A Direct, Plasmin Independent Assay For Plasminogen Activation
A Direct, Plasmin Independent Assay For Plasminogen Activation
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纤溶酶原激活的直接、不依赖纤溶酶的测定
DOI：
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发表时间：
1984
影响因子：
6.7
通讯作者：
D. Loskutoff
中科院分区：
文献类型：
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作者：
L. Mussoni;D. Lawrence;D. Loskutoff
我们已经修改了直接，125 I-纤溶酶原裂解测定纤溶酶原激活剂（PA），并采用它来比较尿激酶（UK），组织激活剂（TA），和培养的牛主动脉内皮细胞产生的PA。该试验基于在SDS和β-巯基乙醇存在下通过聚丙烯酰胺凝胶电泳显示单链纤溶酶原转化为双链纤溶酶。在反应混合物中加入Triton X-100、白蛋白和抑肽酶，减少了反应物的吸附和水解损失，提高了测定的线性和灵敏度。在这些条件下，纤溶酶形成至少6小时呈线性，在20倍UK浓度范围内呈剂量依赖性，并且比先前报道的UK直接测定法灵敏至少100倍（0.01单位/ml）。在初步实验中，我们确定了10 μ M的Km值为UK和纤溶酶原。在不存在纤维蛋白的情况下，TA对纤溶酶原的激活是最小的，并且与激活剂的浓度无关。然而，在存在纤维蛋白的情况下，（1）活化的初始速率显著增加，（2）需要的TA少100-1000倍，和（3）活化与TA和纤维蛋白的浓度成比例。令人惊讶的是，激活UK和细胞PA部分抑制纤维蛋白。表氨基己酸（EACA; 0.5- 100 mM）刺激激活UK在存在和不存在纤维蛋白的情况下的30- 40%。相反，EACA（0.1- 100 mM）在纤维蛋白存在下抑制TA活性超过90%。然而，在不存在纤维蛋白的情况下，即使在高EACA浓度（IOOmM）下，TA也仅被抑制50%。这些结果表明，1251-纤溶酶原的切割可用作各种PA的直接、灵敏和定量测定，并为研究纤溶酶原激活及其刺激或抑制剂提供了一种新的方法。
We have modified the direct, 125I-plasminogen cleavage assay for plasminogen activator (PA) and employed it to compare urokinase (UK), tissue activator (TA), and PAs produced by cultured bovine aortic endothelial cells. The assay is based on conversion of single chain plasminogen to two chain plasmin as revealed by polyacrylamide gel electrophoresis in the presence of SDS andβ-mercapto- ethanol. Inclusion of Triton X-100, albumin and trasylol in the reaction mixture reduced the adsorptive and hydrolytic loss of reactants, and increased the linearity and sensitivity of the assay. Under these conditions, plasmin formation was linear for at least 6 hrs, dose- dependent over a 20-fold range of UK concentrations, and at least 100-fold more sensitive (0.01 units/ml) than previously reported direct assays for UK. In preliminary experiments, we determined a Km value of 10yM for UK and plasminogen. Activation of plasminogen by TA was minimal in the absence of fibrin, and independent of the concentration of activator. However, in the presence of fibrin, (1) the initial rate of activation increased dramatically, (2) 100-1000 fold less TA was required, and (3) activation was proportional to the concentration of both TA and fibrin. Surprisingly, activation by UK and cellular PAs was partially inhibited by fibrin. Epsilon amino caproic acid (EACA; 0.5-100mM) stimulated activation by UK both in the presence and absence of fibrin by 30-40%. In contrast, EACA (0.1-100mM) inhibited TA activity in the presence of fibrin by over 90%. However, in the absence of fibrin, TA was inhibited by only 50%, even at high EACA concentrations (lOOmM). These results indicate that cleavage of 1251- plasminogen can be employed as a direct, sensitive and quantitative assay for various PAs, and offers a new approach for studying plasminogen activation and agents that stimulate or inhibit it.
DOI：
10.1073/pnas.80.10.2956
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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作者：
LOSKUTOFF, DJ;VANMOURIK, JA;LAWRENCE, D
通讯作者：
LAWRENCE, D
培养的正常人内皮细胞和平滑肌细胞中纤溶酶原激活剂形式的免疫学鉴定和比较。
影响因子：
7.5
作者：
Booyse,FM;Scheinbuks,J;Radek,J;Osikowicz,G;Feder,S;Quarfoot,AJ
通讯作者：
Quarfoot,AJ