During acute inflammation, 3 neutrophil subsets are found in the blood: neutrophils with a conventional segmented nucleus, neutrophils with a banded nucleus, and T-cell-suppressing CD62L neutrophils with a high number of nuclear lobes. In this study, we compared the in vivo kinetics and proteomes of banded, mature, and hypersegmented neutrophils to determine whether these cell types represent truly different neutrophil subsets or reflect changes induced by lipopolysaccharide (LPS) activation. Using in vivo pulse-chase labeling of neutrophil DNA with 6,6-H-glucose, we found that H-labeled banded neutrophils appeared much earlier in blood than labeled CD62L and segmented neutrophils, which shared similar label kinetics. Comparison of the proteomes by cluster analysis revealed that CD62L neutrophils were clearly separate from conventional segmented neutrophils despite having similar kinetics in peripheral blood. Interestingly, the conventional segmented cells were more related at a proteome level to banded cells despite a 2-day difference in maturation time. The differences between CD62L and mature neutrophils are unlikely to have been a direct result of LPS-induced activation, because of the extremely low transcriptional capacity of CD62L neutrophils and the fact that neutrophils do not directly respond to the low dose of LPS used in the study (2 ng/kg body weight). Therefore, we propose CD62L neutrophils are a truly separate neutrophil subset that is recruited to the bloodstream in response to acute inflammation. This trial was registered at www.clinicaltrials.gov as #NCT01766414.

However, these direct methods experience a high false-positive rate because of contamination by uncontrolled platelet lysis. Here, we take a reverse approach, monitoring quantitatively the concentration changes of all proteins in the platelets after platelet stimulation, whereby we assume that the released protein content should be detectable in the reduction of its level from the whole platelet proteome. For accuracy, our strategy uses stable isotope-labeling ( Figure 1A) to discriminate the released proteins from uncontrolled lysis products. In other words, most platelet proteins do not change except the ones that are significantly released on activation. Materials and MethodsMaterials and Methods are available in the online-only Supplement. 10-13 ResultsPerforming the reversed releasate quantitative proteomics experiments on 3 individuals resulted in the identification of 4375 unique proteins, of which 2970 (68%) could be quantified in ≥2 of 3 individuals (Table I in the online-only Data Supplement). Identity of the Platelet ReleasateFirst, we set out to identify the releasate on full platelet activation. The releasate was evaluated in each individual by the determination of the dimethyl intensity ratio between the light (resting) and intermediate (activated) labeled peptides ( Figure 1A). As expected, the vast majority of © 2013 American Heart Association, Inc. Objective-Platelet activation and subsequent protein release play an important role in healthy hemostasis and inflammatory responses, yet the identity and quantity of proteins in the platelet releasate are still debated. Here, we present a reversed releasate proteomics approach to determine unambiguously and quantitatively proteins released from activated platelets. Approach and Results-Isolated platelets were mock and fully stimulated after which the released proteins in the supernatant were removed. Using high-end proteomics technology (2D chromatography, stable isotope labeling, electron transfer dissociation, and high collision dissociation fragmentation) allowed us to quantitatively discriminate the released proteins from uncontrolled lysis products. Monitoring the copy numbers of ≈4500 platelet proteins, we observed that after stimulation via thrombin and collagen, only 124 (<3%) proteins were significantly released (P<0.05). The released proteins span a concentration range of ≥5 orders, as confirmed by ELISA. The released proteins were highly enriched in secretion tags and contained all known factors at high concentrations (>100 ng/mL, eg, thrombospondin, von Willebrand factor, and platelet factor 4). Interestingly, in the lower concentration range of the releasate many novel factors were identified. proteins (>95%) had a resting/activated ratio close to 1.0. These ratios were highly reproducible between replicates with a median RSD between ratios of 9.6% (Table I and Figure I in the online-only Data Supplement). Using the statistical criteria described in the Methods section in the online-only Data Supplement, 124 proteins were observed to be ...

Both ELISA on established α-granule proteins and MS-based quantitative proteomics showed that the most abundant α-granule proteins are released in similar quantities from platelets after stimulation with either PAR-1 or PAR-4. Our findings provide evidence against the hypothesis that PAR-1 and PAR-4 stimulation of platelets trigger differential release of alpha-granule, but further studies are needed to draw conclusions for physiological conditions.

Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a two-phase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC-MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC-MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients.