Small-angle synchrotron x-ray scattering reveals distinct shape changes of the myosin head during hydrolysis of ATP.

In the energy transduction of muscle contraction, it is important to know the nature and extent of conformational changes of the head portion of the myosin molecules. In the presence of magnesium adenosine triphosphate (MgATP), fairly large conformational changes of the myosin head [subfragment-1 (S1)] in solution were observed by small-angle x-ray scattering with the use of synchrotron radiation as an intense and stable x-ray source. The presence of MgATP reduced the radius of gyration of the molecule by about 3 angstrom units and the maximum chord length by about 10 angstroms, showing that the shape of S1 becomes more compact or round during hydrolysis of MgATP. Comparison with various nucleotide-bound S1 complexes that correspond to the known intermediate states during ATP hydrolysis indicates that the shape of S1 in a key intermediate state, S1-bound adenosine diphosphate (ADP) and phosphate [S1**.ADP.P(i)], differs significantly from the shape in the other intermediate states of the S1 adenosine triphosphatase cycle as well as that of nucleotide-free S1.

Calcium ion-dependent monoclonal antibody against human fibrinogen: preparation, characterization, and application to fibrinogen purification.

We have produced a high-affinity monoclonal antibody classified as IgG1 with kappa-type light chains that recognizes the calcium ion(Ca2+)-dependent conformation of the D-domain of human fibrinogen. Binding of fibrinogen in solution to the insolubilized antibody increased in the presence of increasing concentrations of up to 2 mM Ca2+, the half-maximal binding being reached at 130 microM Ca2+. The dissociation constant was estimated to be 1.6 x 10(-8) M at 2 mM Ca2+. The antibody was found also to be dependent on other divalent metal ions including Zn2+, Mn2+, Co2+ and Cu2+, but not Ba2+, Mg2+ or Sr2+. The synthetic Gly-Pro-Arg-Pro-amide peptide, which has recently been shown to bind to close proximity to the calcium binding site in the D-domain, was unable to elicit the conformation for the antigen to be recognized by this antibody. This antibody was found to be a suitable ligand for the immunoaffinity chromatography of normal and abnormal fibrinogens directly from citrated plasma depleted of the vitamin K-dependent proteins or heparinized plasma by eliminating the precipitation procedure widely adopted in conventional techniques of fibrinogen purification. Indeed, fibrinogen Marburg I with the A alpha chains depleted of the carboxy-terminal A alpha(461-610) residue segment has been purified by this technique, although this dysfibrinogen was difficult to purify by conventional precipitation techniques.

An alternative elastase-mediated degradation of fibrinogen and fibrin observed in a patient with herpes simplex encephalitis and pneumonia.

A 74-year-old female developed pneumonia following herpes simplex encephalitis. Her white blood cell counts reached 28,400/microliters, about 90% of which consisted of granulocytes. The polymorphonuclear (PMN) elastase/alpha 1-antitrypsin complex levels increased and reached the maximum of 5,019 ng/ml, indicating the release of a large amount of elastase derived from the granulocytes. The mechanism of PMN elastase release was most likely to be granulocyte destruction associated with phagocytosis. The cleavage of fibrinogen and fibrin by PMN elastase, independent of plasmin, was indicated by the presence of the fragments in immunoprecipitated plasma from the patient corresponding to elastase-induced FDP D and DD fragments and the absence of fragments corresponding to plasmin-induced FDP D and DD fragments on SDS-PAGE. These findings suggested that the large amount of PMN elastase released from the excessive numbers of granulocytes in this patient with herpes simplex encephalitis and pneumonia, induced the cleavage of fibrinogen and fibrin without the participation of plasmin.

Relation between QT interval dispersion and heart rate.

Because the relation between QT dispersion (QTd) and heart rate (HR) are different from that between QT interval and HR, QTd could be overadjusted at a high HR and be underadjusted at a slow HR if we use Bazett's formula to adjust QTd. HR adjustment of QTd is not needed to evaluate repolarization dispersion.

Papillary squamous cell carcinoma of the uterine cervix: diagnostic pitfalls.

