乌司他丁对急性脑出血模型兔可溶性血栓调节蛋白、血管性假性血友病因子及脑组织病理的影响

目的：通过对急性脑出血模型兔动脉血可溶性血栓调节蛋白(sTM)、血管性假性血友病因子(vWF)以及脑组织标本病理变化的观察,探讨乌司他丁(UTI)在急性脑出血方面的脑保护作用。方法：将造模成功的30只兔随机平均分为三组,每组10只：脑出血模型组(M组),依达拉奉治疗组(Y组),乌司他丁治疗组(U组)。应用ELISA法测定各组兔急性脑出血后动脉血中sTM、vWF的含量,以及兔脑出血病灶周围脑组织病理变化。结果：与M组[sTM(24h:6.06±0.19ng/ml,72h:6.42±0.48 ng/ml);vWF(24h:10.02 ±0.71ng/ml,72h：11.39±0.63ng/ml) ]相比,Y组vWF含量在24h[9.77±0.46ng/ml] 无明显统计学差异(P>0.05),但其在72h [4.51± 0.10ng/ml] 时下降,有统计学差异(P<0.05);Y组的sTM含量在24h[4.61±0.11ng/ml]、72h[4.51±0.10ng/ml]均有下降,有统计学差异(P<0.05);U组的sTM、vWF含量在24h[sTM(4.82±0.77 ng/ml), vWF(9.09±0.24 ng/ml)] 、72h [sTM (4.79±0.75ng/ml), vWF (8.48±0.73ng/ml)] 均下降,有统计学差异(P<0.05)。与Y组[sTM (24h:4.61±0.11ng/ml),72h:4.51± 0.10ng/ml);vWF (24h:9.77±0.46ng/ml,72h:9.64±0.56ng/ml)]相比,U组sTM含量24h [ 4.82±0.77ng/ml]、72h[ 4.79±0.75 ng/ml] 均无统计学差异(P>0.05);U组vWF含量24h [24h: 9.09± 0.24ng/ml、72h:8.48 ±0.73 ng/ml]均有统计学差异(P<0.05)。病理结果提示：U组脑出血病灶周围脑细胞的线粒体嵴断裂、胞浆内空泡增多、细胞肿胀方面均比Y组延缓。结论：乌司他丁可以降低急性脑出血模型兔血清sTM及vWF含量,可以延缓出血灶周围脑细胞线粒体及细胞核的损害,具有一定的脑保护作用。     

川崎病患儿血浆可溶性血栓调节蛋白的检测及其意义

目的探讨川崎病(KD)患儿治疗前后血浆可溶性血栓调节蛋白(sTM)含量变化及其临床意义。方法2004-07—2006-07入住南京医科大学附属南京儿童医院的KD患儿67例,将患儿分为无冠状动脉损伤组(39例)和冠状动脉损伤组(28例),按病程分为急性期、亚急性期和恢复期;另以28例健康儿童作为正常对照组。应用酶联免疫吸附法检测血浆sTM水平。结果急性期有、无冠状动脉损伤组血浆sTM质量浓度均明显高于正常对照组(均P<0.01),且冠状动脉损伤组血浆sTM蛋白水平亦显著高于无冠状动脉损伤组(P<0.01);治疗后sTM水平降低,冠状动脉损伤组KD患儿的血浆sTM水平急性期[(74.04±16.68)ng/mL]、亚急性期[(46.48±14.12)ng/mL]和恢复期[(38.94±12.93)ng/mL]均高于健康对照组[(14.00±5.58)ng/mL],差异有显著性(P<0.01);无冠状动脉损伤组KD患儿急性期[(37.06±14.55)ng/mL]、亚急性期[(34.04±12.47)ng/mL]sTM水平亦显著升高,与健康对照组相比差异有显著性(P<0.01),恢复期[(16.42±9.16)ng/mL]与正常组差异无显著性(P>0.05)。结论sTM可能参与了KD冠状动脉损伤的病理过程,检测sTM的水平对评估KD冠状动脉病变具有重要意义。     

Haplotype of thrombomodulin gene associated with plasma thrombomodulin level and deep vein thrombosis in the Japanese population

