The Asp(272)-Glu(282) region of platelet glycoprotein Ibalpha interacts with the heparin-binding site of alpha-thrombin and protects the enzyme from the heparin-catalyzed inhibition by antithrombin III.
Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebrand factor and thrombin. Thrombin binds to GpIb via its heparin-binding site (HBS) (De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Picozzi, M., and Landolfi, R. (1997) Thromb. Haemostasis 77, 735-740; De Cristofaro, R., De Candia, E., Croce, G., Morosetti, R., and Landolfi, R. (1998) Biochem. J. 332, 643-650). To identify the thrombin-binding domain on GpIbalpha, we examined the effect of GpIbalpha(1-282), a GpIbalpha fragment released by the cobra venom mocarhagin on the heparin-catalyzed rate of thrombin inhibition by antithrombin III (AT). GpIbalpha(1-282) inhibited the reaction in a dose-dependent and competitive fashion. In contrast, the GpIbalpha(1-271) fragment, produced by exposing GpIbalpha(1-282) to carboxypeptidase Y, had no effect on thrombin inhibition by the heparin-AT complex. Measurements of the apparent equilibrium constant of the GpIbalpha(1-282) binding to thrombin as a function of different salts (NaCl and tetramethyl-ammonium 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 GpIbalpha was confirmed using DNA aptamers, which specifically bind to either HBS (HD22) or the fibrinogen recognition site of thrombin (HD1). HD22, but not HD1, inhibited thrombin binding to GpIbalpha(1-282). Furthermore, the proteolytic derivative gamma(T)-thrombin, which lacks the fibrinogen recognition site, binds to GpIbalpha via its intact HBS in a reaction that is inhibited by HD22. Neither alpha- nor gamma(T)-thrombin bound to GpIbalpha(1-271), suggesting that the Asp(272)-Glu(282) region of GpIbalpha may act as a "heparin-like" ligand for the thrombin HBS, thereby inhibiting heparin binding to thrombin. It was also demonstrated that intact platelets may dose-dependently inhibit the heparin-catalyzed thrombin inhibition by AT at enzyme concentrations <5 nM. Altogether, these findings show that thrombin HBS binds to the region of GpIbalpha involving the Asp(272)-Glu(282) segment, protecting the enzyme from the inactivation by the heparin-AT system.
De Cristofaro
R
R
Haemostasis Research Center, Catholic University School of Medicine, Largo F. Vito, 00168 Rome, Italy. decristofaro@rm.unicatt.it
De Candia
E
E
Rutella
S
S
Weitz
J I
JI
D000990:Antithrombin III / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D004277:DNA, Single-Stranded / Q000494:pharmacology; D004546:Elapid Venoms / Q000201:enzymology; D005340:Fibrinogen / Q000378:metabolism; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004546:Elapid Venoms; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D012965:Sodium Chloride; D000990:Antithrombin III; D005340:Fibrinogen; D006493:Heparin; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A; D013917:Thrombin; D008666:Metalloendopeptidases; C099092:mocarhagin; D002118:Calcium