Presence of active and latent type 1 plasminogen activator inhibitor associated with porcine platelets.
Presence of active and latent type 1 plasminogen activator inhibitor associated with porcine platelets.
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DOI：
10.1182/blood.v80.9.2269.bloodjournal8092269
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发表时间：
1992-11
期刊：
影响因子：
20.3
通讯作者：
Irene M. Lang;James J. Marsh;Kenneth M. Moser;Raymond R. Schleef
中科院分区：
文献类型：
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作者：
Irene M. Lang;James J. Marsh;Kenneth M. Moser;Raymond R. Schleef
来自许多实验室的数据表明，人类血小板主要以潜伏形式含有I型纤溶酶原激活物抑制剂（PAI-1）；然而，一份报告（生化28:5773,1989）表明，它主要是PAI-1的活性形式，存在于猪血小板的硫酸铵沉淀中，并且可以从硫酸铵沉淀中纯化出来。为了澄清这一情况，我们调查并比较了猪和人血小板中PAI-1的状况。对PAI-1与固定化t-PA形成复合物能力的免疫学分析表明，猪和人每10(8)个血小板（n = 6; +/- SD）分别含有3.7 +/- 0.4和1.7 +/- 0.3 U的PAI活性；十二烷基硫酸钠（SDS）活化使PAI-1活性提高到10.8 +/- 3.0和3.8 +/- 0.5 U / 10(8)个血小板。血小板裂解物也用过量的可溶性t-PA处理，t-PA与活性PAI-1形成复合物，而潜伏形式则通过sds -聚丙烯酰胺凝胶电泳和反向纤维蛋白自显影检测。此外，固定化t-PA能够消耗血小板提取物中的活性PAI-1，并且可以通过4 mol/L胍激活来定量吸收提取物中残留的潜伏形式。为了研究我们的观察结果与已发表的数据之间的差异，我们提取了猪血小板，并按照文献中描述的方法部分纯化了PAI-1。为了进行定量分析，猪血小板PAI-1也使用我们实验室优化的内皮PAI-1标准色谱程序纯化到均匀性，纯化的蛋白用于建立猪PAI-1抗原的酶联免疫吸收测定。结果表明：(1)猪血小板裂解液浓缩硫酸铵沉淀物中潜伏PAI-1必须先用变性剂稀释后才能检测到；(2)活性和潜伏猪血小板PAI-1可通过分子筛柱凝胶过滤分离。总之，本报告证明PAI-1在猪血小板中以活性和潜伏形式存在。
Data from a number of laboratories indicate that human platelets contain type I plasminogen activator inhibitor (PAI-1) primarily in a latent form; however, one report (Biochemistry 28:5773, 1989) indicated that it is predominantly the active form of PAI-1 that is present in and can be purified from an ammonium sulfate precipitate of porcine platelets. To clarify this situation, we investigated and compared the status of PAI-1 in porcine and human platelets. Immunologic analysis of the ability of PAI-1 to form complexes with immobilized t-PA indicated that porcine and human platelets contained 3.7 +/- 0.4 and 1.7 +/- 0.3 U of PAI activity per 10(8) platelets (n = 6; +/- SD), respectively; sodium dodecyl sulfate (SDS)-activation of the lysates increased PAI-1 activity to 10.8 +/- 3.0 and 3.8 +/- 0.5 U per 10(8) platelets. Platelet lysates were also treated with an excess of soluble t-PA, which formed complexes with active PAI-1, whereas the latent form was detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography. Furthermore, immobilized t-PA was able to deplete active PAI-1 from the platelet extracts, and the latent form remaining in the absorbed extract could be quantitated by activation with 4 mol/L guanidine. To investigate the differences between our observations and the published data, porcine platelets were extracted, and PAI-1 was partially purified as described in the literature. For quantitative analysis, porcine platelet PAI-1 was also purified to homogeneity using standard chromatographic procedures optimized in our laboratory for endothelial PAI-1, and the purified protein was used to develop an enzyme-linked immunoabsorbent assay for porcine PAI-1 antigen. Our results indicate that: (1) latent PAI-1 in concentrated ammonium sulfate precipitates of porcine platelet lysates cannot be detected unless the precipitates are diluted before treatment with denaturants; and (2) active and latent porcine platelet PAI-1 can be separated by gel filtration over molecular sieving columns. In summary, this report documents that PAI-1 in porcine platelets is present in both an active and a latent form.