Study on the mechanism of the annexin II-mediated Co-assembly of t-PA and plasminogen

In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.     

Effect of Antisense Oligonucleotide to Annexin Ⅱ on the t—PA—mediated Plasminogen Activation in vitro

Summary: In order to study the role of annexin Ⅱ , a recombinant expression vector, pZeoSV2 (+)ANN Ⅱ , containing the annexin Ⅱ cDNA, was developed. The 1.1-kb-length annexin ⅡeDNA was inserted into a expression vector, PZeoSV(+) and transfected into HL-60 cells which had low baseline expression of Ann- I. pZeoSV(+) ANN I was analyzed by restriction mapping and the Ann-I sequence identified. The ability of the transfected cells, non-transfected and mock-transfected cells to stimulate t-PA-depend plasminogen activation was compared. The results showed that HL-60 with pZeoSV (+)ANN I transfection could significantly increase the plasminogen activation (8. 9± 1.2 U) in vitro with the difference being significant as compared with non-transfected (1.5±0. 4 U) and mock-transfected cells (4.2±0. 9 U), respectively. Antiannexin Ⅱ oligonucleotides significantly inhibited the binding ability of t-PA and plasminogen to annexin Ⅱ , and obviously reduced the plasminogen activation in vitro. The above findings showed human umbilical vein endothelial cells (HUVECs) treated with sense or missense oligonucleotides indicated no significant change in binding of t-PA and PLG. Treatment of HUVECs with antian nexin Ⅱ oligonucleotides could significantly reduce the plasminogen activation by 2.4± 0. 3 U as compared with sense oligonucleotide group in binding of t-PA and PLG. These results, therefore,suggest that Ann- Ⅱ can bind plasminogen and participate in the stimulation of t-PA-dependent ac tivation of plasminogen, and that interference with Ann- Ⅱ mRNA by antisense oligonucleotidemay be a new strategy for the therapy of bleeding in patients with hyperfibrinolysis.     

Coagulation and fibrinolytic activity in patients with acute cerebral infarction

Thecoagulationandfibrinolyticactivityinpatientswithacutecerebralinfarctionisabnormal Toinvestigatetheirchanges ,wemeasuredtheconcentrationofD dimer (DD) ,tissueplasminogenactivator (t PA) ,plasminogenactivitorinhibitor 1(PAI 1)andplaminogen (PLG )activityinplasmaandcerebrospinalfluid METHODSSubjectswererandomlyselectedfrom 35consecutiveischemicstrokepatients (2 1menand 14womenwithameanageof 6 3years)admittedtoourhospitalwithin 72hoursafteronsetfromApril 2 0 0 0toFebruary 2 0 0 1 Ofthes…     

以硼替佐米为基础的化疗方案对骨髓瘤患者血小板、凝血及纤溶功能的影响

目的探讨以硼替佐米为基础的化疗方案对多发性骨髓瘤(MM)患者血小板、凝血及纤溶功能的影响。方法选取2014年3月~2017年3月在我院进行治疗的MM病人80例,分别在用药前、用药第1 d、用药第4 d的1 h后对血小板最大凝集率、抗凝血酶(AT)、a PTT、PT、TT、D二聚体(D-D)、纤溶酶原活性(PLG)、组织型纤溶酶原激活物活性(t-PA)、纤溶酶原激活抑制物活性(PAI)、α_2-抗纤溶酶抑制物活性(α_2-PI)进行测定,比较用药前后血小板、凝血、抗凝及纤溶功能的变化。结果与用药前相比,用药第1 d、用药第4 d的1 h后,血小板最大凝集率呈现逐渐下降趋势,差异有统计学意义(P=0.009;P=0.012),AT呈现增长趋势但不明显(P=0.393;P=0.273);a PTT、PT、TT无明显变化,而D-D呈现明显增长趋势(P=0.513;P=0.423)。结论以硼替佐米为基础的化疗方案可使MM患者血小板最大凝集率下降,对其他凝血、抗凝及纤溶功能无明显影响。     

登革病毒诱导血管内皮细胞凝血和纤溶系统相关分子变化

目的为研究登革病毒感染血管内皮细胞所引起的凝血和纤溶分子表达变化.方法应用D2V感染生长良好的第2、3代脐静脉内皮细胞以观察内皮细胞在表达组织纤溶酶原激活物(tPA)、纤溶酶原激活物抑制物1(PAI-1)、凝血酶调节蛋白(TM)等分子变化.结果D2V可以显著上调内皮细胞的t-PA的表达并显著提高血浆可溶性凝血酶调节蛋白(sTM)和IL-6水平,而不影响PAI-1.抗IL-6抗体抑制D2V感染对内皮细胞表达t-PA和sTM的增强效应.结论血管内皮细胞是D2V的靶细胞之一,D2V可以诱导内皮细胞表达t-PA和IL-6并提高血浆中sTM水平,IL-6能有效地增强D2V诱导的纤溶和抗凝分子表达,高水平的t-PA和sTM可诱导纤溶亢进和抗凝效应增强,在IL-6诱导血管内皮细胞渗透性升高的前提下,有助于DHF/DSS患者出现血浆渗漏、血容量丢失甚至出血.     

