MOLECULAR MECHANISMS OF ACTIVATED PROTEIN-C RESISTANCE - PROPERTIES OF FACTOR-V ISOLATED FROM AN INDIVIDUAL WITH HOMOZYGOSITY FOR THE ARG(506) TO GIN MUTATION IN THE FACTOR-V GENE

Resistance to activated protein C (APC), which is the most prevalent p athogenetic risk factor of thrombosis, is linked to a single point-mut ation in the factor V (FV) gene, which predicts replacement of Arg (R) at position 506 with a Gln (Q). This mutation modifies one of three A PC-cleavage sites in the heavy chain of activated FV (FVa), suggesting that mutated FVa (FVa:Q(506)) is at least partially resistant to APC- mediated degradation. To elucidate the molecular mechanisms of APC-res istance and to investigate the functional properties of FV in APC resi stance, FV:Q(506) was purified from an individual with homozygosity fo r the Arg to Gln mutation. Intact and activated FV:Q(506) were demonst rated to convey APC resistance to FV-deficient plasma. Thrombin- or fa ctor Xa-activated FV:Q(506) were found to be approx. 10-fold less sens itive to APC-mediated degradation than normal FVa, at both high and lo w phospholipid concentrations. The degradation pattern observed on Wes tern blotting suggested that FVa:Q(506) was not cleaved at position 50 6. However, it was slowly cleaved at Arg(306), which explains the part ial APC sensitivity of FVa:Q(506). FV is initially activated during cl otting and then rapidly inactivated in a process which depends on the integrity of the protein C anticoagulant system. During clotting of AP C-resistant plasma, FV:Q(506) was activated in a normal fashion, but t hen only partially inactivated. In conclusion, the reduced sensitivity of FVa:Q(506) to APC-mediated degradation is the molecular basis for the life-long hypercoagulable state which constitutes a risk factor fo r thrombosis in APC-resistant individuals.