PI3K调控血小板聚集过程中RhoA活性及其与mDia1相互作用

a 本研究探讨RhoA／mDia1通路在血小板聚集过程中的作用及PI3K抑制剂对该过程的调控作用。分离人外周血中血小板,采用GSTpull-down法及免疫共沉淀法检测血小板聚集过程中RhoA、Rac1及Cdc42活性的变化;采用免疫共沉淀法检测血小板聚集过程中RhoA、Rac1及Cdc42是否与mDia1相互结合并形成复合体,并进一步检测PI3K抑制剂对上述过程的影响。结果表明：凝血酶能够诱导聚集血小板中RhoA活性上调,且与mDial相结合的RhoA-GTP显著增高;渥曼青霉素（wortmnnin）能够阻断由凝血酶诱导的聚集血小板中RhoA活性上调,并阻断凝血酶诱导的RhoA—GTP与mDia1的结合。凝血酶能上调聚集血小板中Rac1和Cdc42生物活性,但并未诱发其与mDia1结合,该过程也不能被wortmannin阻断。结论：PI3K通过RhoA／mDia1参与调控凝血酶诱导的血小板聚集等actin细胞骨架重构过程。     

鸦胆子油对血小板聚集的抑制作用及其机制的体外研究

目的以人洗涤血小板悬液为模型,体外观察鸦胆子油对血小板聚集的抑制作用,并探讨其可能机制。方法以离心法制备人洗涤血小板悬液,以血小板聚集分析仪、SDS-PAGE及流式细胞仪检测鸦胆子油对二磷酸腺苷(ADP)和凝血酶诱导的血小板聚集、肌动蛋白聚合体变化以及血小板膜P选-择素(CD62p)及纤维蛋白原受体(FIB-R)表达的影响。结果(1)鸦胆子油(9%和22.5%)显著抑制血小板聚集和FIB-R表达,显著降低血小板最大聚集水平和FIB-R表达水平;22.5%浓度抑制作用显著高于9%浓度。(2)血小板聚集水平与鸦胆子油浓度呈显著负相关,浓度越高,对血小板聚集的抑制程度越大。(3)鸦胆子油对血小板肌动蛋白变化及CD62p表达无抑制作用。结论鸦胆子油通过抑制活化血小板膜纤维蛋白原受体的表达,从而剂量依赖性地抑制血小板的聚集反应,有抗血栓形成作用。     

凝血酶受体活化与血小板膜糖蛋白Ⅰbα的异位分布机制

目的检测凝血酶受体活化过程中血小板膜糖蛋白(GP)Ⅰbα的动态分布及其细胞骨架蛋白的改变，揭示GPIⅠbα的异位分布机制与凝血酶受体在血小板信号传递中的作用。方法以凝血酶受体活化肽(thrombin receptor activating peplide，TRAP)诱导血小板活化，应用流式细胞术比较刺激前后血小板膜表面GPIbd及P-选择素的表达，并通过转移电泳及免疫沉淀技术，分析细胞骨架中GPⅠbα、肌动蛋白、肌球蛋白的变化。观测细胞松弛素(cytochalasin)D与腺苷三磷酸双磷酸酶(Apyrase)Ⅶ对血小板活化过程的影响。结果TRAP作用后促使血小板活化，流式细胞术检测结果显示其P-选择素水平显著升高，而GPⅠbα先呈现进行性减少，而后逐渐回升的可逆性变化。抑制剂作用后这一过程受到不同程度影响，其中细胞松弛素D能阻滞GPⅠbα的内移，ApyraseⅦ对活化过程巾GPⅠbα的逆转没有影响，只对它的恢复起作用，促使GPⅠbα迅速返回细胞表面。细胞骨架蛋白分析结果也表明肌动蛋白、肌球蛋白与GPⅠbα受刺激后呈先增加后减少的动态变化趋势。细胞松弛素D能阻滞GPⅠbα、肌动蛋白与肌球蛋白向骨架中心的转位，Apyrase Ⅶ则能加快骨架蛋白在骨架中心的消散。免疫印迹证实GPⅠbα的这种可逆性变化与血小板膜骨架紧密相关。结论凝血酶受体活化在血小板信号传递过程中发挥了重要作用，导致GPⅠbα由血小板膜表面向内转移而后又逐渐返回至胞膜外，这种动态变化与细胞骨架重组及二磷酸腺苷(ADP)有关。     

