The complex interplay between the intrinsic and extrinsic pathways of coagulation is incompletely understood. The existence of Factor X variants that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma,

De Stefano et al., Br J Haematol. 1988, 69(3):387–91
), is due to a missense mutation (Thr318→Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3%) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50%) in the extrinsic pathway as measured by PT. Using site-directed mutagenesis and transient transfection in HEK 293 cells, we confirmed this differential activity in extrinsic and intrinsic pathways for a variant human Factor X (FX) recombinant protein with a Thr318→Met change. FX antigen was assessed by ELISA as previously described (
Larson PJ et al., Biochemistry. 1998, 37(14):5029–38
) and FX activity by PT or aPTT; analysis of conditioned medium from FX wild-type and FX Thr318→Met showed respectively: antigen 100%, 82%; intrinsic activity 120 U/mg (100%), 1.8 U/mg (1.5%); extrinsic activity 190 U/mg (100%), 81 U/mg (41%). To further study this variant, we constructed a mouse homozygous for the analogous mutation (Thr318→Met) in the murine FX gene. This residue is conserved in human, murine and canine FX. We utilized mouse ES cells in which exon 8 of the murine FX gene had previously been deleted and replaced with a neomycin resistance gene and a partial HPRT gene through a targeted recombination event. These cells were used for a second electroporation event with a targeting construct carrying an exon 8 cassette containing the Thr318→Met substitution, and the missing portion of the HPRT gene. After selection on HAT medium, correctly targeted ES cells were micro-injected into blastocysts and implanted into pseudopregnant mice to generate chimeric mice. Heterozygous offspring of chimeric males were intercrossed to obtain animals homozygous for the FXRoma mutation. Coagulation testing revealed that FXRoma heterozygotes showed the expected 52.1±5.9% intrinsic activity and 142.8±25.1% extrinsic activity. More importantly, FXRoma homozygotes showed 8.5±2.7% intrinsic activity and 107.7±41.1% extrinsic activity. Thus the murine FXRoma mutation demonstrates a coagulation profile similar to that observed with the human variant. Thus the FXRoma mouse can model the effect of impeded intrinsic pathway more accurately than e.g. a FIX knockout mouse, because both FX and FIX can act as substrates for the Factor VIIa-Tissue Factor complex and all the components of the Xase complex will be essentially at normal or near-normal levels. The ability to carry out studies of long-term effects of an impeded pathway, and to analyze the response of these animals to prothrombotic stimuli, will allow us to determine the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway.

Topics:
culture media, conditioned, gypsies, mice, representational oligonucleotide microarray analysis, activated partial thromboplastin time measurement, antigens, factor x, hypoxanthine phosphoribosyltransferase, bleeding diathesis, blood coagulation tests

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2005, The American Society of Hematology
2005
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