Proteomic characterization of platelet gel releasate from adult peripheral and cord blood

Longo, Valentina; Rebulla, Paolo; Pupella, Simonetta; Zolla, L.; Rinalducci, Sara

doi:10.1002/prca.201500126

Cited by 24 publications

(46 citation statements)

References 71 publications

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“…showing that these proteins had more than two-fold increase in their expression profiles in the "thrombinactivated" vs "resting" platelets. We recovered a high overlap of proteins members with the kinome family reported already by other proteomic analyses, [20][21][22][23][24][25][26][27][28][29][30] that are also indexed in many other research studies related to the proteomic profiling of agonists activated platelets, such as the "Adhesome" and "Platelet Web" databases, as described later in the results section. Other major pathways predicted by IPA to be up-regulated in the proteome of "thrombin-activated platelets" were mapped to the cytoskeleton, actin and RhoA mediated cellular signaling ( Figure 18).…”

Section: Figure 14mentioning

confidence: 88%

“…The "in solution" digestion with three enzymes (trypsin/LysC/Glu-C), enabled the extraction of peptides from the total platelet proteome, including the membrane-derived and microvesicles contained in the releasates, as reported previously by other studies on platelets proteomics. [20][21][22][23][24][25][26][27][28][29][30] The extracted peptides were subjected to nano LC/MS/MS sequencing on a Q Exactive quadrupole orbitrap mass spectrometer, as summarized in the protocol shown in Figure 1. The label-free proteomics analysis employed the label free quantification (LFQ) module provided by PEAKS 8.5 (Bioinformatics Solutions Inc.), In addition, the changes in the protein expression profiles were complemented by an additional analysis involving the Scaffold and per SPECtives software (Proteome Software Inc.).…”

Section: Development Of a Workflow For Implementing The Label-free Phmentioning

confidence: 99%

“…[13][14][15][16] Using highly sensitive and highly specific mass spectrometers (providing mass accuracy and resolution), and proteome fractionations assays involving denaturant, and reducing one-dimensional or two-dimensional protein electrophoretic systems (1-DE and 2-DE, respectively) followed by protein digestions with multiple enzymes (trypsin, LysC, Glu-C and Asp-N, among others), and further fractionations of peptides by means of orthogonal chromatographic systems, thousands of proteoforms can be characterized from highly pure fractions consisting of only 2x10 8 platelets, purified from small volumes of blood, usually in the range of few milliliters. [20][21][22][23][24][25][26][27][28] Consequently, the future advances in the mass spectrometric profiling of protein expression and their corresponding highly-regulated PTM are expected to allow platelet proteomics to be implemented in many research and clinical laboratories as a reliable tool for characterizing the molecular and cellular pathways that affect platelet homeostasis in healthy and disease states. Many original research articles and recent reviews highlighted the wide area of research dedicated to label or label-free, targeted or untargeted proteomics profiling of changes in the platelets purified from patients experiencing cardiovascular, inflammatory, and bleeding disorders.…”

Section: Introductionmentioning

confidence: 99%

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Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

Clement¹,

Gonzalez²,

Babińska³

et al. 2018

MOJPB

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One top research assay used to validate the efficacy of pharmacological agents used in antiplatelets aggregation therapy is mass spectrometry coupled with high throughput profiling of changes in the proteomic expression in the activated platelets exposed to inhibitors or agonists of platelets signaling. Herein we present the development of a new, bottom-up proteomics platform that enabled monitoring of changes in protein expression profiles of healthy human platelets, ex-vivo activated with thrombin, using as reference the proteome of resting platelets. The assay employed the platelets cryogenic lysis and protein solubilization under denaturant conditions, enabling the extraction of key membranebound proteins, in addition to the cytosolic and organelles-derived proteins secreted in microparticles, and found in the platelets releasates. Nano LC-ESI/MS/MS sequencing of tryptic/Glu-C/Lys-C peptides from the "in solution" one step digestion of the whole platelets proteomes (2x10 8 platelets/patient sample) was performed on a Q-Exactive quadrupole orbitrap mass spectrometer coupled with label free quantification (LFQ) analysis. The changes in proteomic landscapes were qualitatively and quantitatively analyzed using a combination of systems biology and bioinformatics approaches empowered by the ingenuity pathway analysis (IPA), and the screening of identified proteins and genes entities against curated databases containing platelets proteomes, including the "Adhesome", "Exocarta", "Reactome" and "Platelet Web". The label-free global proteomics analysis retrieved a total of 924 proteins (FDR <1.3% for proteins and <0.8% for peptides) out of which 330 were shared between resting and thrombin-activated platelets. About 50% of the total proteome of thrombin-activated platelets was represented by up-regulated and previously validated protein biomarkers, involved in the pathways mediating the protease and thrombin activated receptor (PARs), integrin signaling, the actin and rhoA linked to cytoskeleton signaling, and activated kinases pathways regulating the cellular motility, aggregation, procoagulation and degranulation. In conclusion, the systems biology approaches validated our newly developed LFQ proteomic assay as a reliable tool for monitoring the changes in protein expression profiles in thrombin-activated platelets, and further advocate for employment of these approaches in assessing the efficacy of antiplatelets drugs (inhibitors of platelets aggregation) during the prevention and treatment of cardiovascular and cerebrovascular diseases. Citation: Gonzalez J, Babinska A, Ewul ELV, et al. Development of a label-free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin-activated human platelets. MOJ Proteomics Bioinform. 2018;7(4):232-255. Platelets purification and ex-vivo activation with thrombin for proteomic analysisPRP were subjected to the whole platelets purification as described previously by other research groups. [30][31][32][33][34][35] Then, platelets were pelleted at 750xg ...

