Platelet membrane proteomics: a novel repository for functional research

Abstract

Being central players in thrombosis and hemostasis, platelets react in manifold and complex ways to extracellular stimuli. Cell-matrix and cell-cell interactions are mandatory for initial adhesion as well as for final development of stable plugs. Primary interfaces for interactions are plasma membrane proteins, of which many have been identified over the past decades in individual studies. However, due to their enucleate structure, platelets are not accessible to large-scale genomic screens and thus a comprehensive inventory of membrane proteins is still missing. For this reason, we here present an advanced proteomic setup for the detailed analysis of enriched platelet plasma membranes and the so far most complete collection of platelet membrane proteins. In summary, 1282 proteins were identified, of which more than half are termed to be of membrane origin. This study provides a brief overview of gene ontology subcellular and functional classification, as well as interaction network analysis. In addition, the mass spectrometric data were used to assemble a first tentative relative quantification of large-scale data on the protein level. We therefore estimate the presented data to be of major interest to the platelet research field and to support rational design of functional studies. (Blood. 2009;114:e10-e19)

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

“…However, Tspan12 is not as ubiquitously expressed as ADAM10 (16), suggesting that ADAM10 might be regulated by additional tetraspanins. Indeed, an extensive proteomics analysis of the human platelet cell surface did not detect Tspan12 but did identify at least 10 other tetraspanins (26,29). In addition, recent platelet transcriptomics profiling (28) found mRNA for 22 and 21 tetraspanins in human and mouse, respectively, but Tspan12 was not among the human tetraspanins and was only very weakly detected in mouse (supplemental Fig.…”

“…To determine whether one or more platelet tetraspanins could interact with ADAM10, a panel of 10 was selected based on their identification on human platelets by proteomics (26,29). Because antibodies are not available to most of these tetraspanins, a co-transfection system in HEK293T cells was Human platelets were surface-biotinylated and lysed in 1% Brij97 lysis buffer (labeled B), which largely maintains tetraspanin-tetraspanin interactions and hence microdomain integrity, or in 1% Triton X-100 (labeled T), which disrupts tetraspanin microdomains (12).…”

“…Tspan14, Tspan15, and Tspan33 interact with and promote the maturation of ADAM10. A-C, HEK-293T cells were transfected with FLAGtagged mouse forms of a panel of 10 tetraspanins previously found to be expressed by human platelets (26,29) in the absence (Ϫ) or presence (ϩ) of HA-tagged bovine ADAM10. The cells were surface-cross-linked with DTSSP, lysed in 1% Triton X-100, and subjected to anti-FLAG immunoprecipitation (i.p) in 1% Triton X-100 containing 0.1% SDS.…”

What Can Proteomics Tell Us About Platelets?

“…Many drug targets are membrane proteins, and thus there is considerable interest in the plasma membrane subproteome. 122 Dedicated studies used gel-based 99,103,123 or gel-free 108 approaches, with the latter yielding ≈650 membrane proteins, identifying even low abundant 7 transmembrane domain receptors, such as the P2Y 1 receptor for ADP (≈150 copies per platelet). These studies clearly extended our knowledge of plasma membrane proteins and receptors.…”

Proteomics unravels platelet function

“…Technical advances in proteomic technologies and recent achievements in generating protein data repositories hold great promise for important discoveries in platelet research. 4 …”