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 h ad the same cofactor activity in prothrombin activation as normal fact or 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 act ivity 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 v ia a monophasic reaction (k = 1.7 x 10(6) M(-1)s(-1)). Immunoblot anal ysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactiva tion 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 wit h cleavage at Arg(306). Cleavage at Arg(506) in normal factor Va resul ted in accumulation of a reaction intermediate that exhibited 40% cofa ctor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the r eaction intermediate retained virtually no cofactor activity at low fa ctor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg(506) is impaired in its ability to interact with fa ctor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg (506) in membrane-bound factor Va was characterized by a low K-m for f actor 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 inactiv ation 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 ch ain also contributed to factor Va inactivation. Comparison of rate con stants for APC-catalyzed cleavage at Arg(306), Arg(506) and Arg(679) i n the absence and presence of phospholipids indicated that phospholipi ds accelerated these cleavages to a different extent. This indicates t hat the binding of factor Va to phospholipids changes the accessibilit y of the cleavage sites and/or the sequence of peptide bond cleavage b y APC.