Abstract

Upon activation platelets release a myriad of soluble proteins as well as two distinct membrane vesicle populations- exosomes and microparticles (MPs) into the external milieu [1]. Although there have been many studies characterising platelet MPs and their increased circulating numbers in disease [2, 3], platelet exosomal vesicles have been characterised to date only by transmission electron microscopy (TEM) [4]. Here, we characterize the proteome of human platelet exosomes and reveal that a population of these vesicles carry active WNT glycoproteins on their surface that can modulate WNT signalling activity in both endothelial and monocytic cells.

To establish the exosomal proteome of human platelets we compared platelet exosomes isolated from three healthy volunteers by (i) differential ultracentrifugation (DC) and (ii) ExoQuickTM (ExoQ) precipitation. Assessment of both exosomal populations was performed using nanoparticle tracking analysis (NTA), TEM, western blot and proteomics analysis (in triplicate) using a high performance Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer coupled with quantitative computational analysis using MaxQuant software. We concluded that DC was the superior isolation method for platelet exosomes, for example, our NTA revealed a 2-fold greater enrichment (6x108 /ml) of platelet exosomes following isolation by DC in comparison to ExoQ. Proteomic analysis of platelet exosomes identified 704 proteins across 18 MS runs, 119 of which were present in all 18 runs and represented the core human platelet exosomal proteome. Using western blotting and immunogold TEM, we confirmed the presence of numerous well-established exosomal membrane markers (CD63, CD9, and HSP70) in our proteome, as well as several platelet-specific proteins including GPIb, as previously described [4], and GPV.

We also detected several of the 19 human WNT glycoproteins to be specifically secreted in the exosomal fraction upon platelet activation and verified their surface expression on platelet-derived exosomes using immunogold TEM. As we have previously demonstrated, platelets themselves can be modulated directly by WNT glycoproteins [5], hence here, we assessed the paracrine impact of these WNT-positive platelet exosomes on intracellular signalling events in monocytes and endothelial cells, both cell types closely linked to the pathogenesis of atherosclerosis. Using fluorescence microscopy, we found that these platelet exosomes were readily endocytosed by both endothelial (EAHY296) and monocytic (THP-1) cell lines where they specifically modulated beta-catenin nuclear expression and canonical WNT signalling.

In conclusion our proteomic analysis has revealed that platelet exosomes are new players in the regulation of WNT signalling in both endothelial and monocytic cells.

1. Coppinger, J.A., et al., Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood, 2004. 103(6): p. 2096-104.

2. Knijff-Dutmer, E.A., et al., Elevated levels of platelet microparticles are associated with disease activity in rheumatoid arthritis. Arthritis Rheum, 2002. 46(6): p. 1498-503.

3. van der Zee, P.M., et al., P-selectin- and CD63-exposing platelet microparticles reflect platelet activation in peripheral arterial disease and myocardial infarction. Clin Chem, 2006. 52(4): p. 657-64.

4. Heijnen, H.F., et al., Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood, 1999. 94(11): p. 3791-9.

5. Steele, B.M., et al., Canonical Wnt signaling negatively regulates platelet function. Proc Natl Acad Sci U S A, 2009. 106(47): p. 19836-41.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.