Factor Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306, Arg-506, and Arg-679.
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.
van der Neut Kolfschoten
Marijn
M
Hemostasis and Thrombosis Research Center, Department of Hematology, Leiden University Medical Center, 2300RC Leiden, The Netherlands. mneut@lumc.nl
Dirven
Richard J
RJ
Vos
Hans L
HL
Tans
Guido
G
Rosing
Jan
J
Bertina
Rogier M
RM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D019556:COS Cells; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D015944:Factor VIIIa / Q000737:chemistry*; D015943:Factor Va / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry* / Q000378:metabolism; D017293:Protein S / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins; D005973:Glutamine; D015943:Factor Va; D015944:Factor VIIIa; D001120:Arginine; D013917:Thrombin; D005998:Glycine