【作者】 韩悦；

【导师】 阮长耿；

【作者基本信息】 苏州大学 ， 内科血液学， 2002， 博士

【摘要】 血小板的主要生理功能是参与血栓与止血，任何凝血过程都涉及血小板的活化，这是一个复杂的信号级连过程，凝血酶在其中占有核心地位。凝血酶活化血小板主要通过一族G蛋白偶连的蛋白酶活化受体PARs（protease-activated receptors）介导。人类血小板表面表达PAR1与PAR4两种受体，凝血酶能连接并切割PAR1或PAR4，暴露新的N-末端，后者作为一个固定配基与受体进行分子内结合，从而激发跨膜信号传导，引起血小板聚集、释放以及膜糖蛋白的一系列改变。GPIb是血小板表面的重要粘附分子，也能与凝血酶结合，对维持血小板结构功能具有重要意义。凝血酶活化血小板后，GPIb由膜表面进入开放管道系统（OCS）并失去与表面抗体的亲和性，但随着活化时间延长又重新返回血小板表面，出现可逆性的动态变化过程。 为比较两类PAR受体在血小板信号传递中的作用，确定它们是否也能引起GPIb的动态分布，并了解这一过程中细胞骨架所发挥的作用，我们选择25μM PAR1-AP（SFLLRNPNDKYEPF）与250μM PAR4-AP（AYPGKF）分别模拟各自的固定配基活化血小板。结合血小板聚集试验、流式细胞仪、荧光显微镜、蛋白电泳以及免疫共沉淀技术，测定相应的血小板聚集率以及血小板膜表面糖蛋白GPIbα与P-选择素表达，分析细胞骨架中GPIbα、肌动蛋白与肌球蛋白的变化。在这同时，应用Cytochalasin D（肌动蛋白多聚化抑制剂）、BAPTA／AM(螯合胞内Ca²⁺)、wortmannin（抑制磷脂酰肌醇-3-激酶PI3-K及肌球蛋白轻链激酶MLCK）、ApyraseⅦ（低ATP／ADP酶，促使ADP分解）、Ro-31-2220（蛋白激酶C选择性抑制剂）及Y27632（抑制Rho激酶）等多种血小板抑制剂，观测它们对血小板活化过程的影响，以揭示PAR活化的部分机制。 血小板聚集试验显示，PAR1与PAR4途径都能活化血小板但作用强弱不等，PAR1（SFLLRNPNDKY）＞PAR4（AYPGKF）＞PAR4（GYPGKF）＞PAR4（GYPGQV）。另洲人学博士学位论又_中文摘要 PARI一AP（sFLLRNPNDKYEPF）与pAR4一Ap（AYpGKF）都可以引起血_小板膜表面糖蛋白GPIba呈现可逆性的动态分布，达到最大程度50%的膜内逆转，之后都缓慢地返回血小板表面。活化后各时间段两者之间均存在明显差异（P都<0.05）。PARI首先在2分钟达到最低值，而PAR4在5分钟表达最低，随后较PARI缓慢地回升。GPIba的这一内转过程同时伴随血小板形态的收缩。选择足以刺激血小板形态改变的不同活化剂浓度都可以引起Gl，Jb。类似的动态改变。抑制剂CytochalasinD与BAPTA/AM （20林M）则能阻滞GPlba的内转及细胞形态的改变，又以CytochalasinD的抑制效果更完全。然而，活化过程中血小板膜表面P一选择素水平未出现可逆性的改变，!分钟明显增多，2分钟后就一直维持于最高水平。 护AR肤刺激过程中GPIba进入肌动蛋白骨架，肌动蛋白、肌球蛋自在细胞骨架中也明显增多。其中PARI一AP刺激后1分钟达到高峰，PAR4一AP在5分钟达到最高值，以后随着时间延长呈现可逆性的分布规律。CytochalasinD与BAPrrA/AM应用后抑制了GPIb。向骨架中心的移位，肌动蛋白的改变也受到明显阴.滞，而肌球蛋白受BAPTA/AM影响的程度比Cytoehalasilll)张。 共免疫沉淀结果显小，“J GPIba相连的肌球蛋白、肌动蛋自随PAR肤i舌化也竹时增多。PAR卜八P在l分钟已经达到高峰，PAR4则石一1一5分钟出现最大值，之后都逐渐减少，呈现先结合后解离的可逆性变化曲线。其中肌球蛋自比肌动蛋自的改变更明显。cytocllalasinD与BAFI’A/AM作用后同样可以减弱肌球蛋自及肌动蛋自与GPlba的结合。 100，IM一与10、:Mw。:tmallllill处理一血二小板，分别抑制磷脂酞肌醇一3一激酶 （pl3一K）及jJ)七球蛋白轻链激酶（MLCK）活性。发现10林M wortmal飞nil飞能抑制血小板聚集，100nMwortmallnin使血小板容易发生解聚。流式细胞仪及荧光显微镜显示GPlba的动态分布过程中，1 oonMwortmannin能部份抑制GPIb以向胞内移动，}丫口1饵、M wortmallnin对整个PAR活化过程中的GPlba逆转没有任何影响，只对PARI刺激过程中GPlba的恢复起作用，延缓它重新返回111}小板表面的进程。蛋白电泳中1。卜Mwol，t mannin既不能苏州人学博士学位论文中文摘要阻_lLGPIba及肌动蛋白向骨架中J合集中，也不能抑制活化初期肌球蛋白及肌动蛋白与GPIb。的连接，却延缓GPIb。及肌动蛋白在胞质中心的消散，减慢肌球蛋白及肌动蛋白与GPIba的解离速度。 ApyraseVH在一定程度上影响血.小板聚集，增加诱导血‘小板聚集所需要的PAR月太浓度。ApyraseVH不能影响活化初期GPlba向细胞内的逆转，但促使GPIba尽快回复细胞表面，这在PARI的活化过程中更明显（10、3（）分钊，，P值<0.05）。 血小板聚集反应受Ro一3卜222。作用而抑制，但变形波不受影响。10抖MRO一31一2220可以明显抑制PARI一AP引起的GPIb。内陷（1、2分钟，P值均<0.05），对J几PAR4一AP则延长GPIba在胞内的逗留，减缓它返回膜外的进程仁10、3〔)分钊”，P值均<0.05)。 20扒MBAJ，TA/AM能完全抑制PARI一AP与PAR4一AP诱导的血一小板聚集，但变形波没有改变。Y27632不影更多还原

