CHARACTERIZATION OF THE INTRINSIC FIBRINOLYTIC PROPERTIES OF PRO-UROKINASE THROUGH A STUDY OF PLASMIN-RESISTANT MUTANT FORMS PRODUCED BY SITE-SPECIFIC MUTAGENESIS OF LYSINE-158
CHARACTERIZATION OF THE INTRINSIC FIBRINOLYTIC PROPERTIES OF PRO-UROKINASE THROUGH A STUDY OF PLASMIN-RESISTANT MUTANT FORMS PRODUCED BY SITE-SPECIFIC MUTAGENESIS OF LYSINE-158
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DOI：
10.1172/jci113815
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发表时间：
1988-12-01
影响因子：
15.9
通讯作者：
MAO, JI
中科院分区：
文献类型：
--
作者：
GUREWICH, V;PANNELL, R;MAO, JI
两个纤溶酶抗性突变体形式的尿激酶原（pro-UK）的构建通过定点突变的赖氨酸158到Val 158和Met 158被用来评估固有的酶和纤溶特性的pro-UK作为不同于其双链UK（TC-UK）衍生物。这两种突变体，而抵抗纤溶酶激活，是作为敏感的促尿激酶降解凝血酶。由于凝血酶仅从激活位点切割两个残基的肽键，因此在两个突变体中保持了该环的完整性。突变体的酰胺水解活性和纤溶酶原激活活性分别为TC-UK的0.14%和0.12%。Met 158和Val 158突变体的纤维蛋白平板活性分别为2,400 IU/ml和700 IU/mg，约为TC-UK的1.5%。这些发现证明了pro-UK的离散但低的固有活性，并表明文献中报告的较高值可能与测定期间UK污染物或纤溶酶诱导的TC-UK生成有关。突变体的凝块溶解需要比pro-UK高> 100倍的剂量才能诱导相当的效果。由此看来，pro-UK活化是pro-UK发生的凝块溶解速率的主要决定因素。通过纤溶酶预处理纤维蛋白和通过加入少量的TC-UK或组织纤溶酶原激活剂（t-PA），增强突变体的凝块溶解。t-PA和突变体的组合在其纤溶作用中具有协同作用。这些发现反映了以前从亲英国获得的结果。我们的结论是，先前描述的增强促尿激酶诱导的血块溶解的UK或t-PA介导的主要是促尿激酶本身，而不是通过促进其激活。
Two plasmin-resistant mutant forms of pro-urokinase (pro-UK) constructed by site-directed mutagenesis of Lys158 to Val158 and Met158 were used to evaluate the intrinsic enzymatic and fibrinolytic properties of pro-UK as distinct from those of its two-chain UK (TC-UK) derivative. Both mutants, while resistant to plasmin activation, were as sensitive as pro-UK to degradation by thrombin. Since thrombin cleaves a peptide bond only two residues from the activation site, the integrity of this loop was maintained in the two mutants. The amidolytic and plasminogen-activating activities of the mutants averaged 0.14 and 0.12% that of TC-UK, respectively. The fibrin plate activities were 2,400 IU/ml and 700 IU/mg for the Met158 and Val158 mutants or about 1.5% that of TC-UK. These findings attest to a discrete but low intrinsic activity for pro-UK and suggest that the higher values reported in the literature may be related to UK contaminants or plasmin-induced TC-UK generation during the assay. Clot lysis by the mutants required doses > 100-fold higher than those of pro-UK to induce a comparable effect. From this it appears that pro-UK activation is a major determinant of the rate of clot lysis occurring with pro-UK. Clot lysis by the mutants was potentiated by plasmin pretreatment of the fibrin and by the addition of small amounts of TC-UK or tissue plasminogen activator (t-PA). Combinations of t-PA and the mutants were synergistic in their fibrinolytic effects. These findings mirror those previously obtained with pro-UK. We concluded that the previously described potentiation of pro-UK-induced clot lysis by UK or t-PA is mediated primarily by pro-UK itself rather than by a promotion of its activation.