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Results for “coagulation factor VIII”

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At least 217 records · Page 12Linked to original sources
PubMed2003

[Effective and stable in vitro expression of human coagulation factor VIII by retrovirus-based plasmid vector coupled with polyamidoamine dendrimer].

OBJECTIVE: To demonstrate the effectiveness of a retrovirus-based plasmid vector coupled with nanometer material-polyamidoamine (PAMAM) dendrimer in stable gene expression of FVIII in vitro and to study the cytotoxicity of PAMAM. METHODS: The retrovirus-based plasmid vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa - 1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. The complex that contained PAMAM and pLNC-FVIII BD transfer FVIII BD cDNA into NIH3T3 cell line. In day 2, 5, 10, 15, 30 after transferring, the antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of FVIII BD mRNA. Inhibitory percentage of cell vitality was used for cytotoxicity of PAMAM. RESULTS: Human FVIII was expressed for 30 days by transfected cells. The mean procoagulant activity of secreted FVIII in these 30 days was 0.929 U/ml, and the FVIII antigen was 0.188 micro g/ml by 10(6) cells in 24 hours, respectively. The level of FVIII didn't significantly decreased during these days. Inhibitory percent of cell vitality was only 5.32%. CONCLUSION: PAMAM could effectively transfer pLNC-FVIII BD into NIH3T3 cells and FVIII could be stably and effectively expressed by the transfected cells. Cytotoxicity of PAMAM was low.

Animals↗
PubMed2004

[In vivo transfection and expression of human coagulant factor VIII cDNA in mice].

The aim is to observe the expression of human factor VIII gene in mice tranduced in vivo and ex vivo. The vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa-1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. 2 x 10(6) of mouse bone marrow stroma cells transduced by LNC-FVIII BD were infused into 4-week-old BALB/c mice by tail-vein injection. pLNC-FVIII BD was conjugated with PAMAM dendrimer to form complex PAMAM-pLNC-FVIII BD, with which C57BL/6J were injected by tail vein (200 micro l contained 15 micro g/mouse) and sacrificed at days 1, 2, 7, 14, 21 and 28, respectively after injection. Tissue such as liver, spleen, lung and kindney were harvested, with which the transcription were detected by means of RT-PCR. In addition, blood was collected to be measured human FVIII Ag, human FVIIIc and anti-FVIII of human inhibitors. The results showed that the highest level of human FVIII in the recipient BALB/c mice was 8.6 +/- 1.44 ng/ml detected on the first day post-injection; anti-FVIII antibodies were detected from the first week post-injection, and then the level of FVIII Ag decreased and cannot be measured on the fourth week. In the C57BL/6J mice physiological level of human FVIII was expressed in plasma at 48 hours after injection and the average human FVIIIc was 0.62 U/ml and the average human FVIII Ag was 115.5 ng/ml, and gradually reduced later. Anti-FVIII of human inhibitors was not revealed all the time. Syngene image scanning demonstrated that the transcription of the human FVIII BD cDNA occurred mainly in spleen and lung, and secondarily in liver and kidney. No side effects of PAMAM-pLNC-FVIII BD were observed in mice tissue by pathological examination at 4 weeks. In conclusion, retrovirus-transduced bone marrow stroma cells effectively produced human FVIII after ex vivo transduction, but the development of anti-FVIII antibodies in recipient mice influenced the expression level. The human FVIII gene can successfully be transduced in vivo through injecting PAMAM-pLNC-FVIII BD cDNA into mice intravenously. There was physiological level expression of human FVIII in plasma at 48 hours after injection and the average human FVIIIc is 0.62 U/ml and the peak in the six mice was 0.89 U/ml, and gradually reduced later.

Animals↗
PubMed2006

Contribution of low density lipoprotein receptor-related protein genotypes to coagulation factor VIII levels in thrombotic women.

The contribution of low density lipoprotein (LDL) receptor-related protein (LRP) to variance of factor VIII (FVIII) levels in plasma was investigated in thrombotic women through analysis of frequent LRP genotypes. The G allele of the LRP -25C/G polymorphism, predicting increased LRP expression, was associated with 15% and 18% mean reductions of FVIII activity and protein levels, respectively. The combination of -25C/G LRP polymorphism with FVIII D1241E and ABO polymorphisms produced a gradient of FVIII levels, thus supporting the notion that several factors, acting in FVIII biosynthesis, post-translational modification and removal from circulation, have additive effects on the variance of FVIII levels in plasma.