蛋白激酶A抑制剂对血小板膜糖蛋白I bα表达及血小板功能的影响

目的 探讨蛋白激酶A(protein kinase A,PKA)对血小板膜糖蛋白(Glycoprotein,GP)I bα表达的调控作用.方法 用健康人洗涤血小板与PKA抑制剂H89温育.用Westem blot检测血小板悬液中GP I bα的酶切片段糖萼蛋白(glycocalicin,GC),并用流式细胞术检测GP I bα的膜表达量.用Western blot检测血小板骨架蛋白的酶切程度来确定钙依赖蛋白酶(calpain)活性,并用流式细胞术检测血小板表面calpain的表达.用血小板聚集仪检测PKA抑制剂H89对瑞斯托霉素诱导的血小板聚集功能的影响.结果 洗涤血小板经H89处理后,血小板表面GP I bα被酶切并释放于血小板悬液中,使血小板表面GP I bα的表达水平显著降低(P＜0.05).多种calpain抑制剂与血小板预温育均可减少GP I bα的酶切程度,表明PKA抑制剂所致的酶切是calpain依赖性的.肌动蛋白结合蛋白(actin-bindingprotein,ABP)和踝蛋白(talin)的酶切表明calpain可被PKA抑制剂激活,流式细胞术结果 表明calpain在血小板表面有所表达.血小板经PKA抑制剂处理后,瑞斯托霉素诱导的聚集功能受到抑制.结论 抑制血小板PKA活性,可导致calpain依赖性的GP I bα酶切,使GP I bα膜表达量减低,并导致GP I bα依赖的血小板聚集功能减低.     

腺苷对血小板体外激活的抑制作用

本研究的目的主要是研究腺苷对血小板的体外激活抑制作用,为血小板冻干前处理过程筛选血小板功能保护剂提供依据.采用流式细胞术（FCM）测定血小板膜表面CD62P和PAC-1的表达,应用APACT-2聚集仪测定瑞斯托菌素（restocetin）、凝血酶（thrombin）、二磷酸腺苷（ADP）和没食子酸（propyl gallate）诱导的血小板聚集率.研究结果表明,冻干预处理后血小板膜表面CD62P表达显著增加,腺苷浓度为0.75 mmoL/L时可抑制CD62P表达,且呈剂量效应;腺苷可抑制没食子酸诱导的血小板聚集反应,但对瑞斯托霉素诱导的血小板聚集无抑制作用,腺苷浓度≥1.0 mmol/L对凝血酶诱导的聚集有显著抑制作用.因此,腺苷可抑制体外处理导致的血小板激活,对瑞斯托霉素和凝血酶诱导血小板聚集反应无明显抑制.结论：腺苷可作为血小板体外激活抑制剂及冻干前处理的功能保护剂.     

左旋精氨酸和西洛他唑对血小板体外激活的抑制作用

本实验旨在研究左旋精氨酸(L-arginine)和西洛他唑(cilostazol)在体外对血小板激活的抑制和功能保护作用,为可逆性血小板抑制剂在血小板保存中的应用提供依据。采用对血小板CD62p和PAC-1表达及凝血酶激活后再表达率的流式细胞分析(FCMs)、血小板聚集试验以及血小板凝血活性检测等手段,观察血小板激活、血小板功能状态。结果表明,体外处理后血小板CD62p和PAC-1表达率显著增加,西洛他唑和左旋精氨酸对这一过程CD62p和PAC-1表达有较强抑制作用,且随浓度增加而递增。西洛他唑可抑制凝血酶激活后CD62p和PAC-1的表达,左旋经氨酸仅抑制凝血酶激活后的PAC-1表达。左旋精氨酸和西洛他唑对3种诱导剂诱导的血小板聚集呈现不同程度的抑制作用,抑制作用随浓度递增。左旋精氨酸≥15mmol/L时,血小板聚集时间延长甚至不凝集。西洛他唑在浓度1-4mmol/L范围均延长血小板聚集时间。结论:5mmol/L和10mmol/L的左旋精氨酸均可抑制血小板体外处理过程的激活,且血小板的聚集和再表达功能不受影响,5mmol/L作用更佳。1mmol/L浓度西洛他唑抑制血小板体外激活,可保留血小板部分聚集活性和再表达能力。     

PI3K/Akt信号通路对转化生长因子β1诱导的人肺上皮-间质细胞转分化的影响

目的:探讨磷脂酰肌醇3激酶(PI3K)/丝氨酸/苏氨酸蛋白激酶(Akt)通路在转化生长因子β1(TGF-β1)体外诱导的人肺上皮细胞A549间充质化过程中的作用。方法:将体外培养的A549细胞随机分成3组:正常对照组、TGF-β1组及抑制剂组,正常对照组不加入TGF-β1,TGF-β1组加入10ng/mLTGF-β1,抑制剂组加入TGF-β1(10ng/mL)和PI3K的抑制剂Ly294002(10nmol/L),72h后通过RT-PCR检测各组A549细胞上皮标志物E-钙黏蛋白(E-cad)及间充质细胞标志物α-平滑肌肌动蛋白(α-SMA)表达的变化,ELISA方法检测间充质细胞标志物纤连蛋白(Fn)表达的变化,Westernblot检测Akt磷酸化(p-Akt)水平的变化。所有实验至少重复3次。结果:正常体外培养的A549细胞有E-cad表达及微量的α-SMA、Fn、p-Akt表达;TGF-β1组E-cad的表达下调,α-SMA、Fn、p-Akt的表达上调;抑制剂组与TGF-β1组比较E-cad的表达上调,α-SMA、Fn、p-Akt的表达明显抑制。结论:PI3K/Akt途径参与TGF-β1介导的肺上皮-间质细胞转分化过程,PI3K特异性抑制剂Ly294002可有效抑制TGF-β1介导的肺上皮-间质细胞转分化过程。     

人源化抗血小板膜糖蛋白Ⅵ单链抗体的研究

,形成4.1×107个克隆.用辅助噬菌体M13K07援救TG1后产生的抗体滴度为2.62×1010cfu/ml.亲和层析后得到的纯化抗体可抑制胶原诱导的血小板聚集而对二磷酸腺苷(ADP)诱导的血小板聚集无明显影响.结论 利用噬菌体抗体库技术表达的人源化抗血小板GPⅥ抗体ScFv片段可抑制血小板聚集功能.     

磷脂酰肌醇-3激酶在间充质干细胞成肌分化中的表达及肌损伤修复中的作用

目的 以 5-氮杂胞苷( 5-azacytidine, 5-Aza-CR)诱导间充质干细胞( mesenchymal stem cells,MSCs)成肌分化,探讨生肌调控因子 Myf5的表达情况及磷脂酰肌醇-3激酶( phosphoinositide-3 kinases, PI3K)在此分化全过程中的作用. 方法 分离、纯化及扩增 MSCs,以 5-Aza诱导其成肌分化, RT PCR测定 Myf 5的表达,免疫组化检测α-sarcomeric表达; Western blot检测 LY 294002抑制前后磷酸化 Akt蛋白水平的变化. 结果 LY294002作用后, Myf5表达延迟至诱导后 12 h;诱导后 10 d,部分 MSCs表达α-sarcomeric;磷酸化 Akt在 5-Aza诱导后蛋白水平逐渐增强, LY294002抑制明显下降. 结论 MSCs成肌分化过程中, PI3K是重要的正向信号转导通路.     

凝血酶信号传导过程中P13-K与MLCK激酶的作用

本研究比较凝血酶受体活化过程中不同浓度的wortmannin对血小板聚集率以及血小板膜表面活化标记物糖蛋白GPIb表达的影响,探讨脂酰肌醇-3-激酶（P13-K）与肌球蛋白轻链激酶（MLCK）在凝血酶信号传递机制中的作用。分别以凝血酶受体活化肽PAR1-AP（SFLLRN）与PAR4-AP（AYPGKF）诱导血小板活化。检测100nmol／Lwortmannin（抑制P13-K）与10μmol／Lwortmannin（抑制MLCK）作用过程中血小板聚集与血小板膜表面糖蛋白GPIb的改变。结果显示：wortmannin作用对两类凝血酶受体诱导血小板活化的过程均有不同程度的影响。100nmol／L的wortmannin部分抑制PAR1-AP引起的血小板聚集,对PAR4-AP诱导的聚集过程没有影响;10μmol／Lwortmannin明显抑制两类PAR肽引起的血小板活化,抑制程度达到60％-80％,仅出现少部分聚集。流式细胞仪检测显示,GPIba的动态分布过程中100nmot／Lwortmannin能部份抑制GPIbα向胞内移动,PAR1—AP刺激后5分钟内wortmannjn起效明显（1、2、5分钟P〈0．05）,对PAR4-AP的反应则稍延迟,在2到10分钟有意义（2、5、10分钟P〈0．05）。10μmol／Lwortmannin对整个PAR活化过程中的GPIba逆转没有任何影响,只对PAR1刺激过程中GPIbcL的恢复起作用,延缓GPIbα重返血小板膜表面的进程;而PAR4-AP作用后各时间段GPIba的动态分布不受10μmol／Lwortmannin任何影响。结论：P13-K与MLCK在凝血酶受体的活化过程中作用不同,P13-K在GPIbat内转的短暂过程中起着重要作用;而MLCK磷酸化仅仅对PAR1受体介导的GPIbα回复起促进作用。     

Pathways responsible for platelet hypersensitivity in rats with diabetes. I. Streptozocin-induced diabetes

Several pathways are activated when platelets aggregate and undergo the release reaction. We have examined the relative importance of these pathways in the responses to adenosine diphosphate (ADP), thrombin, or collagen of washed platelets from rats with diabetes induced by streptozocin. ADP-induced aggregation was enhanced without the release reaction with platelets from diabetic rats. Collagen-induced aggregation and release, and the adherence of platelets to collagen-coated glass were similar with platelets from diabetic and control rats. Thrombin (1 U/ml) induced more extensive loss of tritium from 3H-arachidonic acid-labeled platelets from diabetic rats than from control rats. Platelet aggregation and the release of 14C-serotonin from prelabeled platelets was greater in response to low concentrations of thrombin (0.04 U/ml). Creatine phosphate-creatine phosphokinase (CP/CPK) and aspirin completely blocked aggregation and partially blocked the release of granule contents from platelets from control and diabetic rats exposed to this low concentration of thrombin. Thus, the enhanced platelet aggregation in response to low concentrations of thrombin was likely mediated in part by released ADP and products formed from arachidonate. In contrast, with a higher concentration of thrombin (0.0625 U/ml), CP/CPK and aspirin did not inhibit the increased sensitivity of diabetic platelets to thrombin-induced aggregation and release; the concentrations of CP/CPK completely blocked aggregation induced by ADP (10 mumol/L), and the aspirin inhibited thromboxane B2 production in response to thrombin (1 U/ml) by 99%. Thus, a thrombin-induced pathway(s) of aggregation and release independent of released ADP and the products of arachidonate metabolism is enhanced in platelets from diabetic rats.     

