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Recently, new missense mutations in the activated protein C (APC) cleavage sites of human factor V (FV) distinct from the R506Q (FV Leiden) mutation have been reported. These mutations affect the APC cleavage site at arginine (Arg) 306 in the heavy chain of activated FV. Whether these mutations result in APC resistance and are associated with a risk of thrombosis is not clear. The main objective of the present study was to identify the APC-resistant phenotype of FV molecules with different mutations in APC cleavage sites. To study this, recombinant FV mutants were reconstituted in FV-deficient plasma, after which normalized APC-sensitivity ratios (n-APC-SRs) were measured in activated partial thromboplastin time-based and Russell's Viper Venom time-based APC-resistance tests. The mutations introduced in FV were R306G, R306T, R506Q, R679A and combinations of these mutations. Based on the APC-sensitivity ratios, we conclude that the naturally occurring mutations at Arg306 (i.e. FV HongKong and FV Cambridge) result in a mildly reduced sensitivity for APC (n-APC-SR, 0.74-0.87), whereas much lower values (n-APC-SR, 0.41-0.51) are obtained for the mutation at Arg506 (FV Leiden). No effect on the n-APC-SR was observed for the recombinant FV mutant containing the single Ala679 mutation. Because reduced sensitivity for APC, not due to FV Leiden, is a risk factor for venous thrombosis, these data suggest that mutations at Arg306 might be associated with a mild risk of venous thrombosis.

The monocyte is the only circulating cell type capable of initiating blood coagulation by the expression of tissue factor (TF). The mechanism and kinetics of TF mRNA and TF activity induction in human peripheral blood monocytes (HPBM) in response to bacterial lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) were investigated. Northern blot analysis showed that both LPS and PMA induce a transient accumulation of TF mRNA in HPBM, that reaches maximum levels after 3-6 h and rapidly declines thereafter. Nuclear run-on experiments demonstrated that the accumulation of TF mRNA requires de novo transcription of the TF gene. Since cycloheximide alone also caused an increase of TF mRNA levels and gene transcription it is concluded that the transcriptional activation of the TF gene does not require protein synthesis. Using specific protein kinase inhibitors, it was further demonstrated that activation of the protein kinase C pathway is involved in the induction of TF mRNA in HPBM. The accumulation of TF mRNA in LPS-stimulated HPBM is followed by an increase of TF activity on the cell surface. The kinetics of TF mRNA induction were found to be very similar in HPBM stimulated with LPS or PMA. However, in the latter case TF activity appeared considerably later on the cell membrane than in the LPS-stimulated cells. Non-stimulated HPBM contain very low levels of mRNA of the tissue factor pathway inhibitor (TFPI). No induction of TFPI (mRNA, activity or antigen) in HPBM after LPS or PMA treatment was demonstrated. This seems to be in contrast with the earlier observation that the human monocyte cell line U937 produces significant amounts of TFPI in response to treatment with LPS and PMA.