Human plasma platelet-derived exosomes: effects of aspirin

Edward Goetzl

Laura Goetzl

Joel Karliner

Norina Tang

Lynn Pulliam

^{Department of Medicine, University of California, San Francisco, California, USA}

^{Department of Laboratory Medicine, University of California, San Francisco, California, USA}

^{Jewish Home of San Francisco, San Francisco, California, USA}

^{Department of Obstetrics, Gynecology, and Reproductive Sciences, Temple University, Philadelphia, Pennsylvania, USA}

^{Veterans Affairs Medical Center, San Francisco, California, USA}

^{Correspondence: Geriatric Research Center, JHSF, 302 Silver Ave., San Francisco, CA 94112, USA., E-mail:}ude.fscu@lzteog.drawde

Received 2015 Nov 4; Accepted 2016 Jan 29.

Abstract

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma platelet-derived exosome number is lower and its chemokine and HMGB1 levels higher after age 65 yr. Aspirin consumption significantly suppressed cargo protein levels of plasma platelet-derived exosomes without altering total levels of exosomes. Cargo proteins of human plasma platelet-derived exosomes may biomark platelet abnormalities and in vivo effects of drugs.— Goetzl, E. J., Goetzl, L., Karliner, J. S., Tang, N., Pulliam, L. Human plasma platelet-derived exosomes: effects of aspirin.

Keywords: chemokines, glycoproteins, HMGB1, intercellular communication, atherosclerosis

Abstract

Human platelets generate 50- to 120- nm-diameter exosomes that are secreted from endosomally derived multivesicular bodies, with lesser contributions directly from α-granules (1, 2). The constitutive release of exosomes and quantities of their protein cargoes loaded at multiple sites increase only modestly after stimulation with thrombin, collagen, or calcium ionophore . In contrast, platelet release of plasma membrane-derived, cytosol-containing 150- to 1000- nm-diameter microvesicles is strikingly enhanced by many stimuli, physicochemical stresses, and apoptosis (3). Furthermore, in contrast to the predominantly phospholipid content of microvesicles, platelet exosomal cargoes may include diverse cytokines, chemokines, growth factors, coagulation factors, lipoproteins, and other lipids, as well as several types of RNA (1 –3). Platelet exosome membrane proteins also reflect those of their platelet source, including the constitutively expressed glycoprotein GPIb, as well as GPVI, αIIbβ3, CD40 ligand, and P-selectin from activated platelets (1, 3, 4).

Interactions of platelets with endothelial cells and monocytes that are important in the pathogenesis of atherosclerosis are mediated, in part, by platelet exosomes. Many studies have not distinguished between total platelet microparticles and the exosome and microvesicle subsets. However, it is platelet exosomes, like exosomes from many other types of cells, that serve as the major mediators of platelet intercellular interactions (5, 6). Preliminary findings suggest that uptake of platelet-derived exosomes by endothelial cells enhances their adhesiveness by both increasing endothelial expression of adherence proteins and decreasing endothelial generation of antiadhesive factors (4, 7, 8). Platelet exosomes also augment platelet adherence to monocytes and monocyte activation to an inflammatory phenotype (9).

Our hypothesis is that sustained platelet activation in vivo in some vascular diseases will elevate loading of cytoadhesive, thrombogenic, and inflammatory factors into platelet exosomal cargo sufficiently to promote their greater delivery to endothelial cells and macrophages at sites of vascular lesions. Augmented delivery of platelet exosomal atherogenic cargo to lesional endothelial cells and macrophages may consequently accelerate development of vascular plaques, clots, and strictures (10, 11).

Each value is the mean ± sem pg/ml (n = 4) for proteins extracted from exosomes released by 10^{platelets into 1 ml Tyrode’s buffer with 1 mM CaCl}₂ in 30 min at 37°C and precipitated by ExoQuick-TC (upper row) or precipitated and immunochemically purified (lower row).

The mean quantity of exosomal CD81 in immunopurified samples is significantly less than in preceding precipitates by a paired Student's t test. For the other 4 proteins, none of the differences in quantity was significant after CD81 normalization to compare for the same amount of exosomes.

Each value is the mean ± sem (pg/ml; n = 6) for platelet-derived exosomes isolated from plasma. Values for GPVI, GXCL4, CXCL7, and HMGB1 were normalized with corresponding levels of CD81. The P values (bottom row) reflect significance of the difference between the 2 age groups.

Acknowledgments

The authors are grateful to Judith H. Goetzl (Geriatric Research Center, Jewish Home of San Francisco) for expert preparation of the graphic illustrations. These studies were supported, in part, by Grant HL129853 (to L.P.) from the U.S. National Institutes of Health, National Heart, Lung, and Blood Institute. E.J.G. has filed a provisional patent application involving the methodology for isolation and analyses of platelet exosomes from plasma, and has also developed the methodology, obtained blood, performed exosome isolations and ELISAs, analyzed data, and drafted the manuscript; L.G. participated in study design and edited the manuscript; J.S.K. participated in study design and edited the manuscript; N.T. performed exosome counts; and L.P. analyzed data and edited the manuscript.

Acknowledgments

Glossary

BSA	bovine serum albumin
DBS	Dulbecco’s balanced salt
GPIb	platelet glycoprotein Ib
GPVI	platelet glycoprotein VI
HMGB1	high-mobility group box 1 protein
PPP	platelet-poor plasma

Glossary

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