不溶性血小板膜颗粒的制备及其功能初探

Preparation and preliminary investigation of infusible platelet membrane

目的制备具有止血功能的不溶性血小板膜(IPM)颗粒。方法过期的浓缩血小板经过差速离心去除红细胞、白细胞和血浆,用生理盐水悬浮,经冻融破碎、离心洗涤、超声匀浆,获得大小较均一的IPM颗粒;检测其理化和功能指标:IPM颗粒大小、表面蛋白受体、聚集功能和凝血酶生成能力。结果制备得到的IPM颗粒大小为140nm左右,其表面含有糖蛋白GPⅠbα(CD42b)、糖蛋白GPⅡb-Ⅲa(CD41/CD61)、血小板内皮细胞粘附分子(CD31)、P-选择素(CD62p);体外功能试验显示:IPM颗粒在瑞斯托霉素和Ⅲ型胶原诱导下能发生聚集;凝血酶生成试验证实:IPM能为凝血因子Ⅹ酶复合物和凝血酶生成提供催化表面,参与凝血级联反应。结论应用冻融和超声相结合的方法成功制备出大小均一的IPM颗粒,其表面保留了与止血功能相关的蛋白受体;在血小板聚集诱导剂的刺激下IPM颗粒可以发生聚集反应,初步表现为止血功能。

Objective To prepare infusible platelet membrane （ IPM ） particles which have hemostatic function. Methods Outdated platelet concentrate was separated from red blood cells,white blood cells and plasma,by differential eentrifugation. Then the platelets precipitate were resuspended in physiological saline, and disrupted by repeated freezing and thawing,three times. The frozen and thawed suspension was centrifuged to remove intracellular residue components. And the lysed platelets were then homogenized by sonicating. The sonicated platelets suspension was centrifuged to get IPM particles. The supernatant was used for the following research ： the size of the IPM particles, surface protein receptor of particles, aggregation function and thrombin generation ability. Results The IPM particles size is about 140 nm,and retain glycoprorein （GP） Ⅰ bα （ CD42b ）, GP Ⅱb- Ⅲ a （ CD41/CD61 ） , CD31, CD62p. In vitro, Ristocetin and Ⅲ -collagen could induce particles aggregation. Thrombin generation experiments showed that IPM particles could provide catalytic surface for tenase complex and thrombin generation. Conclusion By freezing-thawing and ultrasonic sonication,we could obtain IPM particles with uniform particle size. The particle surface still remains GP Ⅰ bα, GP Ⅱ b- Ⅲ a, CD31, CD62p, which were associated with hemostatic function. And IPM particles were activated by the aggregation reagents and it could aggregate together. So,it is expected to become a hemostatic agent.