PMID: 2105933Feb 15, 1990Paper

Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers.

The Journal of Biological Chemistry
V RamakrishnanJ B Baker

Abstract

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 ...Continue Reading

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