Plasmin inhibition of platelet function and of arachidonic acid metabolism

To study interactions between platelets and the fibrinolytic system, we examined the effects of human plasmin on human platelets washed by gel filtration. Plasmin concentrations that did not affect platelet shape change, release, or aggregation (less than 1.0 caseinolytic units [CU]/ml) caused a dose- and time-dependent inhibition of platelet aggregation in response to thrombin, ionophore A23187, and collagen. Complete loss of aggregation occurred at 0.1-0.5 CU/ml of plasmin. In a parallel dose-dependent manner, plasmin likewise inhibited thrombin, ionophore, and collagen-stimulated thromboxane B2 production. In contrast, neither aggregation nor thromboxane B2 formation induced by arachidonate was inhibited by plasmin pretreatment of the platelets. Plasmin blocked the thrombin-induced release of [3H]arachidonic acid from platelet membrane phospholipids and the thrombin-induced platelet oxygen burst. However, plasmin did not inhibit the arachidonate-induced oxygen burst. Inhibition of arachidonic acid release by plasmin was not mediated by increase in platelet cyclic AMP. These results suggest that plasmin inhibits platelet function, at least in part, by blocking the mobilization of arachidonic acid from membrane phospholipid pools. The effects of plasmin on platelets may contribute to the hemostatic abnormalities seen in pathologic and pharmacologic fibrinolysis.     

Protease-activated receptor-induced Akt activation – regulation and possible function

BACKGROUND: Thrombin induces the activation of the platelet serine/threonine kinase Akt. Akt activation is dependent on its phosphorylation at Thr308 and Ser473. The mechanism by which thrombin induces Akt phosphorylation is controversial, as is the role of Akt in platelet function. OBJECTIVES: To investigate how protease-activated receptors (PARs) stimulate Akt and the role that Akt plays in human platelet function. METHODS: Platelets were stimulated through PAR1 or PAR4. Specific inhibitors were used to evaluate, by Western blotting, signaling pathways regulating Akt phosphorylation, and the role of activated Akt was evaluated by aggregometry and flow cytometry. RESULTS: Phospholipase C (PLC) controls Akt phosphorylation elicited by PARs. Stimulation of PAR1 or PAR4 resulted in rapid Akt phosphorylation, independently of secreted ADP and phosphatidylinositol-3-kinase (PI3K) activation. Akt phosphorylation approximately 60 s after PAR1 stimulation became entirely dependent on the purinergic receptor P2Y(12) and the activation of PI3K. In contrast, PAR4 partially sustained Akt phosphorylation independently of P2Y(12) and PI3K for up to 300 s. Pharmacologic inhibition of Akt reduced P-selectin expression and fibrinogen binding in platelets stimulated through PAR1, and delayed platelet aggregation in response to submaximal PAR1 or PAR4 stimulation, although aggregation at 300 s was unaffected. CONCLUSIONS: Platelet PAR stimulation causes rapid Akt phosphorylation downstream of PLC, whereas with continuous stimulation, ADP and PI3K are required for maintaining Akt phosphorylation. Activated Akt regulates platelet function by modulating secretion and alpha(IIb)beta(3) activation.     

Platelet cytoskeleton: immunofluorescence studies on thrombin-activated platelets

Cytoskeletal proteins were isolated from chicken gizzard smooth muscle and from platelets and antibodies prepared against them. It was shown by indirect immunofluorescence technique that actin, alpha-actinin, and vinculin are not present on the surface of platelets. Physiologic concentrations of thrombin (0.04 to 0.5 U/ml) that induce platelet aggregation and release in the presence of calcium from freshly isolated platelets do not induce platelet changes resulting in the availability of the cytoskeleton to antibodies. Because the F(ab')2 fragments of anti-cytoskeletal proteins IgG do not inhibit thrombin-induced aggregation of platelets, the direct role of these proteins in thrombin-induced platelet aggregation, as with ADP and collagen, may be rejected. However, when freshly isolated platelets are treated with thrombin (1 U/ml), antibodies to actin, alpha-actinin, and vinculin stained the platelets; therefore, this demonstrates that thrombin at this high and probably nonphysiologic concentration induces a reorganization of the membrane components with the subsequent exposure of the proteins of the cytoskeleton. We demonstrate interaction between isolated actin and alpha-actinin but not vinculin with fibronectin. After stimulation of platelets by thrombin, certain cytoskeletal proteins may interact with subendothelial fibronectin and thereby promote and consolidate platelet adhesion.     

Inhibition of human platelet aggregation by monovalent antifibrinogen antibody fragments.

