Proteomics unravels platelet function

Platelets have a pivotal role in thrombosis, vascular repair, and inflammatory reactions. They lack nuclei and genomic DNA and are thus not amenable to most of the classical cell, molecular biology, and genomic techniques. For the identification of proteins and novel protein functions, proteomic studies are therefore the method of choice. Proteomic approaches are further favored by easy access to large amounts of platelet proteins via blood donations. Platelets possess a pre-mRNA splicing machinery and also have remnants of megakaryocyte-derived mRNA, which both can result in rapid translation upon platelet activation.^2,3  Thus, proteomic comparisons between resting and activating platelets are expected to reveal differences in abundance of proteins. Technical advances in proteomic technologies and recent achievements in generating protein data repositories hold great promise for important discoveries in platelet research.^4,5 

The proteomic comparison of 2 functional states of platelets by Schulz et al is based on the elegant use of the 2-dimensional difference gel electrophoresis (2D-DIGE).^1,6  Classical 2D gel electrophoresis is hampered by inter-gel variability as well as difficulties in protein quantification. In 2D-DIGE, samples are labeled with different fluorescent dyes (typically Cy3 and Cy5, providing a wide dynamic range of fluorescence quantification) and run on the same 2D gel. Gels are scanned with the excitation wavelength of each dye. This allows a direct comparison to each other and to an additional sample (eg, labeled with Cy2), which is used as an internal control allowing a quantitative comparison between gels. These dyes are designed to bind to lysine-residues, to have the same molecular masses, and to preserve the charge of the labeled proteins. Thus, this provides a true reflection of the protein abundance throughout the investigated proteome.⁶  Together with advances in the rapidly advancing fields of mass spectrometry and protein database content and management, 2D-DIGE is an ideal discovery tool for platelet research.

Mechanistic research on platelet activation is very much driven by the prospect of identifying novel targets for antiplatelet therapy. Although current antiplatelet drugs are among the most successful drugs ever developed (in regard to both direct mortality/morbidity benefits and commercial success), there appears to be a link between higher efficacy and higher rates of bleeding complications. Proteomic discovery approaches that aim to identify novel players in platelet activation are therefore also inherently drug discovery programs in the search for better therapeutic targets.

Tucker et al recently reported such a discovery approach focusing on the membrane-associated proteome of thrombin-activated platelets.⁷  Using biotinylation of the platelet surface, the authors isolated and identified proteins that were abundantly associated with the platelet membrane upon platelet stimulation with thrombin. One of the proteins identified, HIP-55, which is an SH3-binding adaptor protein, becomes associated with Syk and the integrin subunit β₃ upon platelet activation. The authors also demonstrated the functional relevance of this protein in α_IIbβ₃ (GPIIb/IIIa) activation.

Schulz et al chose to study glycoprotein VI (GPVI)–induced platelet activation. GPVI mediates platelet binding to collagen, which itself induces cross-linking (ligation) of the receptor causing outside-in signaling and platelet activation.¹  Genetic deficiency and ex vivo as well as in vivo receptor blocking data suggest a central role of GPVI in platelet adhesion and aggregation as well as in thrombus formation. This makes the receptor as well as the associated signaling pathways attractive as therapeutic targets.⁸  Indeed, one of the 9 proteins found in higher abundance upon GPVI ligation, aldose reductase, is the kind of protein one would like to “discover” with a differential proteomic approach. Functional investigations revealed that inhibition of aldose reductase impaired GPVI-induced platelet aggregation. This property warrants further investigation on its use as a therapeutic target for antiplatelet therapy. Another protein “discovered” on 2D-DIGE upon GPVI ligation was ERp57, a disulfide isomerase, which Schulz et al could show to be released after platelet activation and which converts tissue factor from an inactive to an active form.¹  This finding describes an interesting link between GPVI-induced platelet activation and tissue factor–induced blood coagulation.

The report of Schulz et al is a good example of how modern proteomic technologies can become a driving force to better our understanding of platelet (patho)physiology and to pursue discovery of novel targets for antiplatelet therapy.¹  Using 2D-DIGE with a systematic evaluation of the stimulation of other platelet receptors such as P₂Y₁₂, GP1bα, and protease-activated receptors seems a most promising approach for further discoveries.