P1749 Detection of the Factor V Leiden mutation in septic patients

Abstract

Factor X (FX) is a vitamin K-dependent plasma zymogen, which following activation to factor Xa (FXa), converts prothrombin to thrombin in the blood clotting cascade. It was recently demonstrated that a natural variant of FX carrying the Asp-185 deletion (FX-D185del, chymotrypsinogen numbering) was associated with mild bleeding in a patient with severe FX deficiency. In this study, we expressed FX-D185del in mammalian cells and characterized its properties in appropriate kinetic assays in purified systems. We discovered that while the FX variant can be normally activated by physiological activators; both amidolytic and proteolytic activities of the mutant are dramatically impaired. Interestingly, factor Va (FVa) significantly improved the proteolytic defect when the mutant protease was assembled into the prothrombinase complex. Thus, in contrast to > 50-fold catalytic defect in the absence of FVa, the variant activated prothrombin with only ~ 2.5-fold decreased catalytic efficiency in the presence of the cofactor. The FXa variant dramatically lost its susceptibility to inhibition by antithrombin and tissue factor pathway inhibitor, thus exhibiting ~ 2-3 orders of magnitude lower reactivity with the plasma inhibitors. Further studies revealed that Na+ no longer activates the variant protease, suggesting that the functionally important allosteric linkage between the Na+-binding and the P1-binding sites of the protease has been eliminated. These results suggest that the lower catalytic efficiency of FXa-D185del in the bleeding patient may be partially compensated by the loss of its reactivity with plasma inhibitors, possibly explaining the basis for the paradoxical severe FX deficiency with only mild bleeding tendency for this mutation.