A case of papillary squamous cell carcinoma (PSCC) of the uterine cervix is reported. The patient was a 73-year-old Japanese woman with acute renal failure and bilateral hydronephrosis. A cauliflower-like mass was found in the uterine cervix. A uterine cervical biopsy specimen revealed PSCC in situ, while clinically it was an invasive carcinoma. Uterine cervical biopsy was performed a second time to confirm its stromal invasion. However, only small fragments were obtained because of heavy bleeding from the tumor and they showed PSCC in situ again. Following this, computed tomography of the pelvis revealed a 5 cm mass in the uterine cervix, invading the vagina and urinary bladder. Though deep-wedge biopsy, loop electrosurgical excision, or cone biopsy is recommended to evaluate PSCC, it may be impossible to perform any of these procedures because of bleeding such as that seen in our case. In these circumstances, good communication between pathologists and clinicians is important since lack of communication may cause PSCC to be microscopically misinterpreted as in situ carcinoma rather than invasive carcinoma.

What time is the "biologic zero hour" of circadian variability?

Most ambulatory blood pressure monitoring (ABPM) studies have used a mechanical clock as the reference time, but there is no biologic background for assuming that midnight by the mechanical clock is zero hour by the biologic clock. The aim of this study was to determine the biologic zero hour as the zero reference time by evaluating the circadian rhythm of blood pressure, heart rate, and activity. Twenty healthy medical students (18 men, 2 women, mean age 26 years old) were recruited and blood pressure, heart rate, and physical activity were monitored simultaneously by an ABPM device every 30 min for 48 h. Four concepts of zero time were selected in this study and analyzed regarding biologic zero hour: 24:00 by the mechanical clock (clock time); the time of awakening, based on a diary (diary time); the time of a sudden increment in physical activity in the morning (activity time); and the middle of the total sleeping time, based on the diary (midsleeping time). The awakening time is a better individual index than the mechanical clock, and the midsleeping time as the zero reference point is better than the awakening time. We assessed the reproducibility of the data regarding the circadian troughs between the first and second day. The reproducibility of the day-to-day variation of the blood pressure and heart rate was poor. The reproducibility of physical activity was fairly good, but the magnitude of activity was small. A 48-h monitoring profile is superior to a 24-h monitoring period.

Limited reproducibility of circadian variation in blood pressure dippers and nondippers.

The relation between blood pressure (BP) variation and hypertensive organ damage is controversial. The reproducibility of the circadian variation pattern acceptable as the standard for discriminating between "dippers" and "nondippers" has not yet been evaluated. We evaluated the reproducibility of "dipper" and "nondipper" patterns in essential hypertensives by monitoring BP for 48 h. Noninvasive ambulatory BP and heart rate (HR) monitoring for 48 h every 30 min were performed in 253 untreated patients with mild-to-moderate essential hypertension. Mean daytime (awake) and nighttime (sleeping) systolic BP, diastolic BP, and HR values were analyzed by reviewing the patients' diaries. Patients were divided into two groups by presence (dippers) and absence (nondippers) of a reduction of both systolic and diastolic BP during the night of > 10% of the daytime pressure. A subject who was a dipper on day 1 remained a dipper on day 2 in 41% (n = 103, DD group) and changed to nondipper in 16% (n = 41, DN group). A subject who was a nondipper on day 1 remained a nondipper on day 2 in 30% (n = 75, NN group) and changed to a dipper in 13% (n = 34, ND group). Our findings indicate that there is a high risk of false-positive or false-negative results when 24-h recordings are used to identify dipper and nondipper profiles.

QT dispersion in dipper- and nondipper-type hypertension.

