Comparative Platelet Releasate Proteomic Profiling of Acute Coronary Syndrome versus Stable Coronary Artery Disease.

Upon activation, platelets release a host of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). The contents of this PR play a significant role in haemostasis, inflammation, and pathologic sequelae. Despite this, proteomic studies investigating the PR in coronary artery disease have not been performed. Here, we undertook a comparative label-free quantitative (LFQ) proteomic profiling of the 1 U/ml thrombin-induced PR from 13 acute coronary syndrome vs. 14 stable angina pectoris patients using a tandem mass spectrometry approach. Data are available via ProteomeXchange with identifier PXD009356. 318 PR proteins were identified across both cohorts with 9 proteins found to be differentially released, including tetranectin (CLEC3B), protein disulfide-isomerase-A3 (PDIA3), coagulation factor V (F5), and fibronectin (FN1). Strikingly, these 9 differential proteins were all associated with the gene ontology cellular component term "extracellular vesicle" and reduced levels of EVs were detected in the corresponding plasma of ST-segment elevation myocardial infarction (STEMI) patients. Network analysis revealed 3 proteins either reduced (F5; FN1) or absent (CLEC3B) in the PR of STEMI patients that are strongly connected to both the clotting cascade and major druggable targets on platelets. This moderated proteomic signature may prove useful for non-invasive risk assessment of the progression of coronary artery disease. These data further contribute to the growing evidence-base of using the platelet releasate as a predictor of pathological state and disease severity.

The Platelet Releasate is Altered in Human Pregnancy.

PURPOSE: Healthy pregnancy is characterized by an increase in platelet activation and a decrease in the number of circulating platelets with gestation. Despite this recognized importance, proteomic studies investigating platelets in healthy pregnancy have not been performed. As platelet cargo can be altered in different conditions, it is hypothesized that platelets may store a relevant and bespoke collection of molecules during pregnancy. EXPERIMENTAL DESIGN: Comparative label-free quantitative proteomic profiling of platelet releasates (PRs) is performed from 18 healthy pregnant and 13 non-pregnant women using an MS/MS approach. RESULTS: Of the 723 proteins identified, 69 PR proteins are found to be differentially released from platelets in pregnancy, including proteins only expressed during pregnancy such as pregnancy-specific glycoproteins and human placental lactogen. Moreover, the population of exosomal vesicles present in the PR is also modified in pregnancy. Receiver operating characteristic analysis shows the predictive ability of 11 PR proteins to distinctly classify pregnant and nonpregnant women with an area under the curve of 0.876, a sensitivity of 88.9%, and a specificity of 84.6%. CONCLUSIONS AND CLINICAL RELEVANCE: Taken together this demonstrates that platelets and their released cargo are 'educated' in physiologic stressful conditions such as pregnancy and may represent a promising platform to study pregnancy complications.

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults.

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL-1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

A Protocol for Improved Precision and Increased Confidence in Nanoparticle Tracking Analysis Concentration Measurements between 50 and 120 nm in Biological Fluids.

Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets [the platelet releasate (PR)] and in serum, to increase confidence in NTA particle enumeration. The overall variance and the precision of NTA measurements were quantified by root mean square error and relative standard error. Using a bootstrapping approach, we found that increasing video replicates from 5 s × 60 s to 25 s × 60 s captures led to a reduction in overall variance and a reproducible increase in the precision of NTA particle-concentration quantitation for all three biofluids. We then validated our approach in an extended cohort of 32 healthy donors. Our results indicate that for vesicles sized between 50 and 120 nm, the precision of routine NTA measurements in serum, plasma, and PR can be significantly improved by increasing the number of video replicates captured. Our protocol provides a common platform to statistical compare particle size distribution profiles in the exosomal-vesicle size range across a variety of biofluids and in both healthy donor and patient groups.