Monovalent goat antibody fragments (Fab) that were monospecific for human fibrinogen were isolated by affinity chromatography on fibrinogen-Sepharose and used as a direct probe for the involvement of fibrinogen in platelet aggregation and the release reaction. The antifibrinogen Fab inhibited aggregation of washed human platelets induced by thrombin (0.1-10 U/ml) by 50-95%, but had no effect on (14-C)-serotinin release and only a slight inhibitory effect on 125-I-thrombin binding to platelets. Inhibition of aggregation was not observed with nonimmune goat Fab or rabbit antihuman albumie bound tightly at saturation to surface fibrinogen molecules. After washing the platelets once to remove unbound Fab, aggregation by subsequently added thrombin was no longer inhibited. The antifibrinogen Fab inhibited the clotting of fibrinogen by thrombin but did not effect the rate of fibrinopeptide A release, indicating that the Fab inhibits clotting by interfering with the polymerization of fibrin monomers. Our experiments suggest that fibrinogen released from platelets is directly involved in thrombin-induced aggregation of washed platelets, perhaps through polymerization of fibrin monomers generated by proteolytic cleavage of released fibrinogen.     

Properdin factor D: effects on thrombin-induced platelet aggregation.

Factor D, when preincubated with platelet suspensions, at concentrations as low as 1.2 micrograms/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor D were added to platelets at the same time. No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor D by increasing its concentration. Diisopropyl-phosphorofluoridate-inactivated factor D also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor D was not required for inhibition. Factor D absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 125I-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 micrograms/ml). Factor D is thus a competitive inhibitor of thrombin binding, although the affinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor D. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.     

Pathways responsible for platelet hypersensitivity in rats with diabetes. II. Spontaneous diabetes in BB Wistar rats

The discovery of a group of spontaneously diabetic rats has made it possible to examine changes in diabetic animals in the absence of possible confounding toxic effects of diabetogenic agents. The responses of washed platelets to adenosine diphosphate (ADP), thrombin, or collagen have been compared with platelets from spontaneously diabetic rats (these rats were hyperglycemic), their nondiabetic littermates (normoglycemic), and control rats from the same colony. Platelets from the diabetic rats aggregated more extensively in response to ADP than did platelets from the nondiabetic littermates or control animals. In contrast, platelet aggregation and release of granule contents in response to a low thrombin concentration (0.05 U/ml) were greater with platelets from diabetic rats and nondiabetic littermates than with platelets from control rats. A similar effect of collagen on the release of platelet serotonin was observed. Except at low concentrations of thrombin, the enhanced sensitivity to thrombin-induced aggregation and release of granule contents from platelets from diabetic rats or their nondiabetic littermates could not be inhibited by creatine phosphate-creatine phosphokinase (CP/CPK) and aspirin (CP/CPK used at concentrations that inhibited aggregation induced by ADP [10 mumol/L] and aspirin at concentrations that inhibited thromboxane B2 production induced by thrombin [1 U/ml] by 99%). Loss of radioactivity from platelets labeled with 3H-arachidonic acid and the amount of thromboxane B2 formed in response to high concentrations of thrombin (1 U/ml) was greater from platelets from the diabetic rats or their nondiabetic littermates than from control animals. Thus the effect of diabetes on this aspect of arachidonate metabolism is not primarily determined by blood glucose levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aprotinin: is it prothrombotic?

Controversy continues as to whether aprotinin (Trasylol) is prothrombotic. The recent discovery of the thrombin receptor family, known as the protease-activated receptor family (PAR) has been essential in aiding our understanding of the mechanism of action of aprotinin. Our results show that aprotinin has no effect on platelet aggregation induced by adrenaline, adenosine diphosphate, phorbol-12-myristate-13-acetate, collagen or PAR 1 agonist peptide. However, aprotinin inhibits thrombin-induced platelet activation as assessed by macroaggregation, microaggregation and platelet membrane calcium flux. Aprotinin inhibits proteolytic activation of platelets, but platelets can still be activated by non-proteolytic mechanisms.