The aim of this study was to identify the relationship of QT dispersion on 12-lead electrocardiograms and left ventricular mass index on echocardiograms associated with the circadian rhythm of blood pressure (BP). Heart rate and BP were monitored every 30 min for 48 h in 62 patients with essential hypertension using an ambulatory BP monitoring device. The patients were divided into four groups according to gender and circadian BP pattern (nocturnal BP dipper or nondipper). The patients were classified as dippers if their daytime BP decreased by at least 10% during the night and all the other subjects were classified as nondippers. Age, body mass index, and 48-h mean BP were similar among the four groups. During the night-rest period, the systolic and diastolic BP were significantly decreased in dipper-type hypertensives. The maximum QTc interval and QTc dispersion were longer in nondippers than in dippers. Left ventricular mass index (LVMI) had a tendency to increase in nondippers. The nocturnal reduction of BP significantly correlated with QTc dispersion and LVMI. The QTc dispersion significantly correlated with LVMI and interventricular septum thickness.

Highly increased plasma concentrations of the nicked form of beta(2) glycoprotein I in patients with leukemia and with lupus anticoagulant: measurement with a monoclonal antibody specific for a nicked form of domain V.

beta(2)-Glycoprotein I (beta(2)GPI) consists of five tandem repeated domains (I, II, III, IV, and V). The nicked form of beta(2)GPI (N-beta(2)GPI ) which was cleaved by plasmin in vitro at Lys 317-Thr 318 in domain V, showed reduced affinity for the negatively charged phospholipids, especially cardiolipin (CL). Recently, the N-beta(2)GPI was detected in the plasma of patients with disseminated intravascular coagulation syndrome (DIC) by an immunological method. In the present study, we prepared monoclonal antibodies for the nicked form, and demonstrated that the concentrations of this form of beta(2)GPI, which were analyzed by a sandwich ELISA using two specially prepared monoclonal antibodies, were significantly increased in the plasma of patients with leukemia (n = 51, mean +/- SD: 162.0 +/- 118.3 ng/ml) and with lupus anticoagulant (LA) (n =40, mean +/- SD: 3,041.5 +/- 16,579.7 ng/ml), compared to the normals (n = 33, mean +/- SD: 1.04 +/- 1.54 ng/ml). We found a significant correlation between the concentrations of N-beta(2)GPI and those of typical molecular markers for a fibrinolytic state such as plasmin-alpha(2) plasmin inhibitor complex (PIC) and D-dimer in patients with leukemia, but not in patients with LA. These results suggested that the generation of N-beta(2)GPI was caused by plasmin in the patients with leukemia, and by unknown proteases in the patients with LA. In the patients with LA, the levels of N-beta(2)GPI tended to be higher in those without thrombosis than in those with thrombosis.

Effect of the exposure dose of etoposide on the cell growth and cell kinetics of human ovarian cancer cells.

Although schedule-dependent cytotoxicity of etoposide has been reported, its mechanisms have not been elucidated fully. In this study, we attempted to clarify what concentration, time or exposure dose (ED, concentration of drug x time) of etoposide result in the antitumor effect on human ovarian cancer cells from the standpoint of cell cycle perturbation. The different ED were produced by different drug treatment schedules: 10 microgram/ml x 4 h (ED 40), 1.66 microgram/ml x 24 h (ED 40), 5 microgram/ml x 4 h (ED 20), 0.83 microgram/ml x 24 h (ED 20), 10 microgram/ml x 0.8 h (ED 8), 5 microgram/ml x 1.6 h (ED 8), 2 microgram/ml x 4 h (ED 8), 0.33 microgram/ml x 24 h (ED 8). Cell cycle perturbation on day 5 and the suppression of cell growth were dependent on the drug concentration at the lowest exposure dose (ED 8), but were dependent on ED at the higher EDs (20 or 40). The percentage of cells in the G2/M fraction significantly decreased from day 5 to day 7 in BG-1 cells treated at ED 20 or treated with higher concentrations (10 and 5 microgram/ml) at ED 8, but not in those treated at ED 40 or treated with lower concentrations (2 and 0.33 microgram/ml) at ED 8. Therefore, the cytotoxic mechanism of etoposide may not be explained by simple schedule dependency.

The platelet-sparing effect of paclitaxel is not related to changes in the pharmacokinetics of carboplatin.

