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目的建立冷藏(4℃)保存48 h血小板膜糖蛋白GPⅠbα(CD42b)末端半乳糖基化及唾液酸化的方法。方法冷藏保存48 h后,将半乳糖基化底物UDP-gal及唾液酸化底物CMP-Neu Ac加入血小板保存袋中,37℃复温1h,分别以FITC-s WGA、FITC-ECA标记血小板膜糖蛋白GPⅠbα末端β-Glc NAc及β-Gal糖基,流式细胞术检测糖基化效果。以FITC-CD42 b抗体标记血小板膜糖蛋白GPⅠbα,流式细胞术检测冷藏保存以及糖基化处理对于GPⅠbα蛋白量的影响。以金属蛋白酶(metalloproteinase,MP)抑制剂GM6001抑制MP对GPⅠbα的剪切,验证冷藏保存以及糖基化处理对GPⅠbα的量的影响,并阐明其机制。结果 FITC-s WGA、FITC-ECA标记结果显示,冷藏保存48h后37℃复温1h处理(无论是否加入糖基化底物)可致膜表面GPⅠbα末端糖基暴露(以FITC荧光强度表示)明显降低,FITC-s WGA荧光强度由1918.2±526.2降至1316.2±368.1,FITC-ECA荧光强度由8036.6±1466.3降至5672.6±1522.7(P<0.05);同时可致膜表面GPⅠbα蛋白量降低,由1582.9±123.1降至1380.7±136.7(P<0.05);加入GM6001后,GPⅠbα蛋白量恢复至对照组水平1602.2±153.2。相对荧光强度分析显示,冷藏保存48h与冷藏保存48h后37℃复温1h处理组FITC-s WGA/CD42b比值分别为2.23±0.56与2.11±0.38,FITC-ECA/CD42b比值分别为2.02±0.36与1.91±0.33,二组之间无统计学差异。结论冷藏保存48h后37℃复温1h处理可致血小板膜糖蛋白GPⅠbα量减少;减少系复温处理激活MP所致;通过此处理方式实现血小板糖蛋白GPⅠbα重新糖基化不可行。
Objective To establish a method for the galactosylation and sialylation of GP Ⅰ bα (CD42b) end-platelet glycoprotein (GP42b) stored at 4 ℃ for 48 h. Methods After refrigerated storage for 48 h, galactosylated substrate UDP-gal and sialylated substrate CMP-Neu Ac were added to platelet preservation bag and rewarmed at 37 ℃ for 1 h. FITC-s WGA and FITC-ECA labeled platelets The GPⅠbα-terminal β-GlcNAc and β-Gal glycoproteins were detected by flow cytometry. The platelet membrane glycoprotein GPⅠbα was labeled with FITC-CD42 b antibody, and the effect of cryopreservation and glycosylation on GPⅠbα protein content was detected by flow cytometry. The effects of MP6 on the GPⅠbα level were examined by using the GM6001 inhibitor of metalloproteinase (MP). The effects of cold storage and glycosylation on the amount of GPⅠbα were investigated. The mechanism was also clarified. Results The results of FITC-s WGA and FITC-ECA showed that the GPIbα-terminal glycan exposure (expressed as FITC fluorescence intensity) at 37 ℃ for 48 h after rewarming at 37 ℃ for 1 h The fluorescence intensity of FITC-s WGA decreased from 1918.2 ± 526.2 to 1316.2 ± 368.1, and the fluorescence intensity of FITC-ECA decreased from 8036.6 ± 1466.3 to 5672.6 ± 1522.7 (P <0.05). At the same time, the amount of GPⅠbα protein decreased, ± 123.1 to 1380.7 ± 136.7 (P <0.05). After addition of GM6001, the amount of GPⅠbα protein returned to the control level of 1602.2 ± 153.2. Relative fluorescence intensity analysis showed that the ratio of FITC-s WGA / CD42b was 2.23 ± 0.56 and 2.11 ± 0.38 at 37 ℃ for 1 h and 48 h after storage for 48 h, respectively, and the ratios of FITC-ECA / CD42 b were 2.02 ± 0.36 and 1.91 ± 0.33, no significant difference between the two groups. CONCLUSION: Rewarming at 37 ℃ for 1 h at 37 ℃ after cold storage for 48h resulted in a decrease in the amount of platelet glycoprotein GPⅠbα, and a decrease in MP reactivation to activate MP. Glucoprotein GPⅠbα re-glycosylation was not feasible by this treatment.