妊娠晚期糖尿病孕妇止凝血功能指标检测的临床意义

目的 探讨妊娠晚期糖尿病孕妇的血小板活化状态、血管内皮损伤、抗凝及纤溶系统部分功能指标的变化及其临床意义.方法 检测了正常非孕妇女、正常晚期妊娠妇女各20例和46例妊娠晚期糖尿病孕妇的血管性血友病因子、血小板α-颗粒膜蛋白、抗凝血酶-Ⅲ、蛋白C系统筛选、纤溶酶原、组织纤溶酶原激活物及其抑制物、D-二聚体等指标的含量.结果 与正常非孕组及正常晚期妊娠组比较,妊娠晚期糖尿病孕妇组血管性血友病因子、血小板α-颗粒膜蛋白、组织纤溶酶原抑制物水平均显著增高(P＜0.01),组织纤溶酶原激活物活性明显低于正常晚期妊娠组(P＜0.01);与正常非孕组比较,正常晚期妊娠组和妊娠期糖尿病组空腹血糖、纤溶酶原、D-二聚体等指标均显著增高(P＜0.01).与正常晚期妊娠组比较,妊娠期糖尿病组空腹血糖、纤溶酶原、D-二聚体均显著增高(P＜0.01),抗凝血酶-Ⅲ、蛋白C活性依赖凝固时间在正常晚期妊娠组较非孕组呈下降趋势,妊娠期糖尿病患者AT-Ⅲ水平与正常晚期妊娠组无显著差异.结论 正常晚期妊娠妇女血液处于血栓前状态,而糖尿病孕妇存在着明显的血栓前状态,因此,糖尿病孕妇产前测定凝血和纤溶功能指标,对监测病情、指导治疗、改善预后具有一定的价值.     

肝病患者手术及介入治疗后凝血纤溶指标监测对疗效的评估作用

目的探讨肝病患者手术或介入治疗后凝血及纤溶指标的变化对疗效的评估价值。方法对肝病组（100例）、肝癌组（100例）于手术或介入治疗前与后检测血浆凝血酶原时间（PT）、活化部分凝血酶原时间（AFTT）、纤维蛋白原（FIB）、抗凝血酶Ⅲ（ATⅢ）活性、蛋白C（PC）活性、蛋白S（PS）活性、纤溶酶原（PLG）活性、组织型纤溶酶原活化物（t-PA）、纤溶酶原活化剂抑制物（PAI）、α2-抗纤溶酶（α-PI）活性、纤溶酶-纤溶酶抑制物（PAP）,并与正常对照组100例比较。结果与对照组比较,肝癌组介入治疗前与后PT、APT、t-PA、PAI、PAP均明显升高（P〈0．05或〈0．01）,FIB、ATⅢ活性、PC活性、PS活性、PLG活性、α2-PI活性均明显降低（P〈0．05或〈0．01）;与介入治疗前比较,介入治疗后PT、APTT、t-PA降低,FIB、ATⅢ活性、PC活性、PS活性、PLG活性、α2-PI活性升高（P〈0．05）,而PAI-1和PAP均差异无统计学意义（P〉0．05）。与对照组比较,肝病组手术治疗前与后PT、APTT、t-PA、PAP均明显升高（P〈0．05或〈0．01）,而FIB、ATⅢ、PC活性、PS活性、PLG活性、PAI、α2-PI活性降低（P〈0．01）;与手术治疗前比较,肝病组手术治疗后PT、APTT、t-PA、PAP降低（P〈0．05或〈0．01）,FIB、ATⅢ活性、PC活性、PS活性、PLG活性、PAI、α2-PI活性升高（P〈0．05或〈0．01）。结论肝病组和肝癌组均存在抗凝活性降低及易发纤溶。手术或介入治疗后有所改善,但未完全恢复正常。     

血定安对手术患者纤溶功能的影响

观察了２５例手术患者快速输注血定安（Ｇｅｌｏｆｕｓｉｎｃ）后血浆组织型纤溶酶原激活物（ｔ－ＰＡ）及其抑制物（ＰＡＩ）、Ｄ－二聚体、α－纤溶酶抑制物（α－ＰＩ）活性的变化情况。结果显示：输入血定安１０００ｍｌ后即刻、２ｈ．２４ｈ．对ｔ－ＰＡ．ＰＡＩ．Ｄ－二聚体、α２－ＰＩ活性均无明显影响，Ｐ均＞０．０５。表明血定安不影响手术患者纤溶系统功能。     

Effects of simvastatin on cigarette smoke extract induced tissue-type plasminogen activator and plasminogen activator inhibitor-1 expression in human umbilical vein endothelial cells

Background Cigarette smoking has an influence on both arterial-type and venous-type thrombosis. However, little is known about the direct effect of cigarette smoke extract （CSE） on fibrinolytic activity of human umbilical vein endothelial cells （HUVECs）. Most recently, simvastatin has been marked in its effect on endothelial cells protection and anticoagulation. In this study, the effect of CSE on the expression of tissue-type plasminogen activator （t-PA） and plasminogen activator inhibitor-l（PAl-1） in HUVECs was addressed. The role of simvastatin in CSE-induced fibrinolytic activity changes was investigated as well. Methods The fourth to fifth generation of HUVECs were incubated respectively with 0, 5%, 10% and 20% CSE for 6 hours or exposed to 5% CSE for 0, 4, 6, 8, 12, 24 hours to determine the expression changes of t-PA and PAl-1 protein. Meanwhile, cells were also accordingly exposed either to 5% CSE alone or simvastatin pre-treated and 5% CSE for 24 hours to assess the role of simvastatin in CSE-induced t-PA and PAl-1 protein and mRNA expression in HUVECs. RT-PCR and ELISA techniques were used for detecting the t-PA or PAl-1 mRNA and protein. Results After 6-hour exposure to CSE, the expression levels of t-PA protein in 10% and 20% CSE-treated groups reduced significantly （（0.0365±0.0083） ng/ml, （0.0255±0.0087） ng/ml） when compared with that of control group （（0.0660±0.0120） ng/ml） （P 〈0.05）. In contrast, the levels of PAl-1 protein in 5%, 10% and 20% CSE-treated groups increased remarkably （（13.3225±0.5680） ng/ml, （14.2675±1.5380） ng/ml, （14.4292±1.6230） ng/ml） when compared with that of control group （（8.5193_±0.7537）ng/ml） （P〈0.05）. After stimulation with 5% CSE for 0, 4, 6, 8, 12, 24 hours, the levels of PAl-1 protein increased over time and reached the peak at 24 hours （（14.6400±1.0651） ng/ml）, which was significantly higher than that of control group （（12.0656±0.6148） ng/ml） （P 〈0.05）. Additionally, CSE could up-regulate PAl-1 expression at both the mRNA and the protein levels. The levels of PAl-1 mRNA and protein increased significantly in 5% CSE-treated group （（8.8030±0.4745） ng/ml, （1.8155±0.0412） ng/ml） compared with those of control groups （（5.0588±0.2315） ng/ml, （1.3030±0.0647） ng/ml） （P 〈0.01）, and decreased after 2-hour simvastatin pre-treatment （（5.4875±0.3166） ng/ml, （1.3975-±0.0297） ng/ml） （P 〈0.01）. No significant difference was found at the levels of t-PA protein and mRNA （P 〉0.05）. Conclusions CSE inhibits the fibrinolytic activity of HUVECs in vitro. Simvastatin plays a protective role in CSE-induced fibrinolytic malfunction.     

痰瘀同治对脑梗死急性期t-PA、PAI影响的临床观察

目的观察化痰祛瘀汤对脑梗死急性期患者临床神经功能缺损评分、血浆组织型纤溶酶原激活物(t-PA)及血浆组织型纤溶酶原激活物抑制物(PAI)水平的影响,以及对中医证候的影响。方法42例脑梗死急性期患者随机分为2组,对照组22例,采用中西医常规治疗;治疗组20例,在对照组治疗的基础上加口服中药汤剂化痰祛瘀汤。治疗前后行神经功能评分及中医证候评定,并测定治疗前后血浆t-PA、PAI水平。结果治疗组总有效率达75%,对照组为41%,组间比较有显著性差异(P<0.05)。神经功能评分有明显改善,明显提高t-PA水平,降低PAI水平(P<0.05),对中医证候的改善具极显著意义。结论化痰祛瘀汤可显著改善神经功能缺损,作为脑梗死急性期联合用药可明显提高临床疗效,其作用机制可能与其影响t-PA、PAI活性有关。     

Activity of Ginkgo biloba extract and quercetin on thrombomodulin expression and tissue-type plasminogen activator secretion by human umbilical vein endothelial cells

INTRODUCTION Ginkgo biloba extract (GBE) has been used as a ommonly prescribed drug in traditional Chinese edicine for several thousand years. GBE is still sed in clinics in Europe to alleviate symptoms ssociated with blood circulation disorder. It has be…     

双胸蚓溶栓酶制剂对家兔血浆t—PA与PAI活力的影响

从双胸蚓组织分离、提取的溶栓酶制剂含有纤溶酶类及纤溶酶原激活物,具有良好的溶解血栓作用。动物实验表明该制剂能使体内t-PA活力增加,但不影响PAI活力。     

激肽释放酶激活纤溶酶原机理的研究

作者研究了血浆和组织激肽释放酶激活纤溶酶原的机制。经SDS-PAGE和HPLC分析,提示血浆激肽释放酶裂解纤溶酶原的位点与t-PA、UK的相同,只是前者的酶反应速度明显低于后二者。组织激肽释放酶裂解纤溶酶原的位点除了与血浆激肽释放酶相同的部份外,尚有其它作用位点。因此认为这两种激肽释放酶激活纤溶酶原的机制不完全相同。     

急性高眼压t-PA表达的实验研究

目的:探索血浆组织型纤溶酶原活化因子(t-PA)在青光眼视网膜中的表达变化、可能的作用以及脑源性神经营养因子(BDNF)对其的影响。方法:采用前房灌注法建立兔急性高眼压模型,在玻璃体内注射BDNF或BSS液,用免疫组织化学法检测视网膜t-PA的蛋白表达与定位,用Westernblot分析检测视网膜t-PA的蛋白表达。结果:t-PA与β-actin灰度比值,正常对照组为(57.95±3.79)%、BSS组为(89.36±3.59)%、BDNF组为(66.66±2.16)%;BSS组与正常对照组和BDNF组相比,t-PA表达明显升高(P<0.05)。高眼压后视网膜上t-PA的免疫组化染色明显增强,神经纤维层、RGCs层呈强阳性染色。结论:t-PA与青光眼的病理过程有相关性,BDNF可能部分通过抑制t-PA的表达发挥神经元保护作用。     

脑血栓患者纤溶活性的动态观察

目的　测定脑血栓患者治疗前后纤溶活性的变化 ,探讨其发病机制及诊治方案。　方法　分别采用发色底物法定量检测纤溶酶原 (PLG)、组织纤溶酶原激活物 (t-PA)以及纤溶酶原激活抑制物 (PAI)的活性 ,并用双抗体夹心法测定D -二聚体 (D -D)。　结果　脑血栓患者治疗前t -PA活性较低 ,PLG、PAI活性较高 ,D -D含量增多 ,与正常对照组比较 ,均有显著性差异 (P <0 .0 5 )。治疗后 ,患者t-PA活性增强 ,PLG、PAI活性减弱 ,与治疗前比较 ,差异有显著性 (P <0 .0 5 )。D -D水平与治疗前比较差异无显著性 ,但有升高趋势。　结论　脑血栓患者病程中纤溶系统存在着动态变化 ,测定其纤溶活性的变化 ,将有助于诊治。     

妇科恶性肿瘤患者术后血液血栓前状态的检测

目的：探讨妇科恶性肿瘤患者血栓前状态生物指标的变化。方法：检测38例妇科恶性肿瘤患者手术前后及20例正常非孕妇女的血管性血友病因子（vWF)、血小板α－颗粒膜蛋白（GMP－140）、抗凝血酶（AT－Ⅲ）、蛋白C依赖的活化部分凝血酶原时间（PCAT）、纤溶酶原（PLG）、纤溶酶原激活物抑制物（PAI）、D－二聚体和组织纤溶酶原激活物（t-PA)等指标。结果：妇科恶性肿瘤患者术前vWF含量、GMP－140、AT－Ⅲ、PLG、PAI、D－二聚体均比对照组高（P＜0．01），而术后升高更明显；PCAT手术前、后均明显低于正常非孕妇女；而组织纤溶酶原激活物（t-PA) 则无显著性改变（P＞0．05）。结论：提示妇科恶性肿瘤患者术前血液呈血栓前状态，术后呈更明显的血栓前状态。     

肝硬化患者纤溶活性变化与临床指征关系的探讨

目的:探讨肝硬化患者纤溶活性增高与组织纤溶酶原激活物(t-PA),组织纤溶酶原激活物抑制剂(PAI)的关系。方法:根据Ch ild-Pugh分级把明确诊断为肝硬化的86例患者分为三级,A级26例,B级30例,C级30例。对每例样本检测组织纤溶酶原激活物(t-PA)抗原,组织纤溶液酶原激活物抑制剂(PAI),纤维蛋白(原)降解产物(FDP)和D-二聚体(D-d im er)。结果:46例具有正常纤溶活性(FDP<5 mg/l;D-d im er<0.5 mg/l)的病例,其中A级26例,B级12例,C级8例,t-PA抗原随病情严重程度而显著性升高(P<0.05),而PAI活性在三组结果近似(P>0.05)。t-PA在纤溶活性高的患者略高于正常纤溶患者,但差异没有显著性意义(P>0.05);PAI在两组间结果近似,差异没有显著性意义(P>0.05)。结论:t-PA随病情加重而显著性升高,PAI随病情加重变化不大。     

EXPRESSION AND ROLE OF PLASMINOGEN SYSTEM IN PROCESS OF RESTENOSIS

Objective To study the expression and role of plasminogen system in the process of restenosis. Methods We established a double-injury model of atherosclerotic restenosis in rabbit iliac artery mimicking human arterial restenosis. The time course of tissue plaminogen activator （ tPA ）, urokinase plasminogen activator （ uPA ）, urokinase plasminogen activator receptor （uPAR） and plasminogen activator inhibitor-1 （ PAI-1 ） was investigated by immunohistochemistry. The mRNA expression of uPA and uPAR were detected after vascular procedures by in situ hybridization. Results In uninjured arteries, the weak expression of tPA and PAI-1 was detected in intimal and endothelial cells. The expression of tPA, uPA, uPAR and PAI-1 was significantly induced after double-injury, but after double-injury 14d, the expression of tPA restore to preinjury levels. The expression of uPA and uPAR in intimal was higher than that of media and maintain high levels in intimal within 42d and 56d. Conclusion Whereas t-PA is primarily involved in clot dissolution and play a limited role in the process of restenosis, in plasminogen system, uPA and uPAR play a prominent role in the process of restenosis.     

一个遗传性纤溶酶原缺陷症家系的表型与基因突变分析

目的：对一个遗传性纤溶酶原（PLG）缺陷症家系进行表型和基因变异分析,探讨其发病的分子机制。方法：检测先证者及其家系成员（共3代4人）的凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血酶时间（TT）、纤维蛋白原（FIB）、D-二聚体（D-D）、纤维蛋白（原）降解产物（FDP）、纤溶酶原活性（PLG:A）、纤溶酶原抗原（PLG:Ag）、抗凝血酶活性（AT:A）、蛋白C活性（PC:A）及蛋白S活性（PS:A）等指标以明确诊断。用DNA直接测序法分析先证者PLG基因的所有外显子及侧翼序列、5’和3’非翻译区及家系成员相应的突变位点区域,用反向测序予以证实。应用ClustalX-2.1-win软件将突变氨基酸进行同源物种序列保守性分析;利用PolyPhen-2、PROVEAN、SIFT和MutationTaster 4个在线生物信息学软件分析突变氨基酸对蛋白质功能的影响;用Swiss-PdbViewer软件对突变位点进行蛋白质模型和氨基酸相互作用分析。结果：先证者和其祖父、父亲PLG:A均降低为正常值的50%左右,PLG:Ag含量正常,结果分别为45%、95%,64%、94%和64%、104%;基因分析发现先证者PLG基因第15号外显子存在c.1858G＞A杂合错义突变导致p.Ala601Thr;其祖父和父亲具有同样的基因突变。Ala601在其11个同源物种间高度保守;4个在线生物信息学软件预测结果一致,均为有害突变,可引起相应疾病。结论：该先证者的PLG基因第15号外显子存在c.1858G＞A（p.Ala601Thr）杂合错义突变;祖孙三代均为Ala601Thr杂合子,突变符合孟德尔遗传规律,且与该家系PLG活性降低有关。     

内皮-平滑肌联合培养:细胞间相互作用对组织型纤溶酶原激活剂的影响

作者建立一个新的摹拟动脉壁结构的内皮和平滑肌细胞体外联合培养模型,并以此检测内皮、平滑肌细胞在合成和分泌组织型纤溶酶原激活物(t-PA)中的相互影响。结果表明,不同细胞组分的条件培养液(CM)中,t-PA活性有明显差异。内皮的CM中,t-PA活性最高,而平滑肌的CM中则最低。两种细胞联合培养时,培液的t-PA活性也明显比单纯内皮的降低。提示了细胞间的相互作用对血管壁溶栓机制的影响。