INTRODUCTION: Thrombomodulin (TM) is an essential cofactor in protein C activation by thrombin. Here, we evaluated the contribution of genetic variations in the TM gene to soluble TM (sTM) level and deep vein thrombosis (DVT) in Japanese. PATIENTS AND METHODS: We sequenced the TM putative promoter, exon, and 3'-untranslated region in DVT patients (n=118). Among 17 genetic variations we identified, two missense mutations (R385K, D468Y) and three common single nucleotide polymorphisms (-202G>A, 2487A>T, 2729A>C) were genotyped in a general population of 2247 subjects (1032 men and 1215 women) whose sTM levels were measured. We then compared the frequency of these mutations in DVT patients with that in the age, body mass index-adjusted population-based controls. RESULTS: We identified one neutral mutation (H381) and three missense mutations (R385K; n=2, A455V; n=53 heterozygous, n=14 homozygous, D468Y; n=2) of TM in the DVT patients. Age-adjusted mean values of sTM were lower in C-allele carriers of 2729A>C than in noncarriers in the Japanese general population (women: 16.7+/-0.3 U/ml vs. 17.9+/-0.2 U/ml, p<0.01, men: 19.4+/-0.3 U/ml vs. 20.4+/-0.3 U/ml, p=0.03). Additionally, the CC genotype of this mutation was more common in the male DVT patients than in the male individuals of the general population (odds ratio=2.76, 95% confidence interval=1.14-6.67; p=0.02). This mutation was in linkage disequilibrium (r-square>0.9) with A455V mutation. CONCLUSIONS: TM mutations, especially those with a haplotype consisting of 2729A>C and A455V missense mutation, affect sTM levels, and may be associated with DVT in Japanese.     

益气养阴活血法对2型糖尿病大鼠血浆sTM和sEPCR 的影响

目的：探讨具有益气养阴活血功效的丹蛭降糖胶囊对2 型糖尿病模型大鼠血浆可溶性血栓调节蛋白（sTM）、可溶性内皮细胞蛋白C 受体（sEPCR）的影响及其防治糖尿病血管病变作用机制。方法：将60 只SD 雄性大鼠适应性喂养1 周后,按体重分层随机分为2 组：空白组（N 组）12 只,高脂饲料组48 只。4 周后,高脂饲料组大鼠腹腔注射链脲佐菌素（STZ）,诱发出2 型糖尿病大鼠模型,再根据血糖水平分层随机分为3 组：模型组（M 组）、盐酸吡格列酮组（P 组）、加用丹蛭降糖胶囊组（D 组）。盐酸吡格列酮组和加用丹蛭降糖胶囊组均给予盐酸吡格列酮10 mg·kg-1·d-1 灌胃,加用丹蛭降糖胶囊组在此基础上加用丹蛭降糖胶囊0.47 g·kg-1·d-1 灌胃,空白组与模型组予生理盐水5 mL·kg-1·d-1 灌胃,给药4 周后分别检测各组sTM、sEPCR、PT、APTT、FIB 水平。结果：与对照组比较,模型组、盐酸吡格列酮组和加用丹蛭降糖胶囊sTM、sEPCR、FIB 均明显增高（P<0.05）,PT、APTT 水平明显降低（P<0.05）;与盐酸吡格列酮组相比,加用丹蛭降糖胶囊组大鼠血浆sTM、sEPCR、FIB 水平均显著降低（P<0.01）,APTT 水 平明显升高（P<0.05）,但在升高PT 方面二者无明显差异。结论：益气养阴活血法能降低sTM、sEPCR 水平,有效改善凝血功能。其防治糖尿病血管病变的机制可能是通过降低sTM、sEPCR 含量增强抗凝与纤溶活性,调节凝血功能来改善血液高凝状态,从而保护血管内皮功能。     

