TF mRNA in human platelets and PBMCs. Platelet and PBMC RNAs were extracted before and after stimulation with 5 μM TRAP for 15 minutes. PBMCs were also stimulated for 2 hours with LPS. (A) Amplification of mRNA transcripts in nonstimulated and TRAP-activated platelets. β-actin represents housekeeping transcripts. In this particular experiment, TF mRNA was detected after TRAP stimulation but not in nonstimulated platelets. The platelet origin of TF mRNA was demonstrated by the amplification of GPIbα mRNA in nonstimulated platelets, by the TF mRNA increase after TRAP activation, and by the lack of transcripts for monocyte CD14 mRNA. (B) PCR amplification of cDNAs of nonstimulated and LPS-stimulated PBMC preparations for 2 hours. CD14 mRNA bands are distinctive of monocytes. TF mRNA was barely detectable in unstimulated PBMCs, although markedly increased after 2 hours of stimulation with LPS. Platelet mRNA in the PBMC suspensions was demonstrated by the observation of GP-1bα amplification in nonstimulated and activated samples. (C) Reverse transcriptase–PCR products of nonactivated and 5 μM TRAP-activated PBMC suspensions. TF mRNA was only slightly enhanced after TRAP stimulation when compared with LPS stimulation in panel B. This mild increase was observed in 3 of 8 experiments, whereas in the remaining 5 experiments, it was unnoticed. Barely visible GPIbα mRNA transcripts were detected, confirming platelet contamination in PBMC preparations. The amplification products were run with a 100-bp ladder in 8% polyacrylamide gel and were silver stained. Negative controls without template showed no amplification products (not shown). gDNA products of β-actin, TF, and CD14 (Table 1) were undetected in all the experiments.