CHARACTERIZATION OF THE MOLECULAR DEFECT IN FACTOR V-R506Q

A poor anticoagulant response of plasma to activated protein C is correlated with a single mutation in the factor V molecule (Arg(506) --> Gln), Factor V was purified to homogeneity from plasma of two unrelated patients (patient I, factor V-I, and patient II, factor V-II), who are homozygous for this mutation, The factor V molecule from both patients has normal procoagulant activity when compared with factor V isolated from normal plasma in both a clotting time-based assay and in an assay measuring alpha-thrombin formation, The cleavage and subsequent inactivation by activated protein C (APC) of the alpha-thrombin-activated membrane-bound cofactor (factor Va) from both patients were analyzed and compared with the cleavage and inactivation of normal human factor Va, In normal factor Va, cleavage at Arg(506) generates a M(r) = 75,000 fragment and a M(r) = 28,000/26,000 doublet and is necessary for the optimum exposure of the sites for subsequent cleavage at Arg(306) and Arg(679), Proteolysis at these sites leads to the appearance of M(r) = 45,000 and 30,000 fragments and a M(r) = 22,000/20,000 doublet. Cleavage at Arg(306) is membrane-dependent and is required for complete inactivation, Following 5 min of incubation with APC (5.4 nM) membrane-bound normal factor Va (280 nM) has virtually no cofactor activity whereas under similar experimental conditions factor Va(I) and factor Va(II) retain approximately 50% of their initial activity, After 1 h of incubation with APC, factor Va(I) retains 20% of its initial cofactor activity whereas factor Va(II) has 10% remaining cofactor activity, The initial loss in cofactor activity (similar to 70%) of membrane-bound factor Va(I) and factor Va(II) during the first 10 min of the inactivation reaction is correlated with cleavage at Arg(306) and appearance of a M(r) = 45,000 fragment and a M(r) = 62,000/60,000 doublet, Subsequently, the M(r) = 62,000/60,000 doublet is cleaved at Arg(679) to generate a M(r) = 56,000/54,000 doublet resulting in complete loss of cofactor activity. Both procofactors, factor V-I and factor V-II were inactivated following cleavage at Arg(306) and Arg(679), With APC inactivation rates equivalent to those observed for normal factor V. Our data demonstrate that; 1) cleavage of Arg(506) is required for optimum exposure of the cleavage sites at Arg(306) and Arg(679) and rapid inactivation of membrane-bound factor Va; and 2) cleavage at Arg(306) by APC On membrane-bound factor V occurs at the same rate in both normal and APC-resistant individuals, Thus cleavage at Arg(306) and Arg(679) and subsequent inactivation of the membrane-bound procofactor, factor V, does not require prior cleavage at Arg(506) for optimum exposure.