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

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.

Author information

1
Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, Hopkins Building, The University of Reading, Whiteknights, Reading, United Kingdom RG6 6UB.

Abstract

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.

PMID:
19341245
DOI:
10.1021/pr900027j
[Indexed for MEDLINE]

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