Dataset Information

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS).

ABSTRACT: Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome (CAPS), and to compare the CAPS proteins to human and murine platelet proteomic data. Washed platelets were isolated from healthy dogs, and stimulated with saline (control) or gamma-thrombin (releasate). Proteins were separated by SDS-page, trypsin-digested and analyzed by liquid chromatography and tandem mass spectrometry (MS). CAPS proteins were defined as those with a MS1-abundance ratio of two or more for releasate vs. unstimulated saline control. A total of 1,918 proteins were identified, with 908 proteins common to all dogs and 693 characterized as CAPS proteins. CAPS proteins were similar to human and murine platelet secretomes and were highly represented in hemostatic pathways. Differences unique to CAPS included replacement of platelet factor 4 with other cleavage products of platelet basic protein (e.g. interleukin-8), novel proteins (e.g. C-C motif chemokine 14), and proteins in relatively high (e.g. protease nexin-1) or low (e.g. von Willebrand factor) abundance. This study establishes the first in-depth platelet releasate proteome from healthy dogs with a reference database of 693 CAPS proteins. Similarities between CAPS and the human secretome confirm the utility of dogs as translational models of human disease, but we also identify differences unique to canine platelets. Our findings provide a resource for further investigations into disease-related CAPS profiles, and for comparative pathway analyses of platelet activation among species.

SUBMITTER: Cremer SE

PROVIDER: S-EPMC6853320 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS).

Cremer Signe E SE Catalfamo James L JL Goggs Robert R Seemann Stefan E SE Kristensen Annemarie T AT Brooks Marjory B MB

PloS one 20191113 11

Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome ...[more]

PMID: 31721811

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Project description:Protein secretion can mediate communication of distant cells in a multicellular organism and can control a broad range of physiological functions. Regulation of this process is crucial, especially for cells of the immune system that sense pathogens through specific receptors and release proteins that initiate and orchestrate the immune response. This secretome is today analyzed using antibody-based assays, but mass spectrometry could provide an unbiased and comprehensive alternative. Here we developed a quantitative, high resolution mass spectrometric workflow to detect and quantify proteins that are released from macrophages upon Toll-like receptor 4 (TLR4) activation by the microbial component lipopolysaccharide (LPS)5. We quantified the time-resolved release of 775 proteins, including 52 annotated cytokines, from only 150.000 primary cells per condition. The dynamic range of the secretory induction was at least 10,000 fold and we achieved sensitivity in the low picogram range. Comparison to the transcriptome revealed high correlation for some protein classes but also anti-correlation resulting from the transcriptionally decoupled release of lysosomal proteins. Using gene knockout (KO) mouse strains as genetic models with intracellular signaling adaptor deficiencies, we defined distinct secretory profiles depending on which arms of the signaling pathways are missing. We show that MyD88 dominates over TRIF in the number and fold-induction of secreted proteins6,7. Interestingly, the sum of the two signaling arms does not equal the wild-type response; instead, protein secretion is tuned by synergistic and predominantly redundant mechanisms. The kinetic data reveals that the release of anti-inflammatory proteins dampens the initial pro-inflammatory response. Our study provides a paradigm for the sensitive and time-resolved identification of receptor activation induced cellular secretomes, including proteins with unexpected extracellular presence, and allows the characterization of the underlying signal transduction pathways.

Dataset Information

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS).

Publications

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS).

Similar Datasets

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