A FLUOROGENIC THROMBIN GENERATION TEST (FTGT) FOR FACTOR VIII ACTIVITY DEMONSTRATED SUCCESS OF CELL THERAPY WITH LIVER SINUSOIDAL ENDOTHELIAL CELLS (LSEC) IN HAEMOPHILIA A MICE
Abstract number: O-M-066
Faulkner1 L., Follenzi2 A., Gupta2 S., Raut1 S.
11Haemostasis Section, Biotherapeutics Group, National Institute for Biological Standards and Control, Potters Bar, United Kingdom 22Department of Medicine and Pathology, Albert Einstein College of Medicine, New York, United States
How-to-cite Faulkner L, Follenzi A, Gupta S, Raut S. A FLUOROGENIC THROMBIN GENERATION TEST (FTGT) FOR FACTOR VIII ACTIVITY DEMONSTRATED SUCCESS OF CELL THERAPY WITH LIVER SINUSOIDAL ENDOTHELIAL CELLS (LSEC) IN HAEMOPHILIA A MICE. J Thromb Haemost 2007; 5 Supplement 2: O-M-066
Abstract
Introduction: We developed a sensitive assay (FTGT) to detect low levels of plasma Factor VIII activity (<0.001IU/ml [1-2]. To verify the utility of this assay, we studied haemophilia A mice lacking FVIII activity, wild-type healthy mice and haemophilia A mice after transplantation of healthy LSEC.
Methods: Mouse LSEC were isolated from FVB/N-Tie-2-GFP donors by enzymatic liver digestion followed by immunoselection. 2 x106 LSEC were transplanted intraportally in FVB/N or haemophilia A/NOD-SCID mice treated beforehand with monocrotaline to promote cell engraftment and proliferation. The fate of transplanted cells was studied over up to 3 months and FVIII mRNA was detected by RT-PCR. To assay plasma FVIII activity, thrombin generation was initiated by adding to 40mul plasma, 80mul substrate mix [0.238mM fluorogenic substrate, 5nM FIXa, 3mug/ml phospholipid and 7mM Ca2+] followed by fluorimetric readings every 30s for 1h (Exc 390nm; Em 460nm). Area under curve, peak height and time-to-peak were obtained with controls.
Results: Transplanted LSEC engrafted, proliferated and reconstituted up to 20% of the LSEC compartment. RT-PCR showed FVIII mRNA in treated haemophilia A mice. In 11 of 13 (85%) haemophilia mice treated with LSEC transplantation, FTGT showed >10% normal FVIII activity levels, indicating phenotypic correction.
Conclusions: FTGT effectively demonstrated plasma FVIII activity in healthy mice as well as correction of FVIII deficiency in haemophilia A mice. This assay will be appropriate for measuring FVIII activity, including after therapeutic interventions in haemophilia A.
References: 1. McIntosh J, Owens D, Lee C, Raut S, Barrowcliffe TW. Correction of abnormal thrombin generation in FVIII deficient plasma by low concentrations of FVIII. J Thrombos Haemost 2003 1: 1005-11.
2. James PD, Raut S, Rivard GE, Poon MC, Warner M, McKenna S, Leggo J, Lillicrap D. Aminoglycoside Suppression of Nonsense Mutations in Severe Hemophilia. Blood 2005 106(9), 3043-8.
