Tissue Factor Gene Expression Analysis in Circulating Monocytes*

Background: There is growing evidence that an increase in stimulated and tissue factor(TF)-expressing monocytes drive the coagulation system to a hypercoagulable state. In order to further investigate this phenomenon, methods are needed that allow sensitive, reliable and specific quantification of TF-expressing monocytes. Materials and Methods: Two procedures of one-step multiplex real-time RT-PCR were developed that allow absolute quantification of TF, CD14 and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) mRNA transcripts. In order to minimize preanalytical changes of the mRNA profile, a blood sampling system which is based on integrated RNA stabilization was used. Results: An in vitro lipopolysaccharide (LPS) model was used for initial assay evaluation. LPS-induced TF gene expression patterns were similar to previously described findings. With the use of the whole blood RNA stabilization system, the marker transcripts were preserved for up to 24 h at room temperature and for up to 5 days at 4 °C. Monocytic TF transcription analysis in different patient groups identified significant higher levels in thrombophilic patients than in a representative control population. In all patients and control probands analyzed we found significantly higher levels of TF transcripts in carriers of the G allele of the TF-603A/G promotor polymorphism. Conclusion: The newly developed RT-PCR allows sensitive, reliable and specific quantification of TF transcripts in whole blood samples.