PURPOSE: To determine whether the platelet-sparing effect of paclitaxel is related to changes in pharmacology of carboplatin. METHODS: A group of 32 patients with epithelial ovarian cancer were treated with intraperitoneal (i.p.) carboplatin-based chemotherapy with carboplatin alone or in combination with cyclophosphamide or paclitaxel, and the relationship between the pharmacology of serum platinum and thrombocytopenia was examined. The target AUC of i.p. carboplatin was 6.5 mg min/ml. Cyclophosphamide was administered intravenously at 400 mg/m2 after i.p. carboplatin and paclitaxel at 175 mg/m2 was given before i.p. carboplatin. RESULTS: Ten patients received i.p. carboplatin alone, 10 received cyclophosphamide and 12 received paclitaxel. The ages of the patients, body surface area, serum creatinine, platelet count before chemotherapy, and the total dose of carboplatin in each patient were similar in all groups. The measured AUC, Cmax, T 1/2, and MRT were similar in these groups. The nadir platelet counts were significantly higher (P = 0.0018) in patients treated with i.p. carboplatin with paclitaxel (12.1 +/- 4.3 x 10(4)/mm3) compared with carboplatin alone (5.2 +/- 3.3 x 10(4)/mm3) or with cyclophosphamide (5.2 +/- 4.8 x 10(4)/mm3). The percentage decrease in platelet counts was significantly lower (62.5 +/- 18.2%) in patients treated with paclitaxel than in the other two groups (81.5 +/- 12.6% carboplatin alone, 88.7 +/- 7.9% with cyclophosphamide). CONCLUSION: The addition of paclitaxel or cyclophosphamide to i.p. carboplatin did not alter the pharmacology of serum platinum. Thrombocytopenia was significantly less in patients treated with carboplatin in combination with paclitaxel. The platelet-sparing effect of paclitaxel is not related to changes in the pharmacology of carboplatin.

Prevalence of ventricular preexcitation in Japanese schoolchildren.

BACKGROUND: Wolff-Parkinson-White syndrome is thought to be a congenital disease, however, its exact prevalence is not known. This may be because of the intermittent activity of accessory pathways in some cases and fluctuations in autonomic tone. AIMS: To investigate the prevalence of ventricular preexcitation by electrocardiography and reported symptoms in each school age child in Yamanashi prefecture. METHODS: From 1994 to 1996, answers to a questionnaire, results of physical examination, and electrocardiography were obtained from all schoolchildren in Yamanashi prefecture (n = 92,161; total population 880,000) on admission to elementary school (age 6 to 7 years, n = 28,395), junior high school (age 12 to 13 years, n = 31,206), and high school (age 14 to 15 years, n = 32,837). RESULTS: Elementary and junior high school students had a significantly lower prevalence of preexcitation than high school students (0.073% and 0.070% v 0.174%, p < 0.001). The prevalence of left free wall pathway was highest in high school students (n = 27) compared with elementary (n = 6) and junior high school students (n = 5) (p < 0.005). The only symptom noted in the answers to the questionnaire was palpitations. The symptomatic cases were more frequent in high school (n = 13) than in elementary (n = 1) and junior high school (n = 2) children, but not significantly. No student with preexcitation had associated heart disease or family history of Wolff-Parkinson-White syndrome or sudden death. CONCLUSIONS: The prevalence of preexcitation in younger schoolchildren was less frequent than previously reported. The prevalence of preexcitation and left free wall pathways increased with age. The symptoms were few and there was no significant morbidity.

Alterations of autonomic nervous activity in recurrence of variant angina.