Adult↗
PubMed1991

Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor.

The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.

Binding, Competitive↗
PubMed1991

[Clinical testing of Premofil M SRK, a blood coagulation factor VIII concentrate purified from human plasma using monoclonal an antibodies].

Premofil M SRK, licensed in Switzerland by the IKS since mid-1990, was clinically tested in close collaboration with 7 hemophilia treatment centers. Up to May 1991, we collected results of in vivo recovery from 17 patients, half-life determinations from 12, and safety data from 9 who were exclusively treated with the preparation for several months. The mean in vivo recovery of factor VIII was calculated to be 77 +/- 14%; the rise in factor VIII activity in plasma following injection of one unit/kg body weight was 1.6 +/- 0.3 units/dl. The mean halflife was 11.9 +/- 4.6 hours. No side reactions were registered throughout the study period. None of the patients showed any signs of HIV or hepatitis infection. It can bei concluded that Premofil M SRK fulfills the requirements related to tolerance, efficacy and safety for a factor VIII concentrate.

Adolescent↗
PubMed1990

Ethnic polymorphism of DXS52 (St 14) locus linked to coagulation factor VIII gene in Argentina.

Sixty-five individuals belonging to 16 argentinian families of hemophilia A were studied using the St 14 probe (DXS52 locus). This probe is widely used for carrier detection and prenatal diagnosis, despite the risk of recombination between the factor VIII gene and the DXS52 locus, because of its high informativity. The families are divided in two groups: one group constituted only of metis of Indians according to interview and morphotype and a second group of caucasoids (Spanish essentially and Italian). In this study we have shown some ethnic variations of the TaqI RFLPs in the DXS52 locus. In the allelic system I, (which alleles are numbered from 1 to 8) we have noted an over representation of the larger alleles (2 and 3) and of the allele 8 in both Argentinian groups when compared to the caucasian population already studied in our laboratory. The additional polymorphic TaqI site giving the beta band in the system II (alpha and beta bands) is found more frequently in the Argentinian families than in Caucasians. Some other additional polymorphic sites have been found in generally constant bands giving additional allelic systems, in metis families.

Alleles↗
PubMed1986

DDAVP in type IIa von Willebrand's disease.

1-D-Amino(8-D-arginine)-vasopressin (DDAVP) infusion in three patients with type IIa von Willebrand's disease (vWD) resulted in a normalization of the factor VIII coagulant, factor VIII-related antigen, and von Willebrand factor (vWF) (ristocetin cofactor) activities and the bleeding time. The normalization of these hemostatic parameters persisted for four hours. Over the same time period there was a marked increase in the quantity of the vWF multimers when blood was collected in the presence of protease inhibitors. The vWF multimers present were even larger than the normal. When blood was collected in the absence of protease inhibitors, a smaller increase in the plasma vWF multimers was observed and fewer of the intermediate and larger vWF multimers were seen; multimers larger than those present in normal plasma were not visualized. The platelet vWF multimers and activities did not change with or without inhibitors. These studies suggest that there is a subgroup of patients with type IIa vWD who respond to DDAVP with complete normalization of their hemostatic abnormalities and whose vWF is sensitive to proteolysis.

Antigens↗
PubMed1986

Plasma clearance rates of coagulation factors VIII and IX in factor-deficient individuals.

The plasma clearance rates of factors IX and VIII were determined in patients with hemophilia A and B who had received factor replacement by prolonged, continuous infusion of factor concentrates. The clearance rates were calculated by dividing the factor infusion rates by the steady-state plasma factor activities corrected for base-line factor activities. The mean factor IX clearance rate in eight factor IX-deficient patients was 233 mL/h (range 159 to 340 mL/h). The mean normalized clearance rate was 3.4 mL/h/kg. The mean factor VIII clearance rate in eight factor VIII-deficient patients was 294 mL/h (range 229 to 361 mL/h) and the mean normalized rate was 5.0 mL/h/kg. Both factors show linear relationships between the factor infusion rates and the steady-state plasma factor activities achieved.

Factor IX↗