Potential calcium-dependent cell-adhesion protein. May be involved in the establishment and maintenance of specific neuronal connections in the brain.
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Platelet-released extracellular vesicles: the effects of thrombin activation
Suades R, Padró T, Vilahur G, Badimon L
Cell Mol Life Sci2022 MarPubMed ID: 35288766Read Article
Applications: WB Reactive species: Rabbit
"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." Article snippet: Protein extracts were separated using Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, electrotransferred to nitrocellulose membrane in semi-dry conditions (***, ***), and subsequently blocked with 5% BSA in Tris-bufered saline (TBS) containing 0.1% Tween-20 (TBS-Tween) for 1 h prior to primary antibody incubation (overnight at 4 °C). Membranes were exposed to rabbit antifotillin monoclonal antibody (***) and anti-PCDHA4 polyclonal antibody (Creative-Diagnostics). Following incubation, membranes were washed with TBS-Tween (three times, 10 min each), incubated with horseradish peroxidaselabelled goat anti-rabbit secondary antibody (***) for 1 h at RT and washed again as previously described.
Figure 1. Representative 2D proteome map of proteins in pEVs.
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