Abstract
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.
Highlights
Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebrand factor and thrombin
To identify the thrombin-binding domain on GpIb␣, we examined the effect of GpIb␣1–282, a GpIb␣ fragment released by the cobra venom mocarhagin on the heparin-catalyzed rate of thrombin inhibition by antithrombin III (AT)
It was found that the major GpIb␣ fragment, referred to as glycocalicin, reduces the rate of thrombin inactivation by AT by competing with heparin for binding to the heparin-binding site (HBS) on thrombin [8]
Summary
Materials—Human ␣-thrombin was purified and characterized as previously reported [10]. High molecular weight heparin (lot HF2025; molecular weight ϭ 16,000) was purchased from Enzyme Research Laboratories Inc. The molecular weight of this heparin fraction was confirmed by subjecting the material to gel filtration using a Bio-Silect SEC 250 –5 column (Bio-Rad) fitted to a high performance liquid chromatograph (Perkin-Elmer, series 10) and monitoring the fractions at 205 nm. Purified heparin oligosaccharides of known molecular weights were used to construct the reference curve. Heparin with high affinity for antithrombin was isolated by affinity chromatography using an antithrombin III column [11]. Antithrombin III (lot HATIII 540AL) purchased from Enzyme Research Labs migrated as a single band on 4 –20% SDS-PAGE gels with an apparent molecular mass of 58 kDa. The antithrombin concentration was calculated spectrophotometrically, using an extinction coefficient E280 (1%) ϭ 6.5 [12].
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