OBJECTIVE: To investigate whether autonomic nervous activity is involved in the recurrence of spontaneous coronary spasm in variant angina. DESIGN: Retrospective analysis. SETTING: Cardiology department of a university hospital. PATIENTS: 18 patients with variant angina were divided into single attack group (SA; nine patients) and multiple attack group (MA; nine patients) according to the frequency of ischaemic episodes with ST segment elevation during 24 hour Holter monitoring. METHODS: Heart rate variability indices were calculated using MemCalc method, which is a combination of the maximum entropy method for spectral analysis and the non-linear least squares method for fitting analysis, at 30 second intervals for 30 second periods, from 40 minutes before the attack to 30 minutes after the attack. High frequency (HF; 0.04-0.15 Hz) was defined as a marker of parasympathetic activity, and the ratio of low frequency (LF; 0.15-0.40 Hz) to high frequency (LF/HF) as an indicator of sympathetic activity. The averaged value during the 40 to 30 minute period before an attack was defined as the baseline. RESULTS: Compared with baseline, the HF component decreased in both groups at two minutes before the attack (p < 0.01), and the LF/HF ratio decreased at three minutes before the attack (p < 0.01). The baseline LF/HF was lower in the MA group than in the SA group (p < 0. 01). CONCLUSIONS: A reduction of sympathetic activity may play a key role in determining the recurrence of transient ischaemic events caused by spontaneous coronary spasm in patients with variant angina.

Direct evidence for systemic fibrinogenolysis in a patient with metastatic prostatic cancer.

Although the possible occurrence of systemic fibrinogenolysis has been suggested in patients with metastasising prostatic cancer (MPC), direct evidence is lacking. We report on a patient with MPC whose laboratory data were consistent with hyperfibrinolysis: marked decrease of alpha 2-antiplasmin (AP) level (less than 50% of normal), increase of plasmin-alpha 2-antiplasmin complex, D-fragment of fibrin and fibrinogen degradation products [FDP(D)] and cross-linked fibrin degradation products (XDP). The patient neither showed laboratory nor clinical evidence for consumption coagulopathy except for a slight increase in thrombin-antithrombin III complex level. Immunoblotting of the patient's serum using an anti-fibrinogen antibody revealed the presence of a 250 kDa protein in addition to DD fragments. Following reduction of this protein by 2-mercaptoethanol after extraction from SDS-PAGE gel, gamma-chain of fibrinogen (47 kDa) was found by immunoblotting using a monoclonal antibody recognising a 86-302 residue of the gamma-remnant of fibrinogen. Moreover, the 250 kDa protein did not bind to Sepharose 4B to which a monoclonal antibody recognising the N-terminus of fragment D was conjugated. These findings indicated that this protein was not fragment DY, but rather fibrinogen fragment X. With the retraction of the prostatic tumour by an effective therapy, the patient's AP level increased gradually. When the plasma AP level rose to 60% of normal, the fragment X was no longer detectable. These findings suggested that systemic fibrinogenolysis occurred in the patient with MPC only when AP levels were markedly decreased.

Cytokinetic and morphologic differences in ovarian cancer cells treated with ET-18-OCH3 and the DNA-interacting agent, etoposide.

New antineoplastic agents with different cytotoxic mechanisms are of interest for their ability to overcome resistance to conventional DNA-interacting agents. Ether lipids are known to be active against ovarian carcinoma both in vitro and in vivo, and the cell membrane is believed to be the target of their antitumor activity. In this study we have investigated the different cytokinetic and morphologic responses of human ovarian carcinoma cells (BG-1) to one of the ether lipids (ET-18-OCH3) and to etoposide. Etoposide induced a significantly greater G2/M block. However, the proportion of the cycling cell fraction decreased significantly in cells treated by ET-18-OCH3 and induction of the hypodiploid traction was strongly correlated with reduction of the cycling cell fraction. On the other hand, the hyperdiploid fraction was found to correlate with reduction of the cycling cell fraction in etoposide treated cells. Despite the significant appearance of the hypodiploid fraction, apoptosis was not observed by DNA-gel assay. Microscopic study showed that the hyperdiploid fraction represented cells with multiple nuclei. These observations support the unique lethal effect of ET-18-OCH3 on ovarian carcinoma cells, distinguishing it from the action of a typical DNA-interacting agent. The membrane-targeted ether lipids deserve consideration for the future chemotherapy of ovarian carcinoma, perhaps in combination with the appropriate DNA-interacting agent. New antineoplastic agents with different cytotoxic mechanisms are of interest not only for their unique inhibitory properties but also for their potential of overcoming resistance to conventional DNA-interacting agents. Ether lipids are known to be active against ovarian carcinoma both in vitro (1, 2, 3) and in vivo (4, 5), and the cell membrane is believed to be the target of their antitumor activity. Etoposide, a DNA-interacting agent, is also active against human ovarian cancer cells in vitro (6) or in clinical trials either as a single agent (7) or in combination with cisplatin (8). We have reported that a cytotoxic dose of one of the ether lipids, ET-18-OCH3, induces a G2/M block in BG-1 human ovarian cancer cells, and also a hypodiploid fraction as shown on DNA analysis by flow cytometry (FCM) (9). The G2/M block was also observed in BG-1 cells following etoposide treatment (6). In the present study, we have investigated the differences in the cytokinetic and morphologic responses of BG-1 cells to ET-18-OCH3 and to etoposide.

