INCREASED TISSUE FACTOR-INITIATED PROTHROMBIN ACTIVATION AS A RESULT OF THE ARG(506)-]GLN MUTATION IN FACTOR-V-LEIDEN

The effect of the Arg(506) --> Gln mutation in factor V-LEIDEN on thro mbin generation was evaluated in a reconstituted system using the puri fied components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway, Recombinant full-length tissue fa ctor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the pro tein C pathway on TF-initiated thrombin generation, Thrombin generatio n initiated by 1.25 pM factor VIIa.TF in the absence of the protein C pathway components occurs following an initiation phase, after which p rothrombin is quantitatively converted to 1.4 mu M thrombin, The facto r V-LEIDEN mutation did not influence thrombin generation in the recon stituted model in the absence of the protein C pathway, In the presenc e of 2.5 nM TFPI, 65 nM protein C, and 10 nar recombinant soluble thro mbomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nM thrombin, In contrast, persistent thrombin generation was observed in the presence of factor V-LEIDEN in the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm, The ra te of thrombin generation with factor V-LEIDEN increased with time and ultimately resulted in quanti tative prothrombin activation, When the TFPI concentration was reduced to 1.25 nM, thrombin generation is sti ll curtailed in the presence of normal factor V, In contrast, under si milar conditions using factor V-LEIDEN, 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 V-LEIDEN suggests that the observed synergy between TFPI and the protein C pat hway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg(506). This cleavage appears to have a dramatic regulatory effect in the presence of low concentrations of TFPI, Markedly increased thrombin generation in the presence of both 1 .25 nM TFPI and factor V-LEIDEN was also observed when antithrombin-II I was added to the system to complete the natural set of coagulation i nhibitors, Protein S (300 nM) had a minimal effect in the model on the inhibition of thrombin generation by protein C, Tm, and TFPI, with ei ther normal factor V or factor V-LEIDEN. Protein S also failed to sign ificantly potentiate the action of the protein C pathway in the presen ce of antithrombin-III in reactions employing normal factor V or facto r V-LEIDEN. The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the r eported interactions of protein S with coagulation factors are not dec isive in the reaction, Altogether the data predict that TFPI levels in the lower range of normal values are a risk factor for thrombosis whe n combined with the Arg(506) --> Gln mutation in factor V-LEIDEN.