PEPTIDE-BOND CLEAVAGES AND LOSS OF FUNCTIONAL-ACTIVITY DURING INACTIVATION OF FACTOR VA AND FACTOR VA(R506Q) BY ACTIVATED PROTEIN-C

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg(506) --> Gln (factor V-R506Q). Factor V-R506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (<5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 x 10(7) M(-1)s(-1)), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 x 10(6) M(-1)s(-1)). Inactivation of factor Va(R506Q) proceeded via a monophasic reaction (k = 1.7 x 10(6) M(-1)s(-1)). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg(506) and full inactivation of factor Va required subsequent cleavage at Arg(306). The slow monophasic inactivation of factor Va(R506Q) correlated with cleavage at Arg(306). Cleavage at Arg(506) in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg(506) is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg(506) in membrane-bound factor Va was characterized by a low K-m for factor Va (20 nM) and k(cat) = 0.96 s(-1). For cleavage at Arg(306) in factor Va(R506Q) the kinetic parameters were K-m = 196 nM and k(cat) = 0.37 s(-1). This means that differences between APC-catalyzed inactivation of factors Va and Va(R506Q) become much less pronounced at high factor Va concentrations. When factor Va(R506Q) was inactivated by APC in the absence of phospholipids, cleavage at Arg(679) of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg(306), Arg(506) and Arg(679) in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent. This indicates that the binding of factor Va to phospholipids changes the accessibility of the cleavage sites and/or the sequence of peptide bond cleavage by APC.