Background: Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis.
Objectives: Protein isoforms of platelet-derived thrombospondin-1 (TSP-1) were investigated for their potential to identify in vitro platelet activation when monitoring in vivo processes.
Methods: TSP-1 was determined in plasma, serum or supernatant of purified platelets by ELISA and immunoblotting and was compared with standard markers of platelet activation. A collective of 20 healthy individuals and 30 cancer patients was analyzed.
Results: While in vitro platelet degranulation led to a selective increase in the 200-kDa full-length molecule, an in vivo process involving platelet activation such as wound healing resulted in the predominant rise of the 140-kDa TSP-1 protein. The physiological ratio of circulating TSP-1 variants was determined and a cut-off level at 1.0 was defined to identify plasma samples with artificial in vitro platelet activation exceeding the cut-off level. In contrast, cancer patients known to frequently exhibit increased in vivo activation of platelets presented with a significantly decreased ratio of TSP-1 variants as compared with healthy volunteers.
Conclusions: In comparison to standard platelet markers, TSP-1 constitutes a sensitive and stable parameter suited to monitor in vitro platelet activation. The analysis of TSP-1 protein isoforms further offers a valuable tool to reliably discriminate between in vitro and in vivo effects, to exclude variability introduced during blood processing and improve clinical monitoring.
© 2010 International Society on Thrombosis and Haemostasis.