Tissue plasminogen activator binds to human vascular smooth muscle cells by a novel mechanism. Evidence for a reciprocal linkage between inhibition of catalytic activity and cellular binding.
Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.
Thrombosis Research Institute, Manresa Rd., London SW3 6LR, United Kingdom.
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Werner
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Razzaq
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Ellis
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002478:Cells, Cultured; D002942:Circular Dichroism; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012501:Saphenous Vein / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D005337:Fibrin; D010959:Tissue Plasminogen Activator; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone