Imaging of thrombosis and microcirculation in mouse lungs of initial melanoma metastasis with in vivo cryotechnique.

Microscopic bioimaging of blood flow and distribution of cancer cells in lungs is essential to analyze mechanism of lung metastasis. Such cancer metastasis has been well known to induce hypercoagulable states and thrombosis. In histopathological tissue sections, however, it has been difficult to capture rapid phenomenon of thrombus formation due to technical problems associated with much less retention of soluble serum components as well as dynamic histological features reflecting their living states. In this study, to achieve bioimaging of both hypercoagulable states and thrombosis induced by early metastasis of mouse B16-BL6 melanoma, "in vivo cryotechnique" (IVCT) was used, which retained soluble components at their original sites. Glutathione-coated quantum dots (QDs) were subsequently injected after melanoma cells via right ventricles to examine plasma flow with fluorescence emission. At 5s after the melanoma injection, melanoma cells were mostly stacked and intruded in alveolar capillaries with changing their shapes. Assembly of platelets initially appeared at 1min, and they aggregated around the stacked melanoma cells at 5min. Such aggregated platelets were immunopositive for both phospho-tyrosine 418 and 527 of Src, indicating their partial signal activation. Fibrin monomers were also immunolocalized around both melanoma cells and platelet aggregates, and massive immunoreaction deposits of fibrinogen were also detected near the same areas, but more strongly detected around the melanoma cells, indicating initial thrombus formation. In those areas, QDs were rarely detected, probably because of the lack of blood supply. Thus, IVCT revealed histopathological features of initial thrombosis under their circulatory conditions.

Impact of acute cellular rejection on coagulation and fibrinolysis biomarkers within the immediate post-operative period in pediatric liver transplantation.

We studied restoration of the coagulation and fibrinolysis system in pediatric patients following liver transplantation and biomarkers of blood coagulation and fibrinolysis for suspecting the occurrence of acute cellular rejection. Coagulation activity recovered rapidly within two days following transplantation, but it took approximately 21-28 days for full recovery of the coagulation and fibrinolysis factors synthesized in the liver. PAI-1 levels were significantly higher in patients at the time of acute cellular rejection compared with levels after control of AR, and levels on days 14 and 28 in patients without AR. Plasma protein C and plasminogen levels at the time of rejection were significantly lower than those on day 14 in patients without AR. Statistical analysis suggested that an increase in plasma PAI-1 at a single time point in the post-operative period is a reliable marker among the coagulation and fibrinolysis factors for suspecting the occurrence of acute cellular rejection. These data suggested that appropriate anticoagulation may be required for 14 days after liver transplantation in order to avoid vascular complications and measurement of plasma PAI-1 levels may be useful for suspecting the occurrence of acute cellular rejection in pediatric patients following liver transplantation.

A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma.

BACKGROUND: Soluble fibrin (SF), composed of fibrin monomer (FM) and fibrinogen, is well known to exist in the circulating blood derived from patients with thrombotic diseases, and its quantification is useful to get some information on the state and degree of intravascular coagulation. However, there was no convenient method for the determination of SF. METHODS: We prepared a novel monoclonal antibody (MoAb) (F405) to FM and SF using desAA-fibrin as the immunogen in the presence of anti-polymerant peptide (Gly-Pro-Arg-Pro, GPRP), and the characterization of the F405 was performed by Western blotting analysis and an enzyme-linked immunosorbent assay (ELISA). We also tried to detect SF in human plasma using an ELISA involving the immobilized F405 and horseradish peroxidase (POD)-labeled anti-fibrinogen polyclonal antibody. RESULTS: The antibody reacted with the fibrin degradation products fragments X, Y and E, but not with fibrinogen or its fragments X, Y, D and E, or the fibrin D-dimer. The epitope recognized by F405 appeared to be the alpha-chain N-terminal region exposed upon removal of the A peptide from the Aalpha-chain because F405 was found to bind to the alpha-chain N-terminal oligo-peptide of fibrin (GPRVVERHQ). Since F405 reacted not only with FM in the presence of GPRP peptide, but also with the SF complex prepared by the addition of thrombin-treated FM to human fibrinogen, we attempted to detect SF in human plasma using ELISA. The analytical range of this method was 1-300 microg/ml. The assay detection limit was < 0.5 microg/ml, and the results of intra- and inter-assay precision studies indicated that this method is accurate and yields reproducible results (< 9.4% and < 10%, respectively). When 56 samples of plasma from patients with disseminated intravascular coagulation (DIC) and 117 control samples from healthy individuals were tested, elevated levels of SF complex were detected in the DIC samples: the mean +/- S.D. of the SF concentration in the DIC and control samples were 63.4 +/- 65.3 microg/ml and 1.9 +/- 1.0 microg/ml, respectively. CONCLUSIONS: The ELISA using F405 is useful for the diagnosis of DIC.

