Continuous infusion of recombinant activated factor VII for bleeding control after lobectomy in a patient with inherited factor VII deficiency.

Inherited factor VII (FVII) deficiency is a rare recessive inherited coagulation disorder with limited available information, especially in patients undergoing major thoracic surgery. In addition, an optimal management strategy for the disease has not been defined. We herein report a case involving a 61-year-old man with asymptomatic FVII deficiency who underwent a right middle and lower lobectomy to treat lung cancer. To the best of our knowledge, the present report is the first to describe the use of recombinant activated FVII continuous infusion for bleeding control after a major thoracic surgery in a patient with inherited FVII deficiency.

Properties of a recombinant bovine tissue factor expressed by Silkworm pupae and its performance as an Owren-type prothrombin time reagent for warfarin monitoring.

Tissue factor (TF), or thromboplastin, is a glycoprotein that triggers the extrinsic coagulation pathway. In blood coagulation testing, TF has been used as a natural source for determining Quick prothrombin time (PT) or the Owren PT (OBT). Currently, natural sources are being replaced with recombinant proteins because of their uniform characteristics and the possibility of stable mass production of PT reagents. Because bovine spongiform encephalopathy (BSE)-infected cows are widespread in Japan, we prepared a recombinant bovine TF (rbTF) with a baculovirus expression system using silkworms. To overcome the limitations of natural TF, especially in bovine brain, we expressed a full-length rbTF protein in Silkworm pupae with a baculovirus expression system. Baculovirus inactivation and the presence of DNA fragments in the rbTF fraction were confirmed using Reed-Muench and polymerase chain reaction methods after inactivation with a detergent. The rbTF fraction prepared by an immobilized anti-Silkworm pupae fluid protein Sepharose 4B column was identified as a visible band on western blots with a polyclonal antibody against human TF with cross-reactivity with TFs. The inhibition of the polyclonal antibody against human TF by the clotting assay for PT was identified, and amidolytic biological activity through activated factor VII on S-2288 substrate was observed. In conclusion, the rbTF expressed by the baculovirus system using Silkworm pupae was uniformly specific for bovine TF. The OBT reagent incorporated by this rbTF was similar to those of commercial reagents. It also showed a suitable International Sensitivity Index and reproducibility precision, thereby allowing for diagnostic use.

[The expression of recombinant factor VII variants is influenced by different cells for transfection using expression studies].

To investigate the influence of different transfected cells using expression studies of dysfunctional factor VII variants, we expressed recombinant FVII using COS-1 cells, CHO-K1 cells, BHK cells and HEK293 cells. The mutant type F7 DNAs with Leu-48Pro in the prepeptide domain, with Ser126Phe in the EGF-2 domain and with Gly354Cys in the catalytic domain were transfected in the four kinds of cell lines. Additionally, FVII Ser126Tyr and Ser126Gly were expressed to investigate different substitutions from Phe in position 126 in FVII. The FVII:Ag of recombinant FVII Leu-48Pro in conditioned medium, which were expressed in COS-1 cells, CHO-K1 cells, BHK cells and HEK293 cells, were 37.9% 68.0%, 11.7% and 9.2% respectively, when wild-type FVII was defined as 100%. The levels of FVII:Ag in cell lysates were 26.2%, 44.9%, 25.6% and 50.5%, respectively. The FVII:C of recombinant FVII Gly354Cys in conditioned medium were 3.7%, 3.5%, 1.5% and 1.7%, respectively. The levels of FVII:Ag were similar to FVII:C. The FVII:C of both recombinant FVII Ser126Phe and Ser126Tyr in conditioned medium of all four cell lines were considerably lower than that of FVII wild type. However, levels of FVII:Ag in conditioned medium of recombinant FVII S126G were 86.8% on COS-1 cells, 14.1% on CHO-K1 cells, 28.3% on BHK cells and 114.6% on HEK293 cells. When carrying out expression studies for FVII mutation, we have to consider different results based on the patient's laboratory phenotype when using different kinds of transfected cells.

