Peng Xiang, Ramström Sofia, Kurz Tino, Grenegård Magnus, Segelmark Mårten
Department of Nephrology, Qingyuan City Hospital of Jinan University, Guangdong, China; Department of Medical and Health Sciences, Linköping University, Linköping, Sweden(1).
Department of Experimental and Clinical Medicine, Linköping University, Linköping, Sweden.
Thromb Res. 2014 Aug;134(2):418-25. doi: 10.1016/j.thromres.2014.06.001. Epub 2014 Jun 11.
Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated.
The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry.
PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca(2+) mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca(2+) chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it.
The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca(2+) signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.
蛋白酶3（PR3）从中性粒细胞嗜天青颗粒中释放出来，并对炎症过程产生复杂影响。PR3催化多种大分子的降解，但对血细胞的影响尚不明确。在本研究中，对PR3对人血小板的作用进行了深入研究。
实验使用从健康人类志愿者捐献的血液中新鲜分离的洗涤血小板进行。在Chrono-Log血小板聚集仪上测量血小板形状变化和聚集情况。通过免疫染色观察MYPT1的磷酸化形式。通过流式细胞术进一步评估血小板活化情况。
PR3诱导血小板形状改变，但不诱导聚集。流式细胞术分析表明，PR3不诱导P-选择素表达或纤维蛋白原与血小板结合，并且不改变对PAR1或PAR4激活肽或凝血酶的反应中的活化情况。此外，Fura-2测量和免疫印迹分析分别显示，PR3刺激小的细胞内Ca(2+)动员和肌球蛋白磷酸酶靶亚基1（MYPT1）的Thr696特异性磷酸化。用Rho/Rho激酶抑制剂Y-27632和细胞内Ca(2+)螯合剂BAPTA/AM分别处理血小板可减少PR3诱导的形状改变，而同时处理则完全抑制该改变。
数据表明中性粒细胞蛋白酶PR3是人类血小板的直接调节剂，并通过激活Rho/Rho激酶和Ca(2+)信号通路导致形状改变。这一发现突出了中性粒细胞与血小板复杂相互作用中的另一种机制。