Detection of soluble HFE associated with soluble transferrin receptor in human serum.

Hereditary hemochromatosis is an autosomal recessive disease, and 80-90% of patients exhibit Cys282Tyr or His63Asp mutations in the HFE gene. HFE, also known as major histocompatibility complex (MHC) class I-like molecule, binds to transferrin receptor 1 (TfR1) and ß2-microglobulin at the cell surface, forming a complex. Some MHC class I molecules are known to be soluble, raising the possibility that HFE also has a soluble form. However, it is not known whether soluble HFE (sHFE) is present in human serum, and there has been no report on the possible binding between sHFE and soluble TfR (sTfR), which is the fragment of the extracellular domain of TfR1 released into the blood. In the present study, we purified an sTfR complex from pooled serum collected from healthy volunteers, showing that the main components of the complex are sTfR and transferrin. We also confirmed the existence of sHFE in this complex. This is the first report on the existence of sHFE in human serum.

Latex immunoturbidimetric assay for soluble fibrin complex.

BACKGROUND: Soluble fibrin complex (SFC), composed of fibrin monomer and fibrinogen derivatives, is known to exist in the circulating blood in patients with thrombosis. Its detection and quantification are useful for obtaining information about the condition and degree of intravascular coagulation in early-stage thrombosis, but there is no rapid method to measure SFC in plasma for clinical use. METHODS: We obtained a monoclonal antibody that specifically reacts with SFC, with desAA-fibrin as the immunogen, and developed a rapid and sensitive latex immunoturbidimetric assay (LIA) using latex-immobilized anti-SFC monoclonal antibody. The assay system was based on the increase in turbidity induced by the reaction of the latex-immobilized anti-SFC monoclonal antibody with SFC in plasma, and the assay procedure was fully automated on a Hitachi 911 analyzer. RESULTS: The method had an analytical range of 3-300 mg/L. Intra- and interassay precision studies indicated that this system provided reproducible data (CVs <3.0% and <2.0%, respectively). The assay detection limit was <0.5 mg/L. There was no interference from bilirubin (up to 440 mg/L), hemoglobin (up to 9.6 g/L), Intralipid (up to 10%), D-dimer (up to 200 mg/L), and rheumatoid factor (up to 470 000 IU/L). SFC concentrations in plasma from patients with thrombotic diseases [mean (SD), 48.9 (57.6) mg/L; n = 160) were significantly higher than those in plasma from healthy individuals [1.8 (2.1) mg/L; P <0.001; n = 304]. CONCLUSION: In terms of linearity, precision, and sensitivity, the LIA, performed on a Hitachi 911 automated analyzer, may be useful for measurement of SFC in plasma.

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of gammaAla327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism.

Thrombophilic dysfibrinogen Tokyo V was identified in a 43-year-old man with recurrent thromboembolism. Based on analyses of the patient fibrinogen genes, the amino acid sequence of the aberrant fibrinogen peptide, and deglycosylation experiments, fibrinogen Tokyo V was shown to have an amino acid substitution of gamma Ala327Thr and possibly extra glycosylation at gamma Asn325 because the mutation confers the N-linked glycosylation consensus sequence Asn-X-Thr. The mutation resulted in impaired function and hypofibrinogenemia (hypodysfibrinogen). Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium, resulting in very low clottability. Additionally, a large amount of soluble cross-linked fibrin was formed upon thrombin treatment in the presence of factor XIII and calcium. However, Tokyo V-derived fibrin was resistant to degradation by tissue plasminogen activator (tPA)-catalyzed plasmin digestion. The structure of Tokyo V fibrin appeared severely perturbed, since there are large pores inside the tangled fibrin networks and fiber ends at the boundaries. Taken together, these data suggest that Tokyo V fibrin clots are fragile, so that fibrinolysis-resistant insoluble fibrin and soluble fibrin polymers may be released to the circulation, partly accounting for the recurrent embolic episodes in the patient.