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Section: Figure 14mentioning

confidence: 88%

Section: Development Of a Workflow For Implementing The Label-free Phmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

Clement¹,

Gonzalez²,

Babińska³

et al. 2018

MOJPB

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“…No conclusive evidence is available with regard to a specific pathophysiologic mechanism that would make cord blood–derived platelets preferable to those obtained from adult blood. However, proteomic studies have outlined differences in the proteome of adult versus cord blood platelets, which indicate greater anti‐inflammatory properties in the latter …”

mentioning

confidence: 99%

“…However, proteomic studies have outlined differences in the proteome of adult versus cord blood platelets, which indicate greater anti-inflammatory properties in the latter. 8 During wound-healing processes, platelet-derived growth factors stimulate tissue repair and cell proliferation, influence extracellular matrix deposition, and support cell proliferation and differentiation. Moreover, platelet gel has an analgesic function and can reduce neurologic and neuropathic pain associated with wounds.…”

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confidence: 99%

Impressive tissue regeneration of severe oral mucositis post stem cell transplantation using cord blood platelet gel

et al. 2017

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Generation of a Functioning and Self-Renewing Diaphragmatic Muscle Construct

Trevisan

Fallas

Maghin

et al. 2019

Stem Cells Translational Medicine

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Surgical repair of large muscular defects requires the use of autologous graft transfer or prosthetic material. Naturally derived matrices are biocompatible materials obtained by tissue decellularization and are commonly used in clinical practice. Despite promising applications described in the literature, the use of acellular matrices to repair large defects has been only partially successful, highlighting the need for more efficient constructs. Scaffold recellularization by means of tissue engineering may improve not only the structure of the matrix, but also its ability to functionally interact with the host. The development of such a complex construct is challenging, due to the complexity of the native organ architecture and the difficulties in recreating the cellular niche with both proliferative and differentiating potential during growth or after damage. In this study, we tested a mouse decellularized diaphragmatic extracellular matrix (ECM) previously described by our group, for the generation of a cellular skeletal muscle construct with functional features. The decellularized matrix was stored using different conditions to mimic the off‐the‐shelf clinical need. Pediatric human muscle precursors were seeded into the decellularized scaffold, demonstrating proliferation and differentiation capability, giving rise to a functioning three‐dimensional skeletal muscle structure. Furthermore, we exposed the engineered construct to cardiotoxin injury and demonstrated its ability to activate a regenerative response in vitro promoting cell self‐renewal and a positive ECM remodeling. Functional reconstruction of an engineered skeletal muscle with maintenance of a stem cell pool makes this a promising tool toward future clinical applications in diaphragmatic regeneration. Stem Cells Translational Medicine 2019;8:858&869

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Proteomic characterization of platelet gel releasate from adult peripheral and cord blood

Abstract: Our study highlighted a differential content of proteins supporting tissue repair and regeneration between APB and CB-derived PGs. These findings may help better identifying future appropriate clinical applications.

Cited by 24 publications

References 71 publications

Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

Development of a label‒free nanolc/ms/ms assay for monitoring the changes in the proteomic landscape of thrombin‒activated human platelets

Impressive tissue regeneration of severe oral mucositis post stem cell transplantation using cord blood platelet gel

Generation of a Functioning and Self-Renewing Diaphragmatic Muscle Construct

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