Abstract
Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.
Highlights
The protein C pathway provides a major anticoagulant mechanism for the regulation of hemostasis by nullification of the procoagulant activities of FVIIIa and factor Va (FVa), the cofactors in the tenase and prothrombinase complexes, respectively
Three-dimensional models of activated protein C (APC) docked at the Arg306 and Arg506 cleavage sites in FVa suggest that heparin prevents optimal docking of APC at Arg506 and stimulates cleavage at Arg306 by bridging electropositive regions in FVa and APC [9]
We have recently shown that heparin alters the APC-catalyzed inactivation of FVa by inhibiting cleavage at Arg506 and stimulating cleavage at Arg306 [9]
Summary
Beside its anticoagulant and anti-inflammatory functions, heparin regulates APC activity by stimulating the inhibition of APC by the serpin protein C inhibitor [2, 19, 20]. It has been shown that heparin alters the pathway of APC-catalyzed FVa inactivation by inhibition of the Arg506 cleavage and stimulation of the Arg306 cleavage in FVa [9]. This latter inhibition has been suggested to contribute to the less favorable effects of APC in heparin-cotreated sepsis patients, as were studied in the PROWESS trial [18, 21]. We have used site-directed mutagenesis to show that FVa residues Lys320, Arg321, and Arg400 are important for the heparin-mediated stimulation of FVa cleavage at Arg306 by APC. The most prominent effect was observed for the FVa mutants with mutations in an acidic cluster spanning from residues 659 to 663, which is likely part of an extended FVa:APC binding interface
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