Change in the localization of heat shock protein 27 (HSP 27) in BG-1 human ovarian cancer cells following treatment by the ether lipid ET-18-OCH3.

We have demonstrated a higher nuclear protein content in the hypodiploid fraction of BG-1 human ovarian cancer cells following treatment with one of the ether lipids, ET-18-OCH3. In this study, we have attempted to identify the overexpressed nuclear protein induced in those dying or dead cells in the hypodiploid fraction and its localization before and after ET-18-OCH3 treatment. The pattern of nuclear proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) before and after ET-18-OCH3 treatment. The partial amino acid sequence of the most dominantly and consistently up-regulated protein spot after ET-18-OCH3 treatment was determined and it was found to be heat shock protein 27 (HSP27). Immunofluorescence staining disclosed that HSP27 localizes in the cytoplasm of the BG-1 cells before ET-18-OCH3 treatment. Condensation of HSP27 around the nuclei was observed following treatment by ET-18-OCH3. Ultimately, the nuclei of the cells in the hypodiploid fraction were stained by immunofluorescent HSP27. These results indicate that change of the localization of HSP27 may play an important role as a component of the signal transduction pathways affected by ether lipids.

Phase II dose escalation: a novel approach to balancing efficacy and toxicity of anticancer agents. Japanese Docetaxel Ovarian Cancer Study Group.

BACKGROUND: Small patient numbers in phase I trials may result in a safe but ineffective dose being recommended for phase II trials. A phase II dose escalation study may identify a dose that is both safe and effective. The Japanese phase I recommended dose of 60 mg/m2 of docetaxel (Taxotere) had been ineffective in phase II trials in ovarian carcinoma. PATIENTS AND METHODS: Patients previously treated with one platinum-based regimen for ovarian cancer received docetaxel (Taxotere) every 3 weeks. The first dose tested was 70 mg/m2. If none of the first 5 evaluable patients responded, the dose was increased. If at least one patient responded, 10 more patients were enrolled. Also, if fewer than 3 of these first 15 evaluable patients responded, the dose was increased. If at least 3 patients responded, another 15 patients were scheduled to be enrolled to confirm efficacy. Unacceptable toxicity in 4 of 5, or 10 of 15 patients would stop escalation. RESULTS: Dose escalation from 70 mg/m2 was not required because responses were noted with acceptable toxicity levels. Overall response in 25 evaluable patients treated at 70 mg/m2 was 24.0% (95% CI = 9.4-45.1%). CONCLUSION: Docetaxel 70 mg/m2 without premedication was identified as a safe and effective dose. Further testing of the phase II dose escalation design is worthwhile.

A rapid latex immunoassay for the detection of plasmin-alpha 2-plasmin inhibitor complex. Utilization of two monoclonal antibodies differentially recognizing respective components of the complex.

