Platelet-released extracellular vesicles: the effects of thrombin activation

Abstract

Platelets exert fundamental roles in thrombosis, inflammation, and angiogenesis, contributing to different pathologies from cardiovascular diseases to cancer. We previously reported that platelets release extracellular vesicles (pEVs) which contribute to thrombus formation. However, pEV composition remains poorly defined. Indeed, pEV quality and type, rather than quantity, may be relevant in intravascular cross-talk with either circulating or vascular cells. We aimed to define the phenotypic characteristics of pEVs released spontaneously and those induced by thrombin activation to better understand their role in disease dissemination. pEVs obtained from washed platelets from healthy donor blood were characterized by flow cytometry. pEVs from thrombin-activated platelets (T-pEVs) showed higher levels of P-selectin and active form of glycoprotein IIb/IIIa than baseline non-activated platelets (B-pEVs). Following mass spectrometry-based differential proteomic analysis, significant changes in the abundance of proteins secreted in T-pEVs compared to B-pEVs were found. These differential proteins were involved in coagulation, adhesion, cytoskeleton, signal transduction, metabolism, and vesicle-mediated transport. Interestingly, release of proteins relevant for cell adhesion, intrinsic pathway coagulation, and platelet activation signalling was significantly modified by thrombin stimulation. A novel pEV-associated protein (protocadherin-α4) was found to be significantly reduced in T-pEVs showing a shift towards increased expression in the membranes of activated platelets. In summary, platelet activation induced by thrombin triggers the shedding of pEVs with a complex proteomic pattern rich in procoagulant and proadhesive proteins. Crosstalk with other vascular and blood cells in a paracrine regulatory mode could extend the prothrombotic signalling as well as promote proteostasic changes in other cellular types.

Keywords: Atherosclerosis; Extracellular vesicles; Microvesicles; Platelets; Thrombin; Thrombosis.

Fig. 1

Scatter plots with bars showing size-selected events with expression of phosphatidylserine (PS, annexin V⁺) and platelet activation markers P-selectin (CD62P⁺) and α_IIbβ₃-integrin (PAC1⁺) in the fluorochrome-conjugated gate on pEV surface from baseline non-activated and thrombin-activated platelets (n = 3 independent experiments/group). Data are expressed as mean ± SEM of the labelling percentage of total population. p Value was calculated by 2-sided unpaired Student’s T test

Fig. 2

Representative 2D proteome map of proteins in pEVs. a Proteins were separated in a pH range 3–10 on 12% SDS-PAGE. b Classification of identified proteins by gene ontology (GO) annotations—biological process, subcellular localization, and molecular function. Number of pEV proteins significantly enriched in all obtained GO-Slim categories

Fig. 3

Thrombin-induced effects on pEVs derived from baseline non-activated and thrombin-induced platelets. a Venn diagrams depicting overlap in vesicular protein spots in the different studied groups. b PDQuest differential analysis of pEV identified protein spots. c Distribution of thrombin-induced identified differential proteins by functional categories. Within each group (baseline non-activated and thrombin-stimulated), five individual experiments (biological replicates) were run. A total of ten gels per arm, two from each experiment, were analysed

Fig. 4

Thrombin-induced effects on pEVs derived from baseline non-activated and thrombin-stimulated platelets. Selection of proteins differentially regulated between baseline non-activated and thrombin-induced platelets based on functional groups. Specifically, close-up views of representative 2-DE images corresponding to up- a and down-regulated b protein spots for cytoskeleton, motility and cell organization (I), signal transduction (II), vesicle-associated transport-related proteins (III), and metabolism (IV). Each panel shows scatter plots with bars for each spot showing variations in spot intensity in the different studied groups (B baseline non-activated and T thrombin-stimulated, n = 5 independent experiments/group). Data are expressed in arbitrary units (AU) as mean ± SEM. Differences were analysed by 2-sided unpaired Student’s T test

Fig. 5

Validation of differentially expressed protocadherin α4 protein. Analysis of the cell-adhesion protein protocadherin α4 (PCDHA4) by 2-DE (a) and western blot (b and c). a Enlargement of representative 2-DE images and scatter plots with bars showing variations in PCDHA4 spot intensity in the baseline non-activated and thrombin-stimulated pEVs (n = 5 independent experiments/group). b Western blot analyses against PCDHA4 on pEV and platelet membrane (MB) samples. Scatter dots with bars showing the quantitative variations in band intensity in the baseline non-activated and thrombin-stimulated group (n = 6 independent experiments/group). Data are expressed in arbitrary units (AU) as mean ± SEM. c Flow cytometric analysis of porcine AV⁺-cEVs on plasma samples and western blot analyses against PCDHA4 on porcine circulating EV (cEV) and platelet (PLT) samples before and after clopidogrel administration (n = 11 pigs). Scatter dots with lines showing the AV⁺-cEV numbers (/µl platelet-free plasma [PFP]) before (at baseline) and after clopidogrel treatment. Scatter dots with bars showing the quantitative variations in band intensity (mean arbitrary units [AU] ± SEM) at baseline and after clopidogrel treatment (n = 11 pigs). Total protein normalization of EVs was performed with Ponceau S and of MB and PLT fraction with β-actin. Differences were analysed by 2-sided unpaired (a and b) and paired (c) Student’s T test

Fig. 6

Thrombin-induced effects on clotting factors and their inhibitor proteins of pEVs derived from baseline non-activated platelets and thrombin-stimulated platelets. a The top canonical pathway of proteins differentially regulated between baseline non-activated and thrombin-induced platelets by IPA software is the intrinsic prothrombin activation pathway. b Enlargement of representative 2-DE images corresponding to spots for coagulation factor fibrinogen gamma (FGG), anti-thrombin III (ATIII), and plasma protein Kallikrein (KLK1) with their corresponding scatter plots with bars indicating variations in spot intensity in the different studied groups (B baseline non-activated and T thrombin-stimulated, n = 5 independent experiments/group). Data are expressed in arbitrary units (AU) as mean ± SEM. Differences were analysed by 2-sided unpaired Student’s T test

Fig. 7

Schematic representation of platelet-derived extracellular vesicle protein cargoes upon platelet activation with thrombin and potential associated cellular functions. Summary of the changes in platelet-derived extracellular vesicle proteins after thrombin-platelet activation compiled together with literature-based discovery of their cellular processes. α₂β₁ integrin alpha-2/beta-1, α_IIbβ₃ integrin alpha-IIb/beta-3, α-SNAP alpha-soluble NSF-attachment protein, ACSL acyl-CoA synthetase long chain, Akt protein kinase B, Arp2/3 actin-related proteins-2/3, BICD1 protein bicaudal-D homolog-1, CDCP1 CUB domain-containing protein 1, also known as membrane glycoprotein gp140, CORO1A coronin 1A, Fak focal adhesion kinase 1, KINDLIN-3 fermitin family homolog 3 [FERMT3], PAR protease-activated receptor, PCDHα4 protocadherin α4, PI3K phosphoinositide-3-kinase, PKCδ protein kinase C-delta, pEV platelet-derived microvesicle, Pyk pyruvate kinase, Rac1 Ras-related C3 botulinum toxin substrate 1, Tub tubulin, SRC proto-oncogene tyrosine-protein kinase Src, WAVE Wiskott–Aldrich syndrome [WASP] family verprolin-homologous proteins