Abstract The effect of the Arg506 → Gln mutation in factor VLEIDEN on thrombin generation was evaluated in a reconstituted system using the purified components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway. Recombinant full-length tissue factor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the protein C pathway on TF-initiated thrombin generation. Thrombin generation initiated by 1.25 pm factor VIIa·TF in the absence of the protein C pathway components occurs following an initiation phase, after which prothrombin is quantitatively converted to 1.4 μm thrombin. The factor VLEIDEN mutation did not influence thrombin generation in the reconstituted model in the absence of the protein C pathway. In the presence of 2.5 nmTFPI, 65 nm protein C, and 10 nm recombinant soluble thrombomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nmthrombin. In contrast, persistent thrombin generation was observed in the presence of factor VLEIDEN in the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm. The rate of thrombin generation with factor VLEIDEN increased with time and ultimately resulted in quantitative prothrombin activation. When the TFPI concentration was reduced to 1.25 nm, thrombin generation is still curtailed in the presence of normal factor V. In contrast, under similar conditions using factor VLEIDEN, the protein C pathway totally failed to down-regulate thrombin generation. The dramatic effect of a 50% reduction in TFPI concentration on the inhibitory potential of the protein C pathway on thrombin generation catalyzed by factor VLEIDEN suggests that the observed synergy between TFPI and the protein C pathway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg506. This cleavage appears to have a dramatic regulatory ...