Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances

Abstract

Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-β-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y₁₂ antagonist cangrelor, while inhibition of thromboxane A₂ formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I₂ (PGI₂) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI₂ or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y₁₂ receptors is important for efficient platelet lysosomal exocytosis by thrombin.

• ADP receptors
• endothelium
• exocytosis
• lysosome
• platelet physiology
• protease activated receptors (PAR)
• thrombin

Declaration of interest

This work was supported by grants from the County Council of Östergötland and the Swedish Research Council. The authors declare no competing financial interests. Concept and design: M.G., S.R., K.Ö.; experimental design, experimental execution and analysis of data A.-C.B.S.H. and A.L.S.; Data interpretation and writing of article M.G., S.R., K.Ö., E.G.L., A.-C.B.S.H. and A.L.S. All the authors have approved the final version of the article.

Supplementary material available online.

Figures S1–S5.