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Identification of surface epitopes of human coagulation factor Va that are important for interaction with activated
Kenneth Segers1, Björn Dahlbäck, Jan Rosing
1Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, 6200MD, The Netherlands.
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.
Insights
Activated protein C (APC) inactivates factor Va (FVa) to regulate thrombin. Heparin influences APC cleavage sites on FVa, with specific residues crucial for this interaction.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Factor Va (FVa) inactivation by activated protein C (APC) is critical for controlling thrombin generation.
- APC cleaves FVa at specific sites (Arg306, Arg506, Arg679), reducing its cofactor activity.
- Heparin modulates APC's action on FVa, inhibiting cleavage at Arg506 and stimulating it at Arg306.
Purpose of the Study:
- To investigate the role of specific FVa amino acids in APC-mediated inactivation.
- To elucidate the mechanism by which heparin influences APC cleavage sites on FVa.
- To identify key residues involved in the interaction between FVa and APC, particularly in the presence of heparin.
Main Methods:
- Three-dimensional molecular modeling of APC docked to FVa cleavage sites.
- Site-directed mutagenesis of FVa, specifically targeting residues Lys320, Arg321, and Arg400.
- Assays to measure APC-mediated FVa inactivation rates in the presence and absence of heparin and protein S.
Main Results:
- Mutagenesis of Lys320, Arg321, and Arg400 to Ala increased APC inactivation at Arg306.
- These basic residues (Lys320, Arg321, Arg400) are essential for heparin-mediated stimulation of Arg306 cleavage.
- Mutations near the Arg506 site showed modest effects, suggesting an extended interaction surface for this cleavage site.
Conclusions:
- A cluster of basic residues in FVa (Lys320, Arg321, Arg400) is required for heparin's stimulatory effect on Arg306 cleavage by APC.
- The interaction surface between FVa and APC is extended, particularly around the Arg506 cleavage site.
- Understanding these interactions provides insights into the regulation of coagulation and the role of heparin in thrombosis.
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