GAMMA-CARBOXYGLUTAMIC ACID-36 AND ACID-40 DO NOT CONTRIBUTE TO HUMAN FACTOR-IX FUNCTION

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependen t blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their imp ortance, factor LX species lacking these Gla residues were isolated fr om heterologously expressed human factor IX. Using ion-exchange chroma tography, peptide mapping, mass spectrometry, and N-terminal sequencin g, we have purified and identified two partially carboxylated recombin ant factor IX species; factor IX/gamma 40E is uncarboxylated at residu e 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasm a-derived factor IX. As monitored by anti-factor IX:Ca(II)-specific an tibodies and by the quenching of intrinsic fluorescence, all these fac tor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to p hospholipid vesicles composed of phosphatidylserine, phosphatidylcholi ne, and phosphatidylethanolamine. Endothelial cell binding was also si milar in all species, with half-maximal inhibition of the binding of I -125-labeled plasma-derived factor IX at concentrations of 2-6 nM. Fun ctionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to particip ate in the activation of factor X in the tenase complex both with synt hetic phospholipid vesicles and activated platelets. However, Gla 36 a nd Gla 40 represent part of the epitope targeted by anti-factor IX:Mg( II)-specific antibodies because these antibodies bound factor IX prefe rentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These res ults demonstrate that the gamma-carboxylation of glutamic acid residue s 36 and 40 in human factor IX is not required for any function of fac tor IX examined.