title: 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.
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.
authors.affiliation: Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, Hopkins Building, The University of Reading, Whiteknights, Reading, United Kingdom RG6 6UB.
authors.forename: William J
authors.initials: WJ
authors.lastname: Kaiser
authors.forename: Lisa-Marie
authors.initials: LM
authors.lastname: Holbrook
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Tucker
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Stanley
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Gibbins