Among six monoclonal antibodies raised against the human plasmin-alpha 2-plasmin inhibitor complex (PPI), three antibodies were found to recognize the plasmin part (group 1) and another three the alpha 2-plasmin inhibitor (alpha 2-PI) part (group 2) of the complex. One of the group-1 monoclonal antibodies, designated JIPPI-3, specifically reacted with a segment of plasmin containing kringles 2 and 3. Although all three group 2 antibodies reacted with both alpha 2-PI and PPI on immunoblotting and ELISA, one of them, JIPPI-50, was unable to react with alpha 2-PI, when the antibody had been covalently conjugated to Sepharose 4B gels and tested for reactivity against the antigens in solution. The results indicated that the epitope for this antibody had been buried in nascent alpha 2-PI, but had been exposed by complex formation with plasmin or by possible conformational changes induced in the alpha 2-PI molecule on insolubilization to nitrocellulose membranes or immunoplates. By utilizing a set of JIPPI-3 and JIPPI-50, individually coated onto latex beads, PPI could be measured in plasma in the range of 0.8-100 micrograms/ml without interference by coexisting plasminogen (120-200 micrograms/ml) or alpha 2-PI (70 micrograms/ml). This measurable range seems to cover the level of PPI clinically observed under hyperfibrinolytic states.

Monoclonal antibody specific for tissue factor pathway inhibitor-factor Xa complex: its characterization and application to plasmas from patients with disseminated intravascular coagulation and pre-disseminated intravascular coagulation.

Tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2 and K3), inhibits the initial reactions of the extrinsic blood coagulation pathway through its K1 and K2 domains. We prepared and characterized a monoclonal antibody (Mab8-1) against TFPI-factor Xa (TFPI-Xa) complex. The reactivities of Mab8-1 toward TFPI-Xa complex, TFPI without C-terminal (TFPI-C)-Xa complex, K1K2-Xa complex and K2K3-Xa complex were examined using a surface plasmon resonance analysis (Biacore). The Biacore system allowed a quantitative analysis of antibody-antigen interaction, in real time, from which the association and dissociation rate constants could readily be obtained. The bindings of Mab8-1 to TFPI-Xa complex, TFPI-C-Xa complex and K2K3-Xa complex were each concentration-dependent. However, no binding of Mab8-1 to the K1K2-Xa complex was observed. The binding of Mab8-1 to TFPI or Xa was also not observed. These results suggested that the epitope for Mab8-1 was exposed in the K3 domain of TFPI, which was generated by the conformational change after the formation of TFPI-Xa complex. We then developed an enzyme-linked immunosorbent assay method specific for TFPI-Xa complex using Mab8-1, and we used this assay to measure plasma levels of TFPI-Xa. The normal range assessed from analyses of plasma from 30 normal healthy volunteers was 17.7-66.7 with a mean of 35.5 +/- 11.7 pmol/l. In order to asses the clinical implication of TFPI-Xa complex in the plasma of patients with thrombotic disorders, plasma concentrations were measured in 37 patients with disseminated intravascular coagulation (DIC) caused by a variety of underlying diseases. The TFPI-Xa antigen levels were significantly higher in the patients with DIC (51.9 +/- 21.6 pmol/l) and the 36 patients with pre-DIC (55.1 +/- 20.2 pmol/l) than in the 137 non-DIC patients (37.9 +/- 13.1 pmol/l). In the patients with DIC or pre-DIC, there was no significant correlation between TFPI-Xa complex and the elevated levels of thrombin-antithrombin complex, plasmin-alpha2 plasmin inhibitor complex, D-dimer, soluble fibrin monomer, soluble thrombomodulin or tissue factor. These data indicate that the plasma level of TFPI-Xa seems to be a novel independent molecular marker of DIC and pre-DIC.

Primary serous adenocarcinoma of the retroperitoneum with a response to platinum-based chemotherapy: a case report.

A CAT-scan-guided biopsy of the retroperitoneal tumor of a 54-year-old female revealed a serous adenocarcinoma resembling a serous adenocarcinoma of ovarian origin. Partial response to platinum-based chemotherapy was observed. Exploratory laparotomies and an autopsy found no possible primary lesion for the tumor. Therefore, we concluded that this tumor is a primary serous adenocarcinoma of the retroperitoneum. Although further accumulation of cases is required, it appears that primary treatment for serous adenocarcinoma of the retroperitoneum is platinum-based chemotherapy if surgical removal is incomplete.