Reducing agents induce thrombomodulin shedding in human endothelial cells

The level of thrombomodulin (TM) on cell surfaces reflects its biosynthesis, intracellular turnover, proteolytic cleavage, and release in soluble form (sTM). In the present study we examined the mechanisms mediating and regulating sTM release. Inducers of endothelial protein C receptor (EPCR) shedding, such as proinflammatory cytokines, phorbol ester, and ionomycin did not affect sTM release from human umbilical endothelial cells (HUVECs). In contrast, several natural and synthetic reducing compounds (i.e., glutathione, dihydrolipoic acid, homocysteine, N-acetyl-L-cysteine, dithiothreitol, and non-thiol cell-impermeable reductant, tris-(2-carboxyethyl)phosphine), but not oxidized glutathione or α-lipoic acid effectively up-regulated the release of sTM in endothelial cells. In addition, the direct activator of metalloproteases, 4-aminophenylmercuric acetate (APMA), was an effective inducer of TM shedding. Considerable inhibition of protein C activation was found with APMA, which is consistent with the effects of this agent on TM shedding. In addition to metalloproteases, serine proteases were shown by pharmacological inhibition studies to be involved in a similar degree in basal sTM release; however, serine proteases seem preferentially to be involved in thiol-induced TM proteolytic processing. From comparisons of non-thiol containing synthetic substrate with human recombinant TM it was demonstrated that disulfide bonds within TM are most likely modified by thiols making TM more susceptible to serine protease-mediated cleavage. In summary, the study shows that the extracellular redox state plays a crucial role in the regulation of TM shedding in HUVECs thereby offering new strategies to interfere with diminished activation of protein C during inflammatory diseases.     

血栓调节蛋白及细胞间粘附因子检测在急性冠脉综合征中的临床应用

目的探讨可溶性血栓调节蛋白、可溶性细胞间粘附因子-1水平在急性冠脉综合征发病中的作用。方法分别对40铡不稳定心绞痛、加例急性心肌梗死患者及40例健康对照采用ELISA法测定血清中可溶性血栓调节蛋白、可溶性细胞间粘附因子-1。结果UAP组、AMI组及ACS组sTM水平明显高于对照组;其中AMI组sTM水平与UAP组相比无统计学差异;AMI组、ACS组sICAM-1水平高于对照组,AMI组sICAM-1水平高于UAP组。结论sTM、sICAM-1水平是反映ACS患者内皮细胞损伤程度和范围的良好标志。     

连续性血液净化治疗对全身炎症反应综合征及脓毒症患者内皮细胞功能的影响

目的 :研究连续性血液净化 (CBP)治疗对全身炎症反应综合征 (SIRS)及脓毒症 (sepsis)患者内皮细胞功能的影响。　 方法 :12例SIRS患者原发病均为急性重症胰腺炎 ,其中 5例有明确的感染证据 (sepsis组 ) ,5例伴急性呼吸窘迫综合征 (ARDS) ,3例伴失血性休克。患者均行CBP治疗 72h ,在治疗 0 ,2 ,12 ,2 4 ,4 8,72h时留取血样 10ml。ELISA法检测患者循环中E selectin、血栓调节蛋白 (sTM)水平。二室弥散系统检测内皮细胞通透性 ,应用激光扫描共聚焦显微系统和Fluo 3/AM荧光探针标记技术 ,测定内皮细胞内游离钙离子浓度 [Ca2 ]i。　 结果 :CBP治疗前sepsis患者E 选择素水平较正常对照组明显升高 [(14 8 1± 110 6 ) μg/Lvs (5 1 5± 15 1) μg/L ,P <0 0 5 ],出现ARDS并发症的患者上升趋势更为明显 [(189 4± 94 2 ) μg/L ,P <0 0 1],无感染的SIRS患者也有增高的趋势[(10 8 2± 10 0 3) μg/L],但没有达到统计学差异。经过CBP治疗 ,各组患者E 选择素水平均明显下降。出现休克并发症的患者sTM水平明显升高 [(13 3± 4 4 ) μg/Lvs (5 2± 3 0 ) μg/L ,P <0 0 1],而其他患者 ,如ARDS患者中均未有sTM水平的升高。休克患者经过CBP治疗可明显降低循环中sTM水平 (7 2± 1 7μg/L)。SIRS/sepsis患者血     

Serial changes in neutrophil-endothelial activation markers during the course of sepsis associated with disseminated intravascular coagulation

INTRODUCTION: For systematic elucidation of serial changes in neutrophil-endothelial activation markers as well as to investigate the correlationship among the inflammation markers, disseminated intravascular coagulation (DIC), and multiple organ dysfunction syndrome (MODS) in patients with sepsis, we made this prospective study. MATERIALS AND METHODS: Forty-five patients with sepsis, severe sepsis, and septic shock were subdivided into two groups, 27 with DIC and 18 without DIC. Eight normal healthy volunteers served as control subjects. Serial levels of soluble L-, P-, and E-selectins, intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), thrombomodulin (sTM), and neutrophil elastase were measured within 12 h after the diagnosis of sepsis (day 0) and on days 1-4 after the diagnosis. The numbers of systemic inflammatory response syndrome (SIRS) criteria that patients met and the DIC score were determined simultaneously. RESULTS: Acute Physiology and Chronic Health Evaluation (APACHE) II score was identical between the two groups. In the DIC patients, higher DIC scores, lower platelet counts, and more maximum numbers of SIRS criteria being met were observed compared with the non-DIC patients. The incidence of MODS and the number of the dysfunctioning organs were higher in the patients with DIC than those without DIC, and the DIC patients had poor outcome. Soluble L-selectin (sL-selectin) levels in both groups tended to be lower than those in the control subjects. All other parameters both in the two groups were continuously higher than those in the control subjects during study period. The levels of soluble E-selectin (sE-selectin), sICAM-1, sVCAM-1, neutrophil elastase, and sTM were more elevated in the DIC patients than those in the non-DIC patients. There were no differences in the sP-selectin levels between the two groups; however, more increased sP-selectin levels per platelet were found in the DIC patients compared with the non-DIC patients. Maximum DIC scores in the DIC group positively correlated with the peak levels of neutrophil elastase and sTM and the number of the dysfunctioning organs. CONCLUSIONS: We found close relations among the neutrophil-endothelial cell interactions, DIC, and MODS in patients with sepsis, severe sepsis, and septic shock. The results indirectly confirm the concept that DIC can produce organ dysfunction and that DIC reflects an inflammatory disorder of the microvasculature.     

Fibrin clot properties in women heterozygous for factor V Leiden mutation: Effects of oral contraceptives

Introduction

Oral contraceptives (OC) in the presence of factor V Leiden mutation (FVL) markedly increase the risk of venous thromboembolism (VTE). Little is known about the OC and FVL-related alterations in fibrin clot properties.

Subjects and Methods

Plasma fibrin clot permeability (K_s) and efficiency of lysis, reflected by clot lysis time (CLT) and the rate of D-dimer release from clots (D-D_rate) induced by recombinant tissue plasminogen activator (tPA) were determined in 25 women with a family history of VTE who were heterozygous for FVL [FVL(+/−) - twice, on third-generation OC and after their discontinuation. Female non-carriers of FVL, matched for demographics, using OC and after their discontinuation served as controls (n = 25). All participants had no personal history of VTE.

Results

OC discontinuation in FVL(+/−) women resulted in shortened CLT (− 9%), and increased K_s (+ 4%) and D-D_rate (+ 1.4%; all p < 0.01). Alterations in fibrin clot properties were associated with decreased prothrombin fragments 1 + 2 (F1 + 2) (− 8%), plasminogen activator inhibitor-1 (PAI-1) antigen (− 11%), and thrombin activatable fibrinolysis inhibitor (TAFI) activity (− 20%; all p < 0.01). During OC use FVL(+/−) carriers compared with non-carriers had higher platelet count, activity of PAI-1, TAFI, and tPA, as well as prolonged CLT and higher D-D_max, along with lower D-D_rate and K_s. Multiple regression analysis adjusted for fibrinogen and age, showed that PAI-1 antigen and TAFI activity independently predicted CLT in FVL(+/−) women on OC.

Conclusion

FVL(+/−) is associated with hypofibrinolysis in apparently healthy women and third-generation OC administration unfavorably alters plasma clot characteristics in female FVL(+/−) carriers with a family history of thrombotic events.