Ramakrishnan V, Sinicropi D V, Dere R, Darbonne W C, Bechtol K B, Baker J B
Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1990 Feb 15;265(5):2755-62.
Several groups have demonstrated that radioiodinated tissue-type plasminogen activator (t-PA) binds to saturable sites on human umbilical vein endothelial cells (HUVECs) in culture (Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719; Beebe, D. P. (1987) Thromb. Res. 46, 241-254; Barnathan, E. S., Kuo, A., van der Keyl, H., McCrae, K. R., Larsen, G. L., and Cines, D. B. (1988) J. Biol. Chem. 263, 7792-7799). Here we report that most of the specific binding of 125I-t-PA to our HUVEC cultures is accounted for by binding to (i) plasminogen activator inhibitor type 1 (PAI-1), a t-PA inhibitor produced in abundance by HUVECs; and (ii) specific binding sites present on the plastic culture surface. The contribution of the sites on plastic can be eliminated by taking several precautions. Then, most or all of the specifically bound 125I-t-PA is present in a sodium dodecyl sulfate-stable 110-kDa 125I-t-PA.PAI-1 complex. Interestingly, a radioiodinated mutant form of t-PA, S478A, which is catalytically inactive and therefore unable to form the covalent complex with PAI-1, still binds to HUVECs. In fact, this ligand binds to HUVECs in 10-30-fold greater amounts than does wild-type 125I-t-PA (resulting in greater than 1 x 10(7) S478A 125I-t-PA molecules bound/cell at 12 nM ligand concentration). In contrast, diisopropyl fluorophosphate-treated t-PA binds to HUVECs in much smaller amounts than does wild-type t-PA. Several findings suggest that PAI-1 is a major binding site for S478A t-PA. The vast amount of binding observed with S478A t-PA, compared with wild-type t-PA, may be accounted for by an observed large scale release of wild-type 125I-t-PA.PAI-1 complexes from the solid phase (cells or extracellular matrix) into the culture medium. Immunoprecipitation experiments demonstrate that, in contrast to wild-type t-PA, S478A t-PA does not extract [35S]methionine-PAI antigen from metabolically labeled extracellular matrix. It is proposed that t-PA releases PAI-1 from the solid phase when it forms the irreversible covalent complex with the inhibitor, a process that does not occur with the catalytically inactive mutant form of t-PA.
多个研究小组已证明，放射性碘化的组织型纤溶酶原激活剂（t-PA）可与培养的人脐静脉内皮细胞（HUVECs）上的可饱和位点结合（哈贾尔，K.A.，哈梅尔，N.M.，哈佩尔，P.C.，和纳赫曼，R.L.（1987年）《临床研究杂志》80卷，1712 - 1719页；比贝，D.P.（1987年）《血栓研究》46卷，241 - 254页；巴纳坦，E.S.，郭，A.，范德凯尔，H.，麦克雷，K.R.，拉森，G.L.，和西内斯，D.B.（1988年）《生物化学杂志》263卷，7792 - 7799页）。在此我们报告，125I-t-PA与我们培养的HUVECs的大部分特异性结合是由于与以下物质结合所致：（i）纤溶酶原激活剂抑制剂1型（PAI-1），一种由HUVECs大量产生的t-PA抑制剂；以及（ii）存在于塑料培养表面的特异性结合位点。通过采取一些预防措施，可以消除塑料表面位点的影响。然后，大部分或所有特异性结合的125I-t-PA以十二烷基硫酸钠稳定的110 kDa 125I-t-PA.PAI-1复合物形式存在。有趣的是，一种放射性碘化的t-PA突变形式S478A，它没有催化活性，因此无法与PAI-1形成共价复合物，但仍能与HUVECs结合。实际上，这种配体与HUVECs的结合量比野生型125I-t-PA高10 - 30倍（在12 nM配体浓度下，导致每细胞结合的S478A 125I-t-PA分子数大于1×10⁷）。相比之下，经二异丙基氟磷酸处理的t-PA与HUVECs的结合量比野生型t-PA少得多。多项研究结果表明，PAI-1是S478A t-PA的主要结合位点。与野生型t-PA相比，S478A t-PA观察到的大量结合可能是由于观察到野生型125I-t-PA.PAI-1复合物从固相（细胞或细胞外基质）大规模释放到培养基中。免疫沉淀实验表明，与野生型t-PA不同，S478A t-PA不能从代谢标记的细胞外基质中提取[³⁵S]甲硫氨酸-PAI抗原。有人提出，当t-PA与抑制剂形成不可逆的共价复合物时，它会从固相中释放PAI-1，而催化无活性的t-PA突变形式不会发生这个过程。
