一种水解血小板膜糖蛋白GPIb的眼镜蛇毒组分的分离纯化与鉴定

目的　建立从中华眼镜蛇毒中分离、纯化可水解血小板膜糖蛋白Ｉｂ（ｐｌａｔｅｌｅｔｍｅｍｂｒａｎｅｇｌｙｃｏｐｒｏｔｅｉｎＩｂ,ＧＰＩｂ）组分的方法。方法　肝素亲和层析,分子筛层析,ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳ ＰＡＧＥ）。结果　经Ｈｅｐａｒｉｎ ＳｅｈｐａｒｏｓｅＣＬ ６Ｂ亲和层析及ＳｅｐｈａｄｅｘＧ １５０分子筛层析,从中华眼镜蛇毒纯化出可降解纤维蛋白原和ＧＰＩｂ的活性组分,回收率为０－８８％,ＳＤＳ ＰＡＧＥ证实此组分为相对分子量约４７１００的单肽链蛋白。结论　此方法成功地从中华眼镜蛇毒中纯化出了水解ＧＰＩｂ的组分,具有高效、简便的特点。     

抗血小板膜糖蛋白Ib凝血酶受体单抗的制备及功能研究

目的：制备抗人血小板膜糖蛋白Ib(GPIb)单克隆抗体（单抗）并研究其功能，方法：杂交瘤技术制备单抗，HLISA法筛选与Western blot鉴定单抗识别的抗原，比浊法检测该单抗对血小板聚集的抑制作用。结果：（1）获得一株新的抗人血小板膜糖蛋白单抗SZ16。（2）在非还原状态下，SZ16识别分子量165000的抗原，表明SZ16抗原为GPIb.(3)该单抗为IgG1亚类。（4）FCM检测表明，,SZ16与正常人及血小板无力症（GT）患者血小板结合率很高，用凝血酶处理血小板后，几乎完全阻断SZ16与血小板的结合。（5）该单抗抑制低浓度凝血酶引起的血小板聚集，对ADP，瑞斯托霉素和花生四烯酸引起的血小板聚集无抑制作用。结论：SZ16为一株新的抗GPIb凝血酶受体的单抗。     

磷酯酶C抗血小板功能的研究Ⅰ——对血小板聚集率和粘附率的作用

目的　通过研究PLC对家兔血小板粘附率和聚集率的作用 ,以探讨PLC对血小板功能的影响。方法　家兔麻醉后 ,经十二指肠给予阴性对照辅料 ,阳性对照ASA和 6种剂量的PLC ,于给药前及给药后 1、2、4h颈动脉取血测定以ADP、AA、Collagen为诱导剂时的血小板聚集率 ,同时用玻球法测定血小板的粘附率。结果　PLC 10 0、 2 0 0IU·kg-1对家兔血小板粘附率无明显影响。PLC 4 0 0～ 10 0 0IU·kg-1可显著地降低血小板粘附率 ,并呈现一定的剂量效应依赖关系。同一剂量PLC组给药后 1、2、4h时相比P <0 0 5。PLC10 0～ 10 0 0IU·kg-16种剂量组在用ADP、AA、Collagen诱导时 ,均可显著地抑制血小板聚集 ;与阴性对照辅料组相比P <0 0 5 ,与阳性对照组相比 ,在ADP诱导时 ,10 0IU·kg-1组的抑制较弱 (P <0 0 5 ) ,而 80 0、10 0 0IU·kg-1组的抑制较强 (P <0 0 5 ) ,并呈一定的剂量效应和时间效应关系 ;而在AA诱导时 ,PLC各组在给药后 2、4h时的抑制率与ASA的相当 ,无明显剂量效应依赖关系 ,而有一定的时间效应关系 ;在Collagen诱导时 ,PLC各组的抑制作用均弱于ASA(P<0 0 5 ) ,无明显的剂量效应依赖关系 ,而有一定的时间效应关系。结论　①PLC经十二指肠给药 ,10 0、2 0 0IU·kg-1对家兔血小板粘附性无明显影响 ,而 4     

磷酯酶C对人血小板细胞质游离钙离子浓度的影响

目的　测定磷酯酶C(phospholipaseC ,简称PLC ,下同 )对静息状态和激活状态下人血小板胞质游离钙离子浓度的影响 ,从而进一步阐明PLC抗血小板聚集作用的机制。方法　将健康成人的洗涤血小板用荧光指示剂Fura 2 /AM进行负载 ,分别用生理盐水、ASA和不同剂量的PLC处理荧光负载后的血小板 ,采用双波长荧光分光光度法分别测定经过不同处理后的血小板在静息状态和以ADP为诱导剂时的激活状态下的胞质游离钙离子浓度 [Ca2 + ]i。结果　以健康成人血为样品时 ,经生理盐水、ASA 334μmol·L-1、2 5、3 75、5、10和 2 0UPLC·ml-1处理后的血小板 ,在静息状态下测得的胞质游离 [Ca2 + ]i 浓度 (nmol·L-1)分别为15 2 5 5± 15 0 7,131 6 3± 15 5 8,14 0 2 7± 12 0 3,139 4 8± 1 73,12 1 11± 9 5 8,116 6 2± 15 96和 10 7 2 0± 17 0 7,而以ADP为诱导剂激活血小板后测得的胞质游离 [Ca2 + ]i(nmol·L-1)分别为 90 2 6 2± 94 74 ,6 87 99± 6 2 86 ,810 99± 72 37,70 1 73± 2 1 37,4 2 9 6 7± 71 5 9,342 82± 4 4 86和 2 6 3 2 7± 2 5 4 6。以生理盐水作为对照 ,ASA 334μmol·L-1、2 5、3 75、5、10和 2 0UPLC·ml-1剂量组对激活状态下血小板胞质 [Ca2 + ]i 的抑制率I(% )分别为 2 5 8     

双抗体酶联免疫法测定血小板抗体

双抗体酶联免疫法测定血小板抗体太原市中心医院（０３０００９）辛秋绒，李翠香，贾玉萍，史玉亮我们从１９９１年开始用酶联免疫法对免疫性血小板减少疾患和非免疫性血小板减少疾患的血小板抗体（ＰＡＩｇＧ）进行了检测，并与健康人做了对照。现将检测结果报告如下。对...     

磷酯酶C对家兔血小板聚集和超微结构的影响

目的　应用电镜研究磷酯酶C(phospholipaseC ,PLC)对家兔血小板聚集和超微结构的影响 ,以探讨用药后PLC抗血小板聚集作用的机制。方法　家兔颈动脉插管取血 ,制备PRP ,将其分为 4组 :①空白对照组 ;②生理盐水组 ;③ 0 5UPLC组 ;④ 2 5UPLC组。 2～ 4组分别用ADP诱导聚集 ,测出其聚集抑制率 ,各组分别制成超薄切片样品进行电镜观察、摄片。结果　 0 5、2 5UPLC明显抑制血小板聚集反应 ,聚集抑制率分别为 86 0 3 %± 12 6%和 82 47%±5 49% ;0 5UPLC抑制血小板形成伪足 ,α颗粒和致密颗粒较多 ,OCS管腔较小 ,其超微结构较生理盐水组变化小 ;2 5UPLC处理血小板的形态结构和超微结构与空白对照组无差异 ,血小板呈圆形或椭圆形 ,边缘光滑 ,无伪足样突起 ,α颗粒、致密颗粒、OCS等正常分布于胞质中。结论　PLC明显抑制ADP诱导的血小板聚集反应及其超微结构的改变 ,这可能是PLC抗血小板聚集作用的重要原因之一     

磷脂酶C对ADP诱导的兔血小板IP_3水平的影响

目的测定磷酯酶C(PLC)对ADP诱导的兔血小板三磷酸肌醇(IP3)的影响,从而进一步阐明PLC抗血小板聚集作用的机制。方法取家兔血除去红细胞制备血小板,血小板计数后分为8个组。第1组用生理盐水处理,第2组用ADP处理,第3组用ASP处理以ADP诱导激活,第4~8组分别用不同剂量的PLC处理并以ADP诱导激活。采用三氯醋酸法制备血小板IP3,再用放射免疫分析法分别测定IP3含量。结果家兔血小板经生理盐水处理后的IP3含量为0.29 pmol/108血小板,而经ADP处理后IP3含量为0.39pmol/108血小板。经ASP 668μmol.L-1、5、10、15、20和25 U PLC.ml-1各组处理,并以ADP激活的血小板,测得的IP3含量分别为0.19、0.08、0.15、0.25、0.04和0.18(pmol/108血小板)。ADP组比生理盐水组IP3有明显的提高,而ASP组、不同的PLC组比ADP组IP3有明显的降低(P<0.05)。结论PLC使得ADP激活后的兔血小板IP3水平下降,且无剂量依赖性,表明PLC具有降低血小板中IP3水平的作用,这可能是PLC抗血小板聚集作用的重要原因之一。     

体外循环对血小板膜糖蛋白的影响

目的　探讨体外循环下心内直视手术对血小板膜糖蛋白 GP b、GP a及 GMP140的影响。方法　选择体外循环下心内直视术患者 30例 ,以体外循环转机前及停机即刻为采血点 ,用流式细胞仪测定血小板膜GP b、GP a 和 GMP140的阳性百分率 (% )。结果　血小板膜 GP b、GP a 百分率由体外循环前的 90± 6 ,93± 5分别减少至体外循环后的 70± 12和 76± 8,两者比较差异有显著性 (P值均 <0 .0 0 1) ;而体外循环前后GMP140阳性率差异无显著性 (P>0 .0 5 )。结论　体外循环使血小板膜 GP b 和 GP a 阳性率下降 ,可能是体外循环引起血小板功能异常的重要因素之一。而血小板在体外循环中发生了活化 ,尚需检测血浆 GMP140以进一步探讨。     

血小板相关抗体与血小板输注效果的关系探讨

目的 探讨血小板相关抗体对血小板输注效果的影响.方法 采用酶联免疫吸附试验(ELISA)检测120例长期反复输血并且需要输注血小板患者的血小板相关抗体,检测输血前后血小板计数,采用血小板计数增值考察血小板输注效果.采用简易致敏红细胞血小板血清学技术( SEPSA)对血小板相关抗体阳性患者进行血小板交叉配型后进行血小板输注.结果 反复输血的患者中血小板相关抗体阳性率为65.0％,血小板输注的有效率为61.7％.血小板相关抗体阳性患者和阴性患者输注血小板时输注无效率分别为84.6％和19.0％,差异有统计学意义(x2=46.914,P＜0.01).对血小板相关抗体阳性患者采用SEPSA进行血小板配型后输注血小板有效率为85.9％.结论 血小板相关抗体阳性与血小板输注无效相关,血小板配型可有效提高临床输注效果.     

磷脂酶C对ADP诱导的兔、人血小板肌动蛋白聚合的抑制作用

目的　测定磷脂酶C(PLC)对ADP诱导的兔、人血小板肌动蛋白聚合的的影响 ,从而进一步阐明PLC抗血小板聚集作用的机制。方法　取家兔和健康成人的PRP分别用生理盐水、ASA和不同剂量的PLC处理 ,以ADP诱导激活 ,采用Triton抽提液提取和SDS PAGE分离出肌动蛋白 ,再以分光光度法分别测定其肌动蛋白相对含量。结果　家兔血小板经生理盐水处理后的肌动蛋白相对含量为 1 6 82±0 319。而经生理盐水、ASA 6 6 8μmol·L-1、PLC 5、10、15、2 0和 2 5U·ml-1各组处理 ,并以ADP激活的血小板 ,测得的肌动蛋白相对含量分别为 2 4 5 0± 0 5 6 2 ,1 0 89± 0 32 2 ,1 72 7± 0 4 4 2 ,1 4 5 0± 0 32 4 ,1 16 1± 0 30 6 ,0 85 7± 0 2 4 2和0 6 92± 0 187。以生理盐水组作为对照 ,各处理组血小板肌动蛋白聚合的抑制率I(% )分别为 5 5 5 5 ,2 9 5 1,4 0 82 ,5 2 6 1,6 5 0 2 ,71 76。人血小板经生理盐水处理后的肌动蛋白相对含量为 1 35 8± 0 376。而经生理盐水、ASA 6 6 8μmol·L-1、PLC 5、10、15、2 0和 2 5U·ml-1各组处理并以ADP激活后的血小板 ,测得的肌动蛋白相对含量分别为2 4 4 5± 0 75 0 ,1 0 96± 0 344 ,1 70 5± 0 5 0 7,1 4 37±0 4 16 ,1 16 5± 0 35 5 ,0 84 5± 0 2 5 7?     

Effects of toluene on platelet membrane glycoprotein Ib and actin-binding protein

Effects of the organic solvent toluene on the platelet membrane receptor glycoprotein Ib (GP Ib) and the cytoskeletal protein, actin-binding protein (ABP), were studied and related to the effects of the local anesthetic dibucaine. The glycocalicin-region of GP Ib contains the binding site for von Willebrand factor; intracellularly GP Ib is linked to the cytoskeleton via ABP. Both GP Ib and ABP are substrates for a calcium-dependent protease, calpain. Washed platelets were incubated with toluene or dibucaine. The toluene concentration in the platelet suspension was analysed by gas chromatography. Using 1.5-2.8 mmol/L toluene, calpain was activated, leading to degradation of ABP and release of glycocalicin from GP Ib. The latter phenomenon was paralleled by a reduced von Willebrand factor-induced platelet agglutination. At lower toluene concentrations (0.3-1.4 mmol/L), degradation of ABP was not detected but an initial increased agglutination that declined to the control level with time was observed. These effects of toluene on the GP Ib-ABP complex are similar to those observed with 1 mmol/L dibucaine. The lowest toluene concentrations used correspond to those that have been found in blood from toluene abusers ("sniffers").     

PLC对ADP诱导的兔血小板cAMP的影响

目的　测定磷酯酶C(phospholipaseC,简称PLC,下同)对ADP诱导的兔血小板cAMP的影响,从而进一步阐明PLC抗血小板聚集作用的机制。方法　取家兔的PRP,除去红细胞,分别用生理盐水、ASA和不同剂量的PLC处理,以ADP诱导激活,采用三氯醋酸法制备血小板cAMP,再用放射免疫分析法分别测定cAMP含量。结果　家兔血小板经生理盐水处理后的cAMP含量为 ( 20 16±0 91 ) pmol/109platelets,而经生理盐水、ASA668μmol·L-1、5、10、15、20和25UPLC·ml-1各组处理,并以ADP激活的血小板,测得的cAMP含量分别为 13 85±1 14, 16 27±0 36, 18 74±0 55,19 80±0 52, 20 49±0 43, 22 55±0 61和 24 24±0 85(pmol/109 platelets)。以生理盐水作为对照,ASA668μmol·L-1、5、10、15、20和 25UPLC·ml-1剂量组对激活状态下血小板cAMP的含量有增加的作用,增加率 (% )分别为17 47, 35 31, 42 96, 47 94, 62 82, 75 02。结论　PLC使得ADP激活后的兔血小板cAMP水平提高(P<0 01),且呈剂量依赖性,表明PLC具有提高血小板中cAMP水平的作用。这可能是PLC抗血小板聚集作用的重要原因之一。     

Inhibition of platelet glycoprotein Ib and its antithrombotic potential

The platelet receptor glycoprotein (GP)Ib-IX-V complex plays a dominant role in the first steps of platelet adhesion and arterial thrombus formation. Through its interaction with the multimeric plasma protein von Willebrand factor (VWF), which is bound to the damaged subendothelial structures, GPIb-IX-V tethers the platelets from the flowing blood thereby slowing them down. This step is a prerequisite for the collagen receptors to participate in firm adhesion resulting in the formation of a first platelet layer which is the basis for further thrombus formation. Recently, other ligands for GPIb-IX-V besides the extensively studied VWF have been identified, such as: alpha-thrombin, coagulation factor XII (FXII), high molecular weight kininogen (HMWK), factor XI (FXI), integrin Mac-1 and P-selectin. In this review, the interaction of GPIb-IX-V with its different ligands is described and the anticipated or demonstrated in vivo effects are discussed.     

The change of serum leptin and its relationship with platelet membrane glycoprotein Ib in patients with coronary heart disease

The aim of this paper was to investigate the change of serum leptin and its relationship with platelet membrane glycoprotein Ib (GP Ib) in patients with coronary heart disease (CHD). The enrolled included 50 patients with CHD (CHD group) and 30 patients without CHD (control group) who were diagnosed by coronary angiography. The positive percentage and the average fluorescence intensity of platelet membrane GP Ib were detected by full-blood flow cytometry. Serum leptin was detected by enzyme linked immunosorbent assay. The positive percentage and the average fluorescence intensity of platelet membrane GP Ib in the CHD group were significantly lower than those in the control group (P < 0.05). After correcting the differences of systolic blood pressure, body mass index (BMI), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting glucose, PPBS, fasting insulin and quantitative insulin sensitive index, serum leptin level in the CHD group was significantly higher than that in the control group (P < 0.05). Single factor correlative analysis revealed that serum leptin in CHD patients was negatively correlated with the average fluorescence intensity of platelet membrane GP Ib (P < 0.05). Multifactorial stepwise regression analysis showed that serum leptin in CHD patients was independently negatively correlated with the average fluorescence intensity of platelet membrane GP Ib (P < 0.05). Logistic analysis demonstrated that serum leptin was independently correlated with the risk of CHD (P < 0.05). Hyperleptinemia was verified in CHD patients. The increase of serum leptin could affect blood platelet activation. Hyperleptinemia may play an important role in the pathogenesis of CHD. __________ Translated from Tianjin Medical Journal, 2007, 35(2): 81–83 [译自: 天津医药]     

Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist

1. Agkistin, purified from the snake venom of Formosan Agkistrodon acutus, belongs to the family of C-type lectin GPIb binding proteins. It is a heterodimeric molecule, consisting of alpha- (16.5 kDa) and beta- (15.5 kDa) subunits with a molecular mass of 32,512 Daltons examined by SDS - PAGE and mass spectrometry. 2. In vitro, agkistin concentration-dependently inhibited ristocetin-induced human platelet agglutination and aggregation in the presence of vWF. It also inhibited TXA2 formation and prolonged the latent period in triggering aggregation by a low concentration of thrombin (0.03 u x ml(-1)). 3. 125I-agkistin specifically bound to unactivated human platelets in a saturable manner with a KD value of 223+/-10.6 nM. This binding reaction was rapid and reversible. Monoclonal antibodies, AP1 and 6D1 raised against platelet GPIb, almost completely blocked 125I-agkistin binding to platelets. However, monoclonal antibody 7E3 raised against GPIIb/IIIa complex, trigramin, a GPIIb/IIIa antagonist, ADP and EDTA did not affect 125I-agkistin binding reaction. 4. Agkistin (250 microg x kg(-1)) significantly prolonged the bleeding time and induced transient thrombocytopenia of mice when given intravenously. Furthermore, it markedly inhibited platelet plug formation in irradiated mesenteric venules of fluorescein-treated mice in vivo. 5. In conclusion, agkistin inhibits ristocetin induced platelet aggregation mainly through its specific binding to platelet GPIb, thereby blocking the interaction between GPIb and vWF. In addition, agkistin exhibits antithrombotic activity in vivo.     

奥扎格雷钠对脑梗死急性期患者血小板膜糖蛋白表达和临床疗效的影响

目的研究奥扎格雷钠对患者血小板膜GPⅡb/Ⅲa复合物和P-选择素、NIHSS评分的影响,探讨奥扎格雷钠对血小板活化的影响及与临床疗效的关系。方法对脑梗死急性期患者入院第1天和第14天进行NIHSS评分,并应用流式细胞术检测血小板膜GPⅡb/Ⅲa复合物和P-选择素。将患者分为阿司匹林组、奥扎格雷钠组,对比两组患者NIHSS评分和血小板膜GPⅡb/Ⅲa复合物和P-选择素的差异。结果两组治疗第14天奥扎格雷钠组的血小板膜NIHSS、GPⅡb/Ⅲa复合物和P-选择素均比阿司匹林组下降更明显(P<0.05)。结论阿司匹林和奥扎格雷钠均可降低血小板活化程度,从而缓解脑梗死病情,而奥扎格雷钠作用更强,取得的临床疗效更为明显。