The Asp(272)-Glu(282) region of platelet glycoprotein Ib alpha interacts with the heparin-binding site of alpha-thrombin and protects the enzyme fromthe heparin-catalyzed inhibition by antithrombin III
R. De Cristofaro et al., The Asp(272)-Glu(282) region of platelet glycoprotein Ib alpha interacts with the heparin-binding site of alpha-thrombin and protects the enzyme fromthe heparin-catalyzed inhibition by antithrombin III, J BIOL CHEM, 275(6), 2000, pp. 3887-3895
Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebra
nd factor and thrombin. Thrombin binds to GpIb via its heparin-binding site
(HBS) (De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Pico
zzi, M., and Landolfi, R. (1997) Thromb. Haemostasis 77,735-740; De Cristof
aro, R., De Candia, E., Croce, G., Morosetti, R., and Landolfi, R, (1998) B
iochem. J. 332, 643-650). To identify the thrombin-binding domain on GpIb a
lpha, we examined the effect of GpIb alpha(1-282), a GpIb alpha fragment re
leased by the cobra venom mocarhagin on the heparin-catalyzed rate of throm
bin inhibition by antithrombin III (AT). GpIb alpha(1-282) inhibited the re
action in a dose-dependent and competitive fashion. In contrast, the GpIb a
lpha(1-271) fragment, produced by exposing QpI alpha(1-282) to carboxypepti
dase Y, had no effect on thrombin inhibition by the heparin-AT complex. Mea
surements of the apparent equilibrium constant of the GpIb alpha(1-282) bin
ding to thrombin as a function of different salts (NaCl and tetramethyl-amm
onium chloride) concentration (0.1-0.2 M) indicated a large salt dependence
(Gamma(+/-) = -4.5), similar to that pertaining to the heparin binding to
thrombin. The importance of thrombin HBS in its interaction with GpIb alpha
was confirmed using DNA aptamers, which specifically bind to either HBS (H
D22) or the fibrinogen recognition site of thrombin (HD1). HD22, but not HD
1, inhibited thrombin binding to GpIb alpha(1-282). Furthermore, the proteo
lytic derivative gamma(T)-thrombin, which lacks the fibrinogen recognition
site, binds to GpIb alpha via its intact HBS in a reaction that is inhibite
d by HD22. Neither alpha- nor gamma(T)-thrombin bound to GpIb alpha(1-271),
suggesting that the Asp(272)-Glu(282) region of GpIb alpha may act as a "h
eparin-like" ligand for the thrombin DBS, thereby inhibiting heparin bindin
g to thrombin. It was also demonstrated that intact platelets may dose-depe
ndently inhibit the heparin-catalyzed thrombin inhibition by AT at enzyme c
oncentrations <5 nM. Altogether, these findings show that thrombin HBS bind
s to the region of GpIb alpha involving the Asp(272)-GlU(282) segment, prot
ecting the enzyme from the inactivation by the heparin-AT system.