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
F. Werner et al., 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, J BIOL CHEM, 274(31), 1999, pp. 21555-21561
Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator
(tPA) specifically, saturably, and with relatively high affinity (K-d 25 n
M), and this binding potentiates the activation of cell-associated plasmino
gen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322), We have obs
erved 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 c
ompeted equally well by all forms of tPA, and its inhibition was unaltered.
These effects were shown to extend to the physiological tPA inhibitor, pla
sminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 compl
ex 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 VS
MC by a novel mechanism involving the serine protease domain of tPA and dis
tinct from its binding to fibrin, Furthermore, reciprocally linked conforma
tional changes in tPA appear to have functionally significant effects on bo
th the interaction of tPA with its VSMC binding site and the susceptibility
of bound tPA to inhibition.