title: Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.
abstract: Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506-->Gln (factor VR506Q). Factor VR506Q 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-1s-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-1s-1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 x 10(6) M-1s-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 Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 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 Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s-1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km = 196 nM and kcat = 0.37 s-1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
authors.forename: G A
authors.initials: GA
authors.lastname: Nicolaes
authors.forename: G
authors.initials: G
authors.lastname: Tans
authors.forename: M C
authors.initials: MC
authors.lastname: Thomassen
authors.forename: H C
authors.initials: HC
authors.lastname: Hemker
authors.forename: I
authors.initials: I
authors.lastname: Pabinger
authors.forename: K
authors.initials: K
authors.lastname: Varadi
authors.forename: H P
authors.initials: HP
authors.lastname: Schwarz
authors.forename: J
authors.initials: J
authors.lastname: Rosing
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D015943:Factor Va / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism
chemical_list: D010455:Peptides; D010743:Phospholipids; D011486:Protein C; D015943:Factor Va; D011516:Prothrombin; D001120:Arginine; D005998:Glycine