Identification of the binding site for activated protein C on the light chain of factors V and VIII.
Activated protein C has been observed to bind to the light chains of factor Va and factor VIII. Fragments of the factor VIII light chain were produced by recombinant DNA techniques and expressed in Escherichia coli. Three fragments of the light chain were studied; L4 (residues 1974-2332), L3.2 (residues 1560-1829 and 2046-2332), and L3.3 (residues 1560-2052). Two fragments, L4 and L3.3, which overlapped sequences between residues 1974-2052, inhibited the anticoagulant activity of activated protein C. Comparison of the sequences of factors V and VIII in this region revealed that residues 2005-2018 in the factor VIII sequence were homologous with residues 1861-1874 in the factor V sequence. The peptides Arg-Ala-Gly-Met-Gln-Thr-Phe-Leu-Ile (RAGMQTPFLI; residues 1865-1874) from the factor V sequence and His-Ala-Gly-Met-Ser-Thr-Leu-Phe-Ile-Val (HAGMSTLFIV; residues 2009-2018) from the factor VIII sequence were synthesized. Both peptides were observed to inhibit the anticoagulant activity of activated protein C and its inactivation of factors Va and VIII. Furthermore RAGMQTPFLI quenched the fluorescence of the dansyl-Glu-Gly-Arg-modified protease. Polyclonal antibodies against RAGMQTPFLI bound to factor Va and inhibited the anticoagulant activity of activated protein C and the inactivation of factor Va. These results indicate that a portion of the binding sites for activated protein C on the light chains of factors V and VIII are contained in the sequences RAGMQTPFLI or HAGMSTLFIV, respectively.
Walker
F J
FJ
American Red Cross Blood Services, Farmington, CT 06032.
Scandella
D
D
Fay
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004789:Enzyme Activation; D005165:Factor V / Q000378:metabolism*; D005167:Factor VII / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D017293:Protein S; D011994:Recombinant Proteins / Q000378:metabolism
D006023:Glycoproteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins; D005165:Factor V; D005167:Factor VII