A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

J Proteome Res. 2009 Jun;8(6):2903-14. doi: 10.1021/pr900027j.


Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets / metabolism*
  • Chromatography, Liquid
  • Collagen / metabolism*
  • HSP47 Heat-Shock Proteins / metabolism*
  • Humans
  • Membrane Proteins / metabolism*
  • Phosphorylation
  • Platelet Activation*
  • Platelet Aggregation
  • Platelet Membrane Glycoproteins / metabolism
  • Proteomics / methods*
  • Signal Transduction
  • Tandem Mass Spectrometry
  • p38 Mitogen-Activated Protein Kinases / metabolism


  • HSP47 Heat-Shock Proteins
  • Membrane Proteins
  • Platelet Membrane Glycoproteins
  • SERPINH1 protein, human
  • platelet membrane glycoprotein VI
  • Collagen
  • p38 Mitogen-Activated Protein Kinases