseminated intravascular coagulation (DIC) had a high level complex (0.18-4.2 ng/assay). This assay may be helpful for the diagnosis of DIC.

New rapid assay for factor XIa-α1antitrypsin complex — application to dic —

We developed a new rapid assay for the factor XIa-alpha 1 antitrypsin in complex (F.XIa-alpha 1 AT) in plasma with the use of a newly produced anti-F.XI monoclonal antibody (KMXI-1). This assay was completed within about 5 hours, and the minimum assay range extended sensitivity about 5-fold over the former assay (Thromb. Res. 44, 489-501, 1986). In 20-fold diluted plasma samples, this assay was not affected by co-existing F.XI or nonspecific color development of the plasma. Normal levels of F.XIa-alpha 1 AT (11 +/- 4.1 ng/ml plasma [n = 96]) increased with the aging of healthy adults. The F.XIa-alpha 1 AT levels of patients with disseminated intravascular coagulation (DIC) rose along with the progression of the disease, and the appearance of high levels and the peak of F.XIa-alpha 1 AT developed faster than FDP-E or alpha 2 plasmin inhibitor-plasmin-complex in most patients. These results indicate that, in addition to FDP, F.XIa-alpha 1 AT is a useful molecular marker for DIC.

Accumulation of 125I-factor XI in atheroma of rabbit with hereditary hyperlipidemia (WHHL-rabbit)

We have studied the turnover and accumulation of rabbit factor XI (F.XI) in atherosclerotic lesion in Watanabe-hereditable hyperlipidemic rabbit (WHHL rabbit) to reveal the participation of blood coagulation in atherosclerotic lesion. Rabbit F.XI was iodinated and administered intravenously to WHHL rabbits and Japanese white rabbits. The turnover of 125I-rabbit F.XI was significantly faster in WHHL rabbits (T1/2 = 2.84 +/- 0.44 days) than in normal rabbits (T1/2 = 4.44 +/- 0.42 days). The thoracic aorta of WHHL rabbit was strongly labelled with 125I-rabbit F.XI, in sections obtained after 5 days by en-face autoradiography, whereas no radioactivity was detected in normal aorta. By an immunohistochemical study of WHHL rabbit aorta, we confirmed that many F.XI- and fibrin-related compounds existed in the atheroma, whereas albumin did not in these area. These results suggest that the activation of F.XI proceeds on the atherosclerotic lesions of WHHL rabbits.

Change of membrane fluidity of rat neutrophils accompanying Escherichia coli inoculation.

Membrane fluidity of rat neutrophils was studied following Escherichia coli inoculation, and characteristic changes were observed. Membrane fluidity was assessed by the excimer‐forming lipid technique using pyrenedecanoic acid and flow cytometry and expressed as the fluorescence intensity ratios of excimer and monomer pyrenedecanoic acid (lE/LM ratio). High lE/lM ratios indicated high membrane fluidity. The IE/Im ratio of rat neutrophils (0.50 ± 0.048) increased after E. coli inoculation, reaching a maximum of almost 1.00 after 10‐20 min and then returning to its starting value. Intravenous injection of heat‐killed E.coli or E. coli‐conditioned culture supernatants into rats induced a rapid increase of lE/lM ratios, which returned to initial levels after 20 min. The effect on membrane fluidity of in vitro neutrophil incubation with E. coli, heat‐killed E. coli, or E. coli conditioned culture supernatants was similar to that observed in vivo. Addition of 5 mM ethylenediaminetetraacetic acid (EDTA) did not affect neutrophil membrane fluidity. Addition of either 5 μg/ml cytochalasin B or 10‐5 M colchicine did not directly affect neutrophil membrane fluidity but did block the change observed following incubation with bacteria.

Spontaneous injuries in the aortic endothelium of the inherited cataract rats and their prevention by tocopherol A study by scanning electron microscopy

The aortic endothelium of inherited cataract rats (ICR), which spontaneously develop cataracts and neutrophilia, was examined by scanning electron microscopy using silver nitrate staining and pressure fixation. In ICR aged 4 weeks, the luminal surface of the aorta was similar to that in Wistar rats from which they had been derived. However, 8 weeks after birth, endothelial cells were upraised and partially detached from an underlying tissue. At 16 weeks, morphological changes exhibited by such detaching cells were more evident than at 8 weeks and fibrin was seen to be adhering to the surface of these cells; no platelet involvement was noted, however. Oral administration of DL-alpha-tocopheryl acetate for 2 weeks resulted in a reduction in the extent of endothelial injury and the luminal surface of the aorta became similar to that seen in 4- or 8-week-old animals. We found that the number of age-associated spontaneous injuries occurring in the aortic endothelium of ICR could be reduced by tocopherol administration.