储存的血小板中血小板微粒的获取及其止凝血功能研究

Obtention and investigation of platelet microparticles in stored platelet concentrates

目的从储存的手工浓缩血小板中获得具有止血功能的血小板微粒(PMP)。方法流式细胞仪检测常规储存1、3、5、7、9、11 d浓缩血小板中PMP的含量;2 500 g离心30 min处理浓缩血小板,下层血小板沉淀用于检测离心剪切力对血小板CD62p及磷脂酰丝氨酸(PS)表达的影响,上层液离心获得PMP,流式细胞术及荧光底物法等分析评价所获取PMP颗粒的理化性质和生理功能,包括颗粒大小、表面蛋白受体和PS、凝血酶生成能力。结果常规储存9 d的浓缩血小板中PMP含量(2.51±0.47)%较储存1 d的(1.79±0.29)%明显增加(P<0.05);离心剪切力对储存>8 d的血小板CD62p及PS的表达无明显影响;离心获得储存9 d浓缩血小板中的PMP直径为(619.81±196.15)nm,表面含有GPⅠb(CD42b)、GPⅡb-Ⅲa(CD41/CD61)、P-选择素(CD62p)及PS;凝血酶生成试验证实PMP促进凝血酶生成的能力:总蛋白含量分别为0.1、0.4、0.8 mg/m L的PMP溶液对应的TTP值(min)分别为68.4±4.6、45.8±2.7、38.8±3.4,均明显低于对照组80.0±2.8(P<0.05)。结论储存9 d的浓缩血小板释放的PMP明显增加;通过离心方法可直接获这一储存时间段的PMP;所获得的PMP表面保留了与止凝血功能相关的糖蛋白和PS,初步表现出止血功能。

Objective To obtain hemostatic platelet microparticles（ PMP） from stored manually prepared platelet concentrates. Methods The content of PMP in platelet concentrates stored at day 1,3,5,7,9 and 11 was detected respectively by flow cytometry. After centrifugation,isolated platelets were used to analyze the exposure of CD62 p and phosphatidylserine,while the supernatant was used to obtain PMP. By using flow cytometry and fluorescent substrate method,the obtained PMP was analyzed by following parameters： the size of PMP particles,surface protein receptors,phosphatidylserine and thrombin generation ability. Results Compared with PMP content in platelet concentrates stored for 1 day（ 1. 79 ±0. 29）,PMP content which was stored for 9 days（ 2. 51 ± 0. 47） showed a significant increase（ P 〈0. 05）. Centrifuge shear force had no effect on the exposure of platelet CD62 p and PS after platelet concentrations were stored for 8 days. The diameter of PMP obtained at day 9 was（ 619. 81 ± 196. 15） nm,and the PMP expressed GPⅠb（ CD42b）,GPⅡb-Ⅲa（ CD61）,P-selection（ CD62p） and phosphatidylserine on the surface. Thrombin generation experiments demonstrated that PMP can promote thrombin generation. PMP total protein content 0. 1,0. 4,0. 8 mg / mL which corresponded to the time to peak（ TTP） 68. 4 ± 4. 6,45. 8 ± 2. 7,38. 8 ± 3. 4min were significantly lower than the control group 80. 0 ± 2. 8 min（ P 〈0. 05）. Conclusion PMP significantly increased when platelet concentrations were stored for 9 days,and PMP can be obtained directly by dual centrifugation. The surface of PMP showed the presence of GPⅠ b,GPⅡb-Ⅲa,P-selection and PSwhich were associated with hemostatic function. Therefore,PMP is considered to be a hemostatic agent.