Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases

J Thromb Haemost. 2007 Jul;5(7):1530-7. doi: 10.1111/j.1538-7836.2007.02590.x. Epub 2007 Apr 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM Proteins / blood*
  • ADAM Proteins / genetics
  • ADAM Proteins / metabolism
  • ADAM10 Protein
  • ADAM17 Protein
  • Amino Acid Sequence
  • Amyloid Precursor Protein Secretases / genetics
  • Amyloid Precursor Protein Secretases / metabolism
  • Animals
  • Binding Sites / genetics
  • Blood Platelets / drug effects
  • Blood Platelets / metabolism*
  • Cell Line
  • Dipeptides / pharmacology
  • Humans
  • In Vitro Techniques
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mutagenesis, Site-Directed
  • Platelet Glycoprotein GPIb-IX Complex / genetics
  • Platelet Glycoprotein GPIb-IX Complex / metabolism*
  • Platelet Membrane Glycoproteins / genetics
  • Platelet Membrane Glycoproteins / metabolism*
  • Protease Inhibitors / pharmacology
  • Rats
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transfection


  • Dipeptides
  • Membrane Proteins
  • N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide
  • Platelet Glycoprotein GPIb-IX Complex
  • Platelet Membrane Glycoproteins
  • Protease Inhibitors
  • Recombinant Proteins
  • platelet membrane glycoprotein VI
  • Amyloid Precursor Protein Secretases
  • ADAM Proteins
  • ADAM10 Protein
  • ADAM10 protein, human
  • ADAM17 Protein
  • ADAM17 protein, human
  • Adam17 protein, rat