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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.

Original publication

DOI

10.1021/pr900027j

Type

Journal article

Journal

J Proteome Res

Publication Date

06/2009

Volume

8

Pages

2903 - 2914

Keywords

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