Structure and Function of Placental Anticoagulant Protein (PAP)

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1. Placental Anticoagulant Protein (PAP) was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephdex G-75, and Mono S. The yield of the purified protein was approximately 20 mg from one placenta.2. PAP is a member of "annexin" family of Ca^<2+>-dependent phospholipid binding proteins, and it inhibits the extrinsic and intrinsic pathways of blood coagulation.3. PAP rapidly lost its anticoagulant effect due to photooxidation in the presence of methylene blue at pH7.9 and 8 ^ﾟC. Photooxidized PAP failed to bind the phospholipid vesicle.4. Photooxidized PAP had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged.5. The peptides which contain a histidine residue, SHLRKV and DHTLIR, corresponding to PAP residues 204-209 and 266-271, respectively, are included in the functional site of PAP and exhibit anticoagulant activity, but the peptides in which alanine is substituted for histidine does not. However, the peptide KHALKG, corresponding from Lys-97 to Gly-102, did not exhibit an anticoagulant activity, showing that it is not included in the functional site. It was suggested that His-205 and His-267 should be involved in the Ca^<2+>- or phospholipid-binding site of PAP, but His-98 was not.6. Monoclonal antibody against PAP was prepared according to the method of Kohler & Milstein.7. PAP was detected in human liver, kidney, colon and endotherial cells, and was located in the cell membrane of the cultured carcinoma cells.