Abstract
Platelets store a plethora of different molecules within their granules, modulating numerous pathways, not only in coagulation, but also in angiogenesis, wound healing, and inflammatory diseases. These molecules get rapidly released upon activation and therefore represent an easily accessible indirect marker for platelet activation. Accurate analysis of platelet-derived molecules in the plasma requires appropriate anticoagulation to avoid in vitro activation and subsequent degranulation of platelets, potentially causing artificially high levels and masking biologically relevant differences within translational research studies. However, there is still enormous heterogeneity among anticoagulants used to prevent unwanted platelet activation, so that plasma levels reported for platelet granule contents range over several orders of magnitude. To address this problem and to define the most robust method of plasma preparation to avoid in vitro platelet activation during processing, we compared plasma concentrations of the three platelet-stored factors thrombospondin (TSP-1), platelet factor 4 (PF4), and soluble P-selectin (sCD62P) between human blood samples anticoagulated with either citrate-theophylline-adenosine-dipyridamole (CTAD), acid-citrate-dextrose (ACD), citrate, ethylenediaminetetraacetic acid (EDTA) or heparin. Additionally, we assessed the effect of storage temperature and time between blood drawing and sample processing within the differentially anticoagulated samples. Our data strongly support the use of CTAD as anticoagulant for determining plasma concentrations of platelet-stored molecules, as anticoagulation with heparin or EDTA led to a 12.4- or 8.3-fold increase in plasma levels of PF4, respectively. Whereas ACD was similar effective as CTAD, citrate only showed comparable PF4 plasma levels when plasma was kept at 4°C. Moreover, blood sampling with CTAD as anticoagulant resulted in the most reproducible values, even when samples were processed at ambient temperature or after storage over 6 hours. In the latter case, anticoagulation with heparin or EDTA led to artificially high plasma levels indicative of in vitro platelet activation. Therefore, we want to raise scientific awareness for choosing CTAD as optimal anticoagulant for the detection of platelet-stored molecules in plasma.
Highlights
Precise measurement of plasma parameters is important for accurate diagnosis, and provides a non-invasive, applicable tool to monitor disease progression and to possibly predict clinical outcome [1]
Whereas plasma levels of platelet factor 4 (PF4) and TSP-1 were comparable between CTAD, ACD and citrate plasma, these proteins substantially increased when heparin or ethylenediaminetetraacetic acid (EDTA) were used as an anticoagulant (Fig 2A and 2B)
We want to raise scientific awareness for choosing the optimal anticoagulant, which is important for studies primarily focusing on platelets and coagulation, and applies to a much broader spectrum of research as platelets store a plethora of heterogeneous proteins, affecting inflammation, angiogenesis, wound healing, and coagulation
Summary
Precise measurement of plasma parameters is important for accurate diagnosis, and provides a non-invasive, applicable tool to monitor disease progression and to possibly predict clinical outcome [1]. As platelet granules store over 300 different molecules, degranulation has to be a precisely coordinated process, adjusted to the specific physiological needs [10, 11]. This is assured by distinct granule types (α-granule, dense granule, and lysosomes), and via diverse packaging and agonist-dependent release of granule subtypes [7]. Due to the high sensitivity of platelets and their quick adaption to changes in the microenvironment, they are prone to in vitro activation, which has to be considered when preparing plasma for analysis of platelet-derived mediators. Prevention of platelet activation and complete removal of platelets is important for plasma preparation as preserving protein stability and enzyme activity of plasma components
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