Controlled Shedding of Platelet Glycoprotein (Gp)vi and Gpib-ix-v by Adam Family Metalloproteinases.
From: Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Vic. Australia.
Journal of thrombosis and haemostasis : JTH
- Publish Date: Jul 2007
- ISSN: 1538-7933
- Volume: 5
- Issue: 7
- Pages: 1530-7
- Medium: Print
- Language: English
- Citation (JAMA): Gardiner E E, Karunakaran D, Shen Y, et al. Controlled Shedding of Platelet Glycoprotein (Gp)vi and Gpib-ix-v by Adam Family Metalloproteinases.. J. Thromb. Haemost. Jul 2007;5:1530-7
Abstract
BACKGROUND: Platelet glycoprotein (GP)VI that binds collagen, and GPIb-IX-V that binds von Willebrand factor, initiate thrombus formation. OBJECTIVES: In this study, we investigated the mechanisms of metalloproteinase-mediated ectodomain shedding that regulate the surface expression of GPVI, GPIbalpha (the major ligand-binding subunit) and GPV (that regulates thrombin-dependent activation via GPIbalpha). METHODS AND RESULTS: Immunoblotting human platelet lysates using affinity-purified antibodies against cytoplasmic domains of GPVI, GPIbalpha or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIbalpha, but not GPVI, exists in a cleaved state on platelets, even when isolated in the presence of metalloproteinase inhibitor (GM6001) or EDTA; (ii) the same-sized membrane-associated fragments of GPVI or GPIbalpha are generated by phorbol-ester (PMA), the mitochondrial-targeting reagent CCCP, the calmodulin inhibitor W7, or the thiol-modifying reagent, N-ethylmaleimide, that directly activates ADAM10/ADAM17; and (iii) GPV is shed by both metalloproteinase- and thrombin-dependent mechanisms, depending on the concentration of thrombin. Based on the predicted cleavage area defined by these studies, ADAM10, but not ADAM17, cleaved a GPVI-based synthetic peptide within the extracellular membrane-proximal sequence (PAR;Q(243)YY) as analyzed by MALDI-TOF-MS. In contrast, ADAM17, but not ADAM10, cleaved within the GPIbalpha-based peptide (LRG;V(465)LQ). Both ADAM10 and ADAM17 cleaved within a GPV-based peptide (AQP;V(494)TT). Metalloproteinase-mediated shedding of GPIbalpha from GPIb-IX-transfected or GPVI-transfected cells induced by W7 or N-ethylmaleimide was inhibited by mutagenesis of sequences identified from peptide analysis. CONCLUSIONS: These findings suggest surface levels of GPVI, GPIbalpha and GPV may be controlled by distinct mechanisms involving ADAM10 and/or ADAM17.
Mesh Headings (Keywords): ADAM Proteins, Amino Acid Sequence, Amyloid Precursor Protein Secretases, Animals, Binding Sites, Blood Platelets, Cell Line, Dipeptides, Humans, Membrane Proteins, Mutagenesis, Site-Directed, Platelet Glycoprotein GPIb-IX Complex, Platelet Membrane Glycoproteins, Protease Inhibitors, Rats, Recombinant Proteins, Transfection
Check for Full Text / PubMed Unique Identifier (PMID): 17445093
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