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

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 <i>N</i>-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 <i>S</i>-nitroso-<i>N</i>-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.

已有研究推测，血小板溶酶体内容物的胞吐作用参与血栓清除与血管壁重塑。然而，调控血小板溶酶体胞吐作用的具体机制仍不明晰。本研究旨在明确血小板溶酶体分泌的潜在通路，并阐明该过程如何受血小板抑制剂调控。
我们通过检测释放的N-乙酰-β-氨基葡萄糖苷酶（N-acetyl-β-glucosaminidase, NAG）活性，以及采用流式细胞术分析血小板表面表达溶酶体相关膜蛋白（lysosomal-associated membrane protein, LAMP）-1的比例，证实高浓度凝血酶可诱导部分溶酶体胞吐作用。使用特异性肽段激活凝血酶受体PAR1或PAR4，同样可有效诱导LAMP-1的表面表达。
值得注意的是，若通过P2Y₁₂拮抗剂坎格雷洛抑制ADP介导的次级激活，凝血酶诱导的溶酶体胞吐作用会显著减弱；而通过乙酰水杨酸抑制血栓素A₂生成的作用则相对有限。此外，一氧化氮释放药物S-亚硝基-N-乙酰青霉胺（S-nitroso-N-acetyl penicillamine, SNAP），或是可升高环磷酸腺苷水平的类二十烷酸前列腺素I₂（prostaglandin I₂, PGI₂），均可显著抑制溶酶体胞吐作用。
综上，模拟功能性内皮细胞功能的血小板抑制剂（如PGI₂或一氧化氮）可有效拮抗溶酶体胞吐作用。此外，我们认为ADP的次级释放以及经由PAR1/4与P2Y₁₂受体介导的伴随信号，对于凝血酶诱导的高效血小板溶酶体胞吐作用至关重要。