Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

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De Candia, E., Hall, S.W., Rutella, S. ORCID: 0000-0003-1970-7375, Landolfi, R., Andrews, R.K. and De Cristofaro, R., 2001. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. Journal of Biological Chemistry, 276 (7), pp. 4692-4698. ISSN 0021-9258

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Official URL: http://doi.org/10.1074/jbc.M008160200

Abstract

The activation of human platelets by α-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibα also has a high affinity binding site for α-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbα may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by α-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparentk cat/K m value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 ± 0.1 × 107 m −1sec−1. The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbα on platelet membranes by mocarhagin treatment reduced the k cat/K m value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k cat/K m value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbα, reduced the apparentk cat/K m value by about 5-fold. 4) Displacement of α-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparentk cat/K m value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparentk cat/K m value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a “cofactor” for PAR-1 activation by thrombin.

Item Type:

Journal article

Publication Title:

Journal of Biological Chemistry

Creators:

De Candia, E., Hall, S.W., Rutella, S., Landolfi, R., Andrews, R.K. and De Cristofaro, R.

Publisher:

American Society for Biochemistry and Molecular Biology

Date:

16 February 2001

Volume:

276