In Vitro Spontaneous Thrombin Generation in Human Factor-IX Concentrates

A series of in vitro studies, designed to ascertain the potential in vivo thrombogenicity of human factor IX containing concentrates, is described. Using concentrates obtained from several different centres the fibrinogen clotting time with some preparations was less than 6 hours and/or the recalcification time of normal plasma was shortened. In some preparations however, the plasma recalcification time was lengthened.Further studies revealed that all diluted factor IX concentrates generated thrombin after recalcification, and that the rate of thrombin generation appeared to be characteristic of a particular preparation. This characteristic we have expressed as the TGt50 which is the incubation period in minutes required, after recalcification, to obtain a 50 second clotting time of a fibrinogen substrate. The TGt50 was found to correlate most strongly with recalcification time of celite exhausted plasma (p < 0.001), but no correlation was observed between it and the immunological factor VIII or antithrombin III levels. However some factor IX concentrates contained antithrombin III in a free (biologically active) and in a complexed (inactive) form.Evidence is presented which suggests that the thrombin generation test and recalcification time of celite exhausted plasma may represent suitable in vitro quality control assays for the potential thrombogenicity of factor IX concentrates.

Download Full-text

Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽

10.1182/blood.v73.2.438.bloodjournal732438 ◽

1989 ◽

Vol 73 (2) ◽

pp. 438-445

Author(s):

TD Palmer ◽

AR Thompson ◽

AD Miller

Keyword(s):

Human Factor ◽

Normal Skin ◽

Human Fibroblasts ◽

Retroviral Vectors ◽

Factor Ix ◽

Skin Fibroblasts ◽

Hemophilia B ◽

Clotting Factor ◽

Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Download Full-text

Adenovirus-mediated regulatable Expression of human Factor IX in vitro and in vivo

32nd Hemophilia Symposium Hamburg 2001 ◽

10.1007/978-3-642-18150-4_9 ◽

2003 ◽

pp. 72-80

Author(s):

M. A. Srour ◽

H. Fechner ◽

X. Wang ◽

U. Siemetzki ◽

T. Albert ◽

...

Keyword(s):

Human Factor ◽

Factor Ix ◽

Human Factor Ix

Download Full-text

Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽

10.1182/blood.v87.12.5095.bloodjournal87125095 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5095-5103 ◽

Cited By ~ 114

Author(s):

G Hortelano ◽

A Al-Hendy ◽

FA Ofosu ◽

PL Chang

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Ex Vivo ◽

Cost Effective ◽

Factor Ix ◽

Hemophilia B ◽

Therapy Strategy ◽

Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

Download Full-text

Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽

10.1182/blood.v73.2.438.438 ◽

1989 ◽

Vol 73 (2) ◽

pp. 438-445 ◽

Cited By ~ 121

Author(s):

TD Palmer ◽

AR Thompson ◽

AD Miller

Keyword(s):

Human Factor ◽

Normal Skin ◽

Human Fibroblasts ◽

Retroviral Vectors ◽

Factor Ix ◽

Skin Fibroblasts ◽

Hemophilia B ◽

Clotting Factor ◽

Human Factor Ix

Abstract Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Download Full-text

Construction, Rescue, and Characterization of Vectors Derived from Ovine Atadenovirus

Journal of Virology ◽

10.1128/jvi.77.22.11941-11951.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 11941-11951 ◽

Cited By ~ 17

Author(s):

Peter Löser ◽

Christian Hofmann ◽

Gerald W. Both ◽

Wolfgang Uckert ◽

Moritz Hillgenberg

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Human Factor ◽

Insertion Site ◽

Factor Ix ◽

Vector System ◽

Virus Rescue ◽

Human Factor Ix

ABSTRACT Gene transfer vectors derived from ovine atadenovirus type 7 (OAdV) can efficiently infect a variety of mammalian cells in vitro and in vivo to deliver and express transgenes. However, early OAdV vectors were designed on human mastadenovirus principles prior to the complete characterization of OAdV genes and transcripts. The distinctive arrangement of the OAdV genome has suggested ways to improve OAdV vector design and utility. We therefore developed a cosmid-based approach that allows efficient construction of recombinant ovine atadenovirus genomes in which the transgene is inserted at one of three sites. Viruses were rescued by transfection of viral DNA into a new ovine fetal skin fibroblast producer cell line, HVO156. The suitability of the three insertion sites was compared with respect to virus rescue efficiency, gene expression levels, and genetic stability of the vectors. We found that one vector with a transgene inserted at site 1, between the pVIII and fiber genes, was unstable. Only one vector that carried a transgene at site 2, near the right end of the genome, together with a nearby deletion was rescued. In contrast, several vectors with different transgenes inserted in site 3, between the E4 and RH transcription units, were repeatedly rescued, and these vectors were stable over at least four passages. Transgene orientation in site 3 had only little effect on expression. Finally, a vector carrying a human factor IX cDNA at site 3, when administered intravenously, produced nearly physiological levels of human factor IX in mice. The availability of an efficient method for vector construction and the identification of a new insertion site for virus rescue and gene expression substantially enhance the utility of the OAdV vector system.

Download Full-text

Pharmacokinetics of Human Factor IX in a dog with Severe Hemophilia B.

10.1055/s-0039-1682760 ◽

1977 ◽

Author(s):

P.A. Gentry ◽

A.R. Thompson ◽

A.W. Forrey

Keyword(s):

Human Factor ◽

Numerical Procedure ◽

Factor Ix ◽

Hemophilia B ◽

High Yield ◽

Severe Hemophilia ◽

Activity Data ◽

Human Factor Ix

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.

Download Full-text

In vitro and in vivo study of an Italian highly purified human factor IX concentrate

Thrombosis Research ◽

10.1016/0049-3848(93)90312-c ◽

1993 ◽

Vol 70 ◽

pp. S32

Keyword(s):

Human Factor ◽

Factor Ix ◽

In Vivo Study ◽

Human Factor Ix

Download Full-text

Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure of the first growth factor-like domain of human factor IX.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.10.3609 ◽

1989 ◽

Vol 86 (10) ◽

pp. 3609-3613 ◽

Cited By ~ 44

Author(s):

R. S. Gronke ◽

W. J. VanDusen ◽

V. M. Garsky ◽

J. W. Jacobs ◽

M. K. Sardana ◽

...

Keyword(s):

Growth Factor ◽

Synthetic Peptide ◽

Human Factor ◽

Factor Ix ◽

Human Factor Ix

Download Full-text

Neutrophil Elastase Cleavage of Human Factor IX Generates an Activated Factor IX-Like Product Devoid of Coagulant Function

Blood ◽

10.1182/blood.v92.4.1287 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1287-1296 ◽

Cited By ~ 22

Author(s):

John A. Samis ◽

Eunice Kam ◽

Michael E. Nesheim ◽

Alan R. Giles

Keyword(s):

Active Site ◽

Neutrophil Elastase ◽

Human Factor ◽

Sodium Dodecyl ◽

Factor Ix ◽

Human Neutrophil Elastase ◽

Human Factor Ix ◽

Sds Page

Abstract In preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa -(Arg145) and β-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC). © 1998 by The American Society of Hematology.

Download Full-text