Factor Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306, Arg-506, and Arg-679.

Abstract

Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV.

• Authors: Marijn van der Neut Kolfschoten, Richard J Dirven, Hans L Vos, Guido Tans, Jan Rosing, Rogier M Bertina
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 8 Pg. 6567-75 (Feb 20 2004) ISSN: 0021-9258 [Print] United States
• PMID: 14660667 (Publication Type: Journal Article)

Chemical References

• Antibodies, Monoclonal
• Epitopes
• Phospholipids
• Protein C
• Protein S
• Recombinant Proteins
• Glutamine
• Factor Va
• Factor VIIIa
• Arginine
• Thrombin
• Glycine

Topics

• Animals
• Antibodies, Monoclonal (chemistry)
• Arginine (chemistry)
• Binding Sites
• COS Cells
• Electrophoresis, Polyacrylamide Gel
• Epitopes
• Factor VIIIa (chemistry)
• Factor Va (chemistry)
• Glutamine (chemistry)
• Glycine (chemistry)
• Humans
• Immunoblotting
• Kinetics
• Mutagenesis
• Mutation
• Phospholipids (chemistry)
• Protein Binding
• Protein C (chemistry, metabolism)
• Protein S (chemistry)
• Protein Structure, Tertiary
• Recombinant Proteins (chemistry)
• Temperature
• Thrombin (chemistry)
• Time Factors