Analysis of fibrinolytic activation mechanism by staphylokinase in gene deficient mice

葡激酶（SAK）通过与纤溶酶原（Pig）或纤溶酶形成复合物来表达纤溶酶原激活物（PA）活性。虽然这种PA活性被α 2-抗纤溶酶（α 2-AP）抑制，但在纤维蛋白存在下，其活性不受α 2-AP的抑制。利用突变体SAK、合成的SAK肽、几种动物的纯化猪和基因缺陷小鼠血浆研究了SAK与猪的相互作用。突变体SAK与猪的相互作用：SAK氨基端氨基酸缺失突变体（14 SAK、18 SAK和25 SAK）由于缺失前14、18和25个氨基酸而收缩。SAK的11 -14个氨基酸区域与Glu-plg结合后是表达PA活性所必需的。合成的SAK肽与猪的相互作用，合成的SAK肽由SAK氨基酸序列的10-30个氨基酸组成。合成的SAK肽不显示PA活性。然而，SAK的合成十九肽（Glu 22-Leu 4 O）与Glupig猪结合并增强天然SAK对猪的激活。α 2-AP和猪α 2-AP基因缺陷小鼠：通过克隆和靶向鼠α 2-AP基因获得α 2-AP基因缺陷小鼠（α 2-AP-/-）。建立了猪基因缺陷小鼠（Pig-/-）的繁育方法和猪基因在小鼠体内的检测方法。SAK与纯化猪在缓冲液中及在α 2-AP或猪缺陷小鼠血浆中的相互作用。在缓冲液和α 2-AP-/-或猪-/-小鼠血浆中研究了SAK和纯化猪之间的相互作用。这些发现表明（1）鼠或牛plg不与SAK反应，（2）SAK-人纤溶酶复合物激活鼠或牛plg，和（3）这种鼠plg激活被鼠α 2-AP抑制。

Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasminogen (Pig) or plasmin. Although this PA activity is inhibited by alpha2-antiplasmin (alpha2-AP), its activity is protected against the inhibition by alpha2-AP in the presnce of fibrin. The interaction between SAK and Pig were investigated using mutant SAKs, synthetic SAK peptides, purified pigs of several animals and gene deficient mice plasma.1). The interaction between mutant SAKs and pig.The deleted mutants of NH2-terminal amino acid region of SAK contracted by lacking the first 14, 18 and 25 amino acid region ( 14 SAK, 18 SAK and 25 SAK). The11-14 amino acid region of SAK was necessary to express PA activity after binding with Glu-plg.2). The interaction between synthetic SAK peptides and pig.The SAK peptides synthesized peptides consisted 10-30 amino acid in SAK amino acid sequence. The synthetic SAK peptides did not show PA activity. However, the synthetic nonadecapeptide (Glu22-Leu4O) of SAK bound to Glupig pig and enhanced pig activation by native SAK.3). The alpha2-AP and Pig gene deficient mice.The alpha2-AP gene deficient mice (alpha2-AP-/-) obtained by cloning and targeting of murine alpha2-AP gene. The pig gene deficient mice (Pig-/-)established breeding method and assay methods of pig gene in mice.4). The interaction between SAK and purified pigs in buffer and in alpha2-AP or Pig deficient mice plasma.The pigs purified from plasma of human, mice, rabbit or bovine. The interaction between SAK and purified pigs investigated in buffer and in alpha2-AP-/- or Pig-/- mice plasma. These findings indicate that (1) murine or bovine plg dose not react with SAK, (2) SAK-human piasmin complex activates murine or bovine plg, and (3) this murine plg activation is inhibited by murine alpha2-AP.