HUMAN PLACENTAL ANTICOAGULANT PROTEIN - ISOLATION AND CHARACTERIZATION
HUMAN PLACENTAL ANTICOAGULANT PROTEIN - ISOLATION AND CHARACTERIZATION
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DOI：
10.1021/bi00391a053
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发表时间：
1987-08-25
期刊：
影响因子：
2.9
通讯作者：
FUJIKAWA, K
中科院分区：
文献类型：
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作者：
FUNAKOSHI, T;HEIMARK, RL;FUJIKAWA, K
用硫酸铵沉淀、DEAESepharoseSephadex G-75和Mono S(Pharmacia)柱层析，从人胎盘可溶性部分中分离纯化出一种抗凝蛋白。从一个胎盘中纯化的蛋白质产量约为20 mg。纯化后的蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法鉴定为单一条带，相对分子质量为36,500。这种蛋白可延长正常血浆的凝血时间，当凝血是由脑血栓活酶或高岭土在脑磷脂和钙离子存在下引起的。还可延长凝血因子Xa诱导的富含血小板血浆的凝血时间，但不影响凝血酶诱导的纤维蛋白原向纤维蛋白的转化。纯化的胎盘蛋白可完全抑制凝血酶原酶的激活，凝血酶原酶是Xa因子Va磷脂钙离子的复合体。去掉磷脂后，胎盘抑制物对凝血酶原激活无影响。此外，它既不抑制Xa因子的氨解活性，也不与Xa因子结合。然而，在钙离子存在的情况下，胎盘抑制剂确实能与磷脂小泡(20%磷脂酰丝氨酸和80%磷脂酰胆碱)特异性结合。这些结果表明，胎盘抗凝蛋白(PAP)通过与磷脂小泡结合而抑制凝血。PAP的三个溴化氰片段的氨基酸序列与Lipocortin I和Lipocortin II的两个不同区域的氨基酸序列具有高度的同源性，表明PAP是Lipocortin家族的成员。
An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospoholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospoholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.