Localization of functionally important epitopes within the second C-type domain of coagulation factor V using recombinant chimeras.
Coagulation factor V, an integral component of the prothrombinase complex, possesses two C-type domains at the carboxyl-terminal end of the molecule. Homologous C-type domains are present in factor VIII as well as several non-coagulation proteins. Deletion of the second C-type domain of factor V results in the loss of procoagulant activity and the ability to bind phosphatidylserine. We now report the effect of substitution of all or a portion of the C2 domain of factor V with the corresponding regions of factor VIII or the human breast carcinoma protein BA46. Substitution of the entire domain with a heterologous C2 domain does not restore significant procoagulant activity, although smaller, exon-size substitutions do result in chimeras with partial activity (approximately 10% of factor Va). Using chimeras with partial substitutions, we determined that the amino-terminal region of the domain is involved in binding to phosphatidylserine. In contrast, the central region of the domain is not involved in phosphatidylserine binding, but an antibody binding at or near this site inhibits procoagulant activity, suggesting that this region is involved in a separate function. Lastly, the molecular basis for the light chain doublet, which is important in the expression of full procoagulant activity, is located within the carboxyl-terminal region of the C2 domain.
Ortel
T L
TL
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Quinn-Allen
M A
MA
Keller
F G
FG
Peterson
J A
JA
Larocca
D
D
Kane
W H
WH
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005165:Factor V / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000145:classification; D046911:Macromolecular Substances; D008970:Molecular Weight; D011817:Rabbits / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D005165:Factor V