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 ...

Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly.