弥散性血管内凝血中敏感实验室检测指标的观察

目的：对反映弥散性血管内凝血（DIC）敏感的实验室检测指标进行比较分析，方法：检测120例Pre-DIC患者，30例DIC患者及30例健康志愿者血浆中PT，TT，KPTT，Fbg,D-Dimer,sP-选择素，凝血酶调节蛋白及BPC的变化，结果：Pre-DIC组及DIC患者组BPC，Fbg,D-Dimer,sP-选择素及TM与正常对照组比较有统计差异（P＜0．05或P＜0．01），对Pre-DIC组患者进行回顾性分析发现，对于诊断早期DIC的敏感实验室检测指标依次为；sP-选择素，TM、D－Dimer,BPC,Fbg,KTPP,TP,TT.敏感度依次为：96．7％，90．8％，81．7％，80．8％，76．6％，63．3％，56．7％，50．8％，而其特异性分别为60．5％，55．3％，73．6％，47．4％，26．3％，44．7％，42．1％，36．8％，虽然Pre-DIC组患者sP-选择素，TM较正常对照组有不同程度的升高，但其升高程度不及DIC组，随后发生DIC的76例患者sP-选择素，TM较非DIC患者升高明显，结论：sP-选择素，TM为DIC时出现异常最早的实验室指标之一。它们反映了患者体内高凝状态及内皮损伤程度，可作为诊断DIC得首选指标。     

急性白血病患者的蛋白C活性改变

用KPTT法测定33例正常人和25例各型AL血浆PC:C,结果表明:AL血浆PC:C明显低于正常人(P<0.001)。AL中尤以明显出血症状组的PC:C降低更为显著。AL的出血常呈白血病性凝血酶血症。凝血酶消耗蛋白C,引起PC:C显著降低。     

Endothelial cells lining polyester fabric express pro-coagulant phenotypein vitro

The paper deals with the in vitro assessment of endothelial cell (EC) phenotype covering an albumin- and chitosan-coated polyester fabric and shows that resting ECs express a pro-coagulant phenotype by releasing a high von Willebrand factor level and expressing low thrombomodulin surface activity, despite maintaining an adequate response to stimulating agents.     

重型颅脑损伤患者血清血栓调节蛋白和血管性假血友病因子动态变化及其临床意义

目的 探讨重型颅脑损伤患者回顾性分析血栓调节蛋白(TM)和血管性假血友病因子(vWf)血清水平的变化及其临床意义.方法 选择62例重型颅脑损伤患者的病历资料,将62例患者分为弥漫性轴索伤组(28例)和局限性脑损伤组(34例).然后将62例患者再分成青年组(16～30岁)20例、中年组(31～65岁)20例和老年组(＞65岁)22例.分别于伤后1,3和7 d时测定TM及vWf的血清水平.TM及vWf血清水平采用酶联免疫吸附法(ELISA)测定.结果 局限性脑损伤组在伤后1～7 d的vWf值明显高于弥漫性轴索伤组(P＜0.05);与中、青年组比较,老年组在伤后1 d TM、vWf血清水平明显升高(P＜0.05);发生迟发性外伤性颅内血肿(delayed traumatic intracerebral hematoma,DTICH)患者的TM水平明显高于未发生DTICH患者的TM水平(P＜0.05).结论 在重型颅脑损伤的急性期,不同损伤类型、不同年龄患者脑内皮细胞损伤和活化的程度不同;TM是反映脑血管内皮细胞损伤的敏感指标之一,通过测量TM和vWf血清水平来评估颅脑损伤预后以及把测量TM作为预测DTICH发生的指标是非常有意义的.     

血栓调节蛋白内皮生长因子样结构域和富含丝氨酸/苏氨酸结构域在大肠埃希菌中的表达

目的:通过构建血栓调节蛋白(thrombomodulin, TM)的内皮生长因子样结构域(D2)、富含丝氨酸/苏氨酸结构域(D3)及绿色荧光蛋白(green fluorescent protein,GFP)的大肠埃希菌表达体系,以获得TM-D23-GFP融合蛋白。方法:从美国国家生物技术信息中心查询TM的D23结构域、GFP的氨基酸序列,进行密码子优化和全基因合成,利用双酶切法将目标基因插入表达载体PET30a中,转化DE3菌株、诱导表达,通过镍离子-亚胺二乙酸-琼脂糖凝胶树脂(Ni-iminodicitic acid-sefinose resin,Ni-IDA)亲和层析、Q Sepharose TM FF离子交换层析纯化,最终得到TM-D23-GFP融合蛋白。结果:经酶切和测序鉴定证实,TM-D23片段已正确插入PET30a表达载体中;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)和蛋白质印迹(Western blotting)检测结果证实,含His-tag标签的TM-D23-GFP融合蛋白成功表达,且其相对分子质量约为60 000。结论:构建的TMD23-GFP融合蛋白可在DE3表达菌株中进行表达,为后期融合蛋白的功能研究打下基础。     

Women with unexplained recurrent pregnancy loss do not have evidence of an underlying prothrombotic state: Experience with calibrated automated thrombography and rotational thromboelastometry

Introduction

Where unexplained recurrent pregnancy loss (RPL) is attributed to an underlying maternal prothrombotic state, empirical prophylactic anticoagulation may be recommended.

Materials and Methods

In the present study we used calibrated automated thrombography and rotational thromboelastometry to determine the procoagulant potential of these women as a rationale for anticoagulation. Fifty women with ≥ three consecutive unexplained losses prior to 14 weeks’ gestation or one loss after this time were compared with forty-one parous women with no miscarriages. Exclusion criteria included antiphospholipid syndrome, inherited thrombophilia and prior venous thromboembolism. Thrombin generation in platelet poor plasma and whole blood thromboelastometry was performed outside pregnancy to determine the presence or not of an underlying prothrombotic state.

Results

Peak thrombin and endogenous thrombin potential were not significantly increased in subjects relative to controls. The use of low tissue factor (1 pM) to better reflect physiological conditions and assay modification to better assess the protein C pathway (5 pM in the presence of thrombomodulin) provided no additional discrimination. Consistent results were shown with thromboelastometry; mean parameters were equivalent between subjects and controls.

Conclusions

These data demonstrate that global coagulation assays provide no evidence of an underlying hypercoagulable state in women with unexplained RPL; this is in keeping with the results of recent randomised controlled trials and strengthens the evidence base against use of anticoagulants in this setting.     

Comparison of markers of coagulation activation and thrombin generation test in uncomplicated pregnancies

Introduction

Pregnancy is a well-established risk factor for venous thromboembolism, and is associated with a state of hypercoagulability or parameters of thrombin generation. Currently, there is a lack of consensual data on thrombin generation during pregnancy. This study aimed to find a sensitive and specific biological marker of coagulation activation and to identify parameters of thrombin generation.

Patients and methods

The population included 101 women with uncomplicated pregnancies. The objective of this study was to correlate thrombin generation test (measured at 5pM tissue factor, 4 μM lipids and without thrombomodulin), with fibrinogen and markers of blood coagulation activation: D-dimer, prothrombin fragments 1+2 (F1+2), thrombin-antithrombin complexes (TAT) and fibrin monomer complexes (FMC) in these women. Internal quality control was performed in each set of experiments.

Results

Fibrinogen, D-dimer, F1+2, and TAT concentrations increased significantly throughout pregnancy, and were correlated with term of pregnancy. In our study, thrombin generation seemed to increase early on, and then remained stable throughout normal pregnancy, in contrast with other markers of blood coagulation activation, excepting FMC. The latter are subject to large inter-individual variations, especially during second trimester. No correlation was demonstrated between thrombin generation parameters and other activation markers.

Conclusion

While markers of coagulation activation significantly increased during pregnancy, thrombin generation increased only early on and remains stable during pregnancy. Finding a sensitive and specific biological marker for vascular pregnancy complications, such as FMC and thrombin generation levels, requires further investigation.     

人血栓调节蛋白基因编码片段的克隆及在真核细胞中的表达

目的 构建人血栓调节蛋白（hTM）基因真核表达质粒，并导人两种真核细胞[非洲绿猴肾母细胞（COS-7细胞）和脐静脉内皮细胞（HUVECs）]中表达，为临床血栓病的基因治疗提供实验依据和物质基础。方法采用PCR法扩增hTM基因的表达片段，HindⅢ＋EcoR Ⅰ酶切后与pcDNA3．1（＋）／neo质粒连接成pcDNA3．1／hTM。经鉴定确认后，由阳离子脂质体包裹转染COS-7细胞及HUVECs，RT-PCR和免疫组化法分别检测转染后细胞hTM的mRNA及蛋白的表达。结果双酶切及测序鉴定均证实hTM基因表达片段被成功克隆，pcDNA3．1／hTM经脂质体介导能有效地转染COS-7细胞和HUVECVs。结论成功构建pcDNA3．1／hTM重组质粒，并且能在两种真核细胞中高效表达，为进一步的基因治疗和对TM抗凝机理研究提供了物质基础。     

Potential Mechanisms of Vascular Thrombosis after Microwave Ablation in an in Vivo Liver

Purpose

To evaluate potential biologic and thermal mechanisms of the observed differences in thrombosis rates between hepatic vessels during microwave (MW) ablation procedures.

肝硬化患者血栓调节蛋白、抗凝及纤溶系统的变化与Child-Pugh分级的关系

目的探讨肝硬化患者血栓调节蛋白（TM）、抗凝及纤溶系统指标的变化及其与Child-Pugh分级的关系。方法肝硬化患者77例，按Child-Pugh分级进行分组：A级组12例，B级组32例，C级组33例；对照组20例。均检测TM、抗凝血酶-Ⅲ（AT-Ⅲ）、蛋白-C（PC）、蛋白-S（PS）、D-二聚体、组织纤溶酶原激活物（t-PA）和纤溶酶原激活物抑制剂-1（PAI-1）。结果在对照组、A级组、B级组、C级组中，TM随病情加重而升高，分别为（4．24±0．81）μg／L、（4．36±2．56）μg／L、（6．66±3．30）μg／L、（6．79±3．69）μg／L；AT-Ⅲ随病情加重而逐渐降低，分别为（102．35±8．12）％、（105．46±8．71）％、（102．09±8．39）％、（94．32±9．85）％；PC和PS随病情加重而有逐渐减低的趋势；D-二聚体随病情加重而逐渐增高，分别为（0．27±0．13）mg／L、（0．42±0．26）mg／L、（0．79±0．35）mg／L、（0．93±0．43）mg／L；t-PA和PAI-1随肝功能恶化而变化的现象不明显。结论肝硬化患者的TM、抗凝和纤溶系统指标存在明显的异常且和Child-Pugh分级相关。检测以上指标有助于对肝功能损害程度及预后的判断。