Tissue Factor Gene Expression Analysis in Circulating Monocytes*

Abstract

Background: There is growing evidence that an increasein stimulated and tissue factor(TF)-expressing monocytesdrive the coagulation system to a hypercoagulablestate. In order to further investigate this phenomenon,methods are needed that allow sensitive, reliable andspecific quantification of TF-expressing monocytes. Materialsand Methods: Two procedures of one-step multiplexreal-time RT-PCR were developed that allow absolutequantification of TF, CD14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA transcripts.In order to minimize preanalytical changes of the mRNAprofile, a blood sampling system which is based on integratedRNA stabilization was used. Results: An in vitrolipopolysaccharide (LPS) model was used for initialassay evaluation. LPS-induced TF gene expression patternswere similar to previously described findings. Withthe use of the whole blood RNA stabilization system, themarker transcripts were preserved for up to 24 h at roomtemperature and for up to 5 days at 4 °C. Monocytic TFtranscription analysis in different patient groups identifiedsignificant higher levels in thrombophilic patientsthan in a representative control population. In all patientsand control probands analyzed we found significantlyhigher levels of TF transcripts in carriers of the G allele ofthe TF-603A/G promotor polymorphism. Conclusion: Thenewly developed RT-PCR allows sensitive, reliable andspecific quantification of TF transcripts in whole bloodsamples.