High precision platelet releasate definition by quantitative reversed protein profiling--brief report.

ABSTRACT

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. CONCLUSIONS: Our reversed releasate dataset forms the first unambiguous, in depth repository for molecular factors released by platelets.