[Investigation on the affinity between questions in the national examination for medical technologists and student level of education in the course of laboratory medicine--nine years history of the committee evaluating appropriate questions].

In order to evaluate the each question in the National Examination for Medical Technologist by comparison with the educated level in the course of laboratory medicine and the practical level of medical technologists, the investigation has been carried out by our committee established in the National University Association for Education of Laboratory Medicine during 9 years since 1997. The committee has asked each school of the 20 members of the Association to pick up good and/or inappropriate questions with the reasons why they are classified as good or improper ones. Some questions were considered as good ones by a large number of schools, while the others were considered improper. The reasons for the judgment have been sometimes very controversial and opinions uncommonly run counter to each other. It is suggested that a good question may become to be improper when the questionnaire is carelessly arranged even if the concept of the question is initially well considered. It is presumed that the annual reports of our committee may have played a role to gain common recognition that the numbers of the inappropriate questions have been decreasing in the recent National Examination for Medical Technologist.

[Education for medical teamwork in Shinshu University].

Both students of health sciences (medical technology, nursing science, physical therapy, and occupational therapy) and medical students learn medical teamwork in the primary stage by joint practice in Shinshu University. The aim of this class is for students that will become medical staff to increase their necessary communication skills for medical teamwork in addition to understanding the mutual medical professional fields in a medical institution. The 242 students of the medical department (147 students of health sciences and 95 students of medicine) take 15 classes during their first term as freshers. One teacher takes charge of a group consisting of 14 students for tutorials by mutually cooperation between teachers of medicine and health sciences. Positive relationships are expected to develop in the group, raising sociality and ethics so that both students of health science and medicine experience interdisciplinary discussion in small groups as an ideal method for continuing health care in times of poor knowledge of medicine and health care.

Factor XI deficiency with a novel homozygous mutation Trp599Arg near the C-terminal region.

We identified a novel mutation in an asymptomatic 72-year old Japanese woman with severe factor XI (FXI) deficiency. Sequence analysis showed a homozygous missense mutation Trp599Arg (g.234T-->C according to Genbank accession number M20218). This residue belongs to a region conserved in human FXI and the FXI of several animals. Molecular modeling showed that the Trp599 residue is positioned in an alpha helix in the C-terminal region of the FXI molecule.

Molecular mechanism of dysfunctional factor VII associated with the homozygous missense mutation 331Gly to Ser.

We have identified a Japanese homozygous FVII deficiency associated with the mutation G331S (c184 [in chymotrypsin numbering]), and have determined the mechanisms responsible for the dysfunctional FVII variant by expressing the mutant recombinant FVII protein. In addition, the recombinant proteins FVIIG331D, G331W and G331F were expressed. The purified recombinant FVII proteins ran as a single chain form on SDS-PAGE having a molecular mass of approximately 50 Kda. The recombinant FVIIG331S expressed the level of the recombinant wild type FVII at 2.0%, and this mutant form was also similar to FVII in the patient's plasma. However, the amidolytic activity of FVIIa using peptidyl substrate S-2288 differed little between the wild type and the four mutant FVII molecules. We suggest that the functional defect found in these mutants is not directly associated with peptidyl substrate recognition or catalysis. The Km values of FX and FIX for the mutant proteins were approximately 7.6- to 15-fold and 13- to 19-fold higher than those for the wild-type protein, respectively. Molecular modelling indicated that the side chain of the S331 mutant is oriented close to the side chain of D338 (c189) at the bottom of the specificity pocket of FVIIa. We show that the replacement of G331 with a serine likely results in a steric hindrance of macromolecular substrate binding, leading to a loss of FVIIa enzymatic activity.

A patient homozygous for a Gly354Cys mutation in factor VII that results in severely impaired secretion of the molecule, but not complete deficiency.

