Structure and Function of Placental Anticoagulant Protein (PAP)

1. 采用硫酸铵沉淀和DEAE-Sepharose、Sephdex G-75和Mono s柱层析的方法，从人胎盘的可溶性部分中纯化出胎盘抗凝蛋白（PAP），从一个胎盘中纯化出的蛋白产量约为20 mg。PAP是Ca^<2+>依赖性磷脂结合蛋白“膜联蛋白”家族的成员，它抑制血液凝固的外在和内在途径。在pH7.9和8 ^ C条件下，亚甲基蓝的存在导致PAP的光氧化作用迅速丧失。3 .光氧化的PAP不能结合磷脂囊泡。光氧化的PAP显著降低了组氨酸的含量，而其他氨基酸的含量基本保持不变。含有组氨酸残基的肽SHLRKV和DHTLIR，分别对应PAP残基204-209和266-271，被包括在PAP的功能位点并表现出抗凝血活性，而丙氨酸取代组氨酸的肽则没有。然而，从Lys-97到Gly-102对应的肽KHALKG没有表现出抗凝血活性，表明它不包括在功能位点。提示His-205和His-267可能参与PAP的Ca <2+>-或磷脂结合位点，而His-98则不参与。根据Kohler & milstein方法制备抗PAP单克隆抗体。PAP在人的肝、肾、结肠和内皮细胞中均有检测到，并位于培养的癌细胞的细胞膜上。

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.