Annexin V binding to platelets is agonist, time and temperature dependent

Abstract

Platelets bind annexin V when stimulated with combinations of platelet agonists such as collagen and thrombin. Previous studies have demonstrated significant heterogeneity of platelets binding annexin V. The relative role of the thrombin protease-activated receptors (PARs), PAR1 and PAR4, together with different methods of platelet preparation on annexin V binding to platelets is unclear. We therefore investigated the role of PAR1- and PAR4-activating peptides in combination with collagen-related peptide on annexin V binding. In diluted whole blood, PAR1- and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding. However, in washed platelets, PAR-activating peptides were less potent than thrombin at inducing annexin V binding. This difference was more pronounced when experiments were performed at 37°C compared to room temperature. In studies of diluted whole blood, platelet rich plasma and washed platelets, platelets incubated at room temperature bound more annexin V than platelets incubated at 37°C. We also saw a significant effect of time on annexin V binding, in that more annexin V bound to platelets with longer incubation times. In conclusion, PAR1 and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding in combination with collagen-related peptide in diluted whole blood and platelet rich plasma, but not in washed platelets. In addition, incubation temperature and time has a strong influence on annexin V binding to platelets. Thus variations in these conditions may explain the differences observed between previous studies.

• Blood platelets
• annexin V
• flow cytometry
• PAR1
• PAR4
• GPVI

Acknowledgements

This work is supported by Science Foundation Ireland under Grant No. 05/CE3/B754. We would also like to thank Prof. Tomas L. Lindahl at the Department of Clinical Chemistry, Linköping University Hospital, Sweden for allowing some of the experiments to be performed in the laboratory in Linköping.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.