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Molecular characterization of an extended binding site for coagulation factor Va in the positive exosite of activated
Andrew J Gale1, Alexander Tsavaler, John H Griffin
1Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. agale@scripps.edu
Abstract:
The anticoagulant human plasma serine protease, activated protein C (APC), inhibits blood coagulation by specific inactivation of the coagulation cofactors factor Va (FVa) and factor VIIIa. Site-directed mutagenesis of residues in three surface loops of a positive exosite located on APC was used to identify residues that play a significant role in binding to FVa. Eighteen different residues were mutated to alanine singly, in pairs, or in triple mutation combinations. Mutant APC proteins were purified and characterized for their inactivation of FVa. Three APC residues were identified that provide major contributions to FVa interactions: Lys(193), Arg(229), and Arg(230). In addition, four residues made significant minor contributions to FVa interactions: Lys(191), Lys(192), Asp(214), and Glu(215). All of these residues primarily contribute to APC cleavage at Arg(506) in FVa and play a small role in the interaction of APC with the Arg(306) cleavage site. In conjunction with previously published work, these results define an extensive FVa binding site in the positive exosite of APC that is primarily involved in binding and cleaving at Arg(506) on FVa.
Insights
Activated protein C (APC) inhibits blood coagulation by inactivating factors Va and VIIIa. Key residues Lys193, Arg229, and Arg230 on APC are crucial for binding and cleaving factor Va, defining its binding site.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Activated protein C (APC) is a crucial anticoagulant serine protease.
- APC regulates blood coagulation by inactivating factors Va and VIIIa.
- Understanding APC's interaction with factor Va is vital for thrombosis research.
Purpose of the Study:
- To identify specific residues in APC's positive exosite responsible for binding factor Va (FVa).
- To elucidate the role of these residues in the inactivation of FVa by APC.
- To characterize the FVa binding site on APC.
Main Methods:
- Site-directed mutagenesis was employed to alter 18 residues in APC's surface loops.
- Mutant APC proteins were purified and characterized.
- The inactivation of FVa by mutant APC was assessed.
Main Results:
- Three APC residues (Lys193, Arg229, Arg230) were identified as major contributors to FVa binding.
- Four additional residues (Lys191, Lys192, Asp214, Glu215) showed significant minor contributions.
- These residues primarily mediate APC cleavage at Arg506 on FVa.
Conclusions:
- An extensive FVa binding site on APC's positive exosite has been defined.
- This site is critical for APC's anticoagulant function through FVa inactivation.
- The findings advance our understanding of coagulation regulation and potential therapeutic targets.