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese woman who suffered occasional epistaxis. The patient had low levels of both FVII coagulant activity (FVII:C) and antigen (FVII:Ag) (5.0% and 7.3% of normal controls respectively). DNA sequence analysis of the FVII gene showed that the patient was homozygous for a mutation that resulted in a Cys for Gly354 substitution, a novel missense mutation in the catalytic domain. Haplotype analysis showed that this missense mutation was inherited from her consanguineous parents. Transient expression experiments showed that secreted FVII Cys354, FVII:C and FVII:Ag levels in conditioned media were reduced to 4% and 5%, respectively, of levels secreted from wild-type FVII. However, the intracellular FVII Cys354 was 67% that of normal recombinant protein. Immunohistochemical analysis showed that intracellular FVII:Ag from FVII 354Cys was present diffusely throughout the cytoplasm. Substitution of FVII 354Gly with amino acids other than Cys (Arg, Asp, Ser and Phe), did not produce a phenotype similar to FVII Cys354Gly. Molecular modelling indicated that FVII Gly354 was located outside the FVII heavy chain, and that Cys135 in the EGF2 domain, Cys262 in the catalytic domain and Cys127 all exist within 10 A of Gly354. Therefore, we propose that the introduction of an additional free cysteine residue in the FVII molecule results in the formation of illegitimate disulphide bonds and a mis-folded domain, leading to defective secretion.

A non-immunological phospholipid-dependent coagulation inhibitor associated with IgGlambda-type multiple myeloma.

We investigated the rare case of a patient with IgGlambda multiple myeloma for whom both prothrombin time and APTT were significantly prolonged. The IgG inhibited coagulation reactions upstream from prothrombin when coagulation was initiated by mRVVT, but not by FXa, as indicated by a chromogenic substrate for FXa. The mPT and the mAPTT showed inhibition of FXa generation in both the intrinsic and extrinsic pathways. The IgG inhibited both protein C (indicated by APTT) and FX (indicated by RVV) but not amidolysis for either activated protein C or FXa. The addition of excess phospholipid significantly shortened the prolonged RVVT of the patient. It inhibited the coagulation reactions of normal plasma and was dependent on decreasing the PS concentration in the APTT reagent. It was suggested that the IgG showed lupus anticoagulant (LA)-like activity that inhibited phospholipid-dependent coagulation reactions in the intrinsic, extrinsic, and common pathways. However, the IgG did not bind cardiolipin-beta2GPI complex, beta2GPI, or prothrombin in ELISA assays. The IgG did not bind to either PS or phospholipid complexes in the presence or absence of prothrombin, FX, or FXa. Interestingly, the IgG lost its LA like-activity when it was degraded to F(ab')2 and Fc fragments by pepsin. We suspected that the IgG might inhibit the interaction between coagulation factors and acid phospholipid non-immunologically and that this process requires an intact IgG conformation, although the reaction mode is still not clear.

Human factor VII deficiency caused by S339C mutation located adjacent to the specificity pocket of the catalytic domain.

This report documents our identification of a novel factor VII (FVII) gene mutation in a Japanese boy with FVII deficiency. The proband's FVII activity was 34% and his FVII antigen level was 40% of normal controls. DNA sequence analysis of the proband's FVII gene identified a C to G point mutation at nucleotide position 10 933 in exon 8, which results in the substitution of Cys (TGC) for Ser339 (TCC). Hinf I digestion results indicate the proband and his mother were heterozygous for the mutation. Both wild-type and mutant FVIIs were transiently expressed in COS-1 cells. FVII levels measured in the culture medium of FVII Ser339Cys mutants were markedly reduced as compared to those of cells with FVII wild-type. The amount of intracellular FVII in FVII Ser339Cys mutants was 80% of that in wild-type. In the wild-type FVII, Ser339 is juxtaposed to Asp338, which is positioned at the bottom of the substrate-binding pocket in the protease domain and located adjacent to FVII Cys340, that forms a disulphide bond with Cys368. We suspect that the creation of a novel unpaired cysteine through this mutation leads to abnormal disulphide bonding during protein folding, thereby reducing the secretion of FVII.