Abstract
Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively. We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation. Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg306 (20-fold). Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg506. Inactivation of factor VaR506Q, which was isolated from the plasma of a homozygous APC-resistant patient and which lacks the Arg506 cleavage site, was also stimulated by protein S but was not affected by factor Xa. This confirms that the target sites of protein S and factor Xa involve Arg306 and Arg506, respectively. Factor Xa completely blocked APC-catalyzed cleavage at Arg506 in normal factor Va (1 nM) with a half-maximal effect (K1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va. In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor VaR506Q were almost annihilated in the presence of factor Xa and protein S. This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition.
Highlights
Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively
We have investigated the effects of protein S and factor Xa on APCcatalyzed factor Va inactivation
Effect of Protein S on Activated protein C (APC)-catalyzed Inactivation of Membrane-bound Factor Va—Inactivation of membrane-bound factor Va by APC occurred via a biphasic reaction that consisted of a rapid phase, which resulted in the formation of a reaction intermediate with partial cofactor activity (40%) that was fully inactivated during the subsequent slow phase
Summary
From the ‡Cardiovascular Research Institute Maastricht, University of Limburg, 6200 MD Maastricht, The Netherlands and ¶Immuno AG, A-1220 Vienna, Austria. In the present study we have determined the effects of protein S and factor Xa on apparent second order rate constants for APC-catalyzed inactivation of normal factor Va and of factor Va that was purified from the plasma of a patient who is homozygous for the mutation Arg506 Gln. The loss of functional activity of factor Va was correlated with the effects of protein S and factor Xa on the cleavage of APC-susceptible peptide bonds in the heavy chain of factor Va by immunoblot analysis.
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