【Abstract】 Platelets play a major role in haemostasis and thrombosis. Platelet activation in vivo results from injury of the vessel wall, which is followed by spreading, secretion of granules, aggregation and procoagulant activity. This latter response leads to the generation of thrombin that is involved in the activation of platelets arriving in the blood flow at the site. Then, thrombin activates platelets at least in part through protease-activated receptors (PARs) which belong to the family of G protein-coupled receptor. Human platelets express PAR1 and PAR4, and activation of either is sufficient to trigger platelet secretion and aggregation. Thrombin activates human platelets by cleavage of the amino-terminal exodomain of PAR1 and PAR4, thereby-exposing a new amino terminus, which serves as a tethered peptide ligand. Binding of the tethered peptide ligand to the body of the receptor induces transinembrane signaling, leading to platelet secretion and aggregation. Another receptor for thrombin on platelets is the glycoprotein complex Ib-iX-V. Glycoprotein (GP) Ib is a sialoglycoprotein, which is implicated in platelet adhesion to the subendothelium expressing von willebrand factor (vWf). The critical roles played by GP Ib is emphasised by the Bernard-souliers syndrome where either a impaired expression of a component of the GP Ib-IX-V complex or a unfunctional complex is associated with a bleeding disorder. Platelet GP Ib moves into the open canalicular system (OCS) upon thrombin activation, and then returned to the platelet surface along time.showing a reversible translocation of GP Ib.In order to compare the participation of PAR-1 and PAR-4 during platelet activation, to determine whether they can induce GPIb redistribution, and then to investigate the role of the cytoskeleton in GP Ib centralisation, we use two peptides corresponding to PAR1-AP (SFLLRNPNDKYEPF) and PAR4-AP(AYPGKF) for stimulation. Using these peptides to stimulate platelets, we follow the redistribution of GP Ib and the link of GP Ibu, with actin and myosin. To study by which mechanism PARs induce the redistribution of GP Ib and the role of actin and myosin. a series of inhibitors were used in our experiments, among them Cytochalasin D (inhibitor of actin polymerization). BAPTA/AM (calcium chelator). Wortmannin (inhibitor of PB-kinasc and myosin light chain kinase). Apyrase VII (low ATP/ADPase). Ro-31-2220 (inhibitor of protein kinase C) and Y27632 (inhibitor of Rho kinase).Stimulation of either PAR1 or PAR4 leads to platelet aggregation with different efficiency. PAR-l-AP being the more potent, the following sequence was found: PAR 1 (SFLLRNPNDKY)>PAR4 (AYPGKF)>PAR4 (GYPGKF)> PAR4 (G YPGQV). In the following study, we used the more potent peptide for PAR1 and PAR4. Concomitant with a shrinkage of platelets, both flow cytoinctry and immtinofluorescence microscopy showed a transient redistribution of GP Ib in response to either PAR1-AP or PAR4-AP. detected as a decrease of approximately 50% of platelet surface GPIb. All over the time course experiment a different kinetic of redistribution of GP Ib was detected for the two peptides (p<0.05). Although an initial reduction of GP Ibalpha is apparent at 1 minute for both peptides. a maximum decrease is detected at 2 minutes upon PAR1-AP and 5 minutes upon PAR4-AP incubation, followed by a return of GP Iba to the surface, but slower for PAR4-AP. Similar results were obtained by various concentrations of PAR1-AP or PAR4-AP. but in a condition of enough concentration to induce platelet shape change. GP Ibainternalisation and shape change were blocked by Cytochalasin D or BAPTA/AM, although the former had a more dramatic effect. For P-selectin expression, there is a significant increase at 1 minute and maintained at a maximum state at 2 minutes.Following PAR stimulation, GP Iba became incorporated into the insoluble cytoskeleton. The fraction of GP Iba detected in the cytoskeleton reached a maximum at 1 minute in response to PAR1-AP, whereas it peaked at 5 minutes upon PAR4-AP activation, to更多还原