纤维蛋白沉积与静脉性溃疡形成的研究

以26例下肢静脉瓣膜关闭功能不全患者和15例正常者为对象,通过局部皮肤病理和超微结构观察,下肢静脉血tPA、PAI:检测发现1.静脉性溃疡及皮肤色素沉着区皮下毛细血管周围纤维蛋白沉积伴成纤维细胞钙盐沉着,坏死;2.患者皮肤营养障碍越严重,tPA越低,PAI越高,即纤溶活性越弱。结果表明:下肢静脉功能不全患者毛细血管周围纤维蛋白沉积形成的弥散屏障是静脉性溃疡形成的主要因素;血液纤溶活性降低造成的清除纤维蛋白能力减退加重了该病理过程。两者导致皮肤营养障碍性改变,直至溃疡形成。     

Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M(r) of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of approximately 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.     

tPA和PAI-2在兔角膜碱烧伤修复过程中的作用

目的　研究兔角膜碱烧伤后修复过程中 ,组织型纤溶酶原激活剂 (tPA)和 2型纤溶酶原激活剂抑制剂 (PAI 2 )的作用。方法　在构建离体和在体兔角膜碱烧伤模型的基础上 ,采用细胞培养、组织切片、免疫细胞化学以及图象分析等技术。结果　tPA的表达在碱烧伤后 2 4小时左右达到峰值 ,而PAI 2则在伤后4 8小时表达最高 ,离体表达部位位于胞浆 ,并相对靠近胞膜和核膜 ,在体表达部位主要位于角膜上皮层。结论　tPA和PAI 2的作用在上皮组织中可能存在着较强的共性     

Procedure-induced inflammation and endothelial cell activation in an artificially ventilated and circulated porcine double-lung model

Systemic inflammation is induced during extracorporeal circulation, resulting in an increased bleeding tendency and endothelial cell activation. Lungs from seven piglets were perfused by autologous blood in an extracorporeal circuit, where the lungs and the left atrium were attached to polyvinyl chloride (PVC) tubings and the blood circulated by a roller pump. The trachea was intubated and attached to a ventilator. The lungs maintained good gas exchange, despite a slight increase in lactate levels. Plasma tPA increased slightly over time, suggesting endothelial cell activation. Activation of inflammatory systems was reflected in increased levels of plasma interleukin (IL)-6 and IL-10. A model for the study of lung endothelial activation during extracorporeal circulation has been shown to be reproducible. The lung tissue was shown to be capable of gas exchange and activation of endothelial cells and procedure-induced inflammation were noted.     

Tissue-Type plasminogen activator (tPA) in breast cancer: relationship with clinicopathological parameters and prognostic significance

Summary Background. Tissue-type plasminogen activator (tPA) is a serine protease primarily involved in the intravascular dissolution of blood clots. High intratumoral tPA levels are associated with prognosis in several human tumors. In addition, tPA has been shown to be an estrogen-inducible protein in human breast cancer cell lines. The aim of the present study was to analyze the cytosolic tPA content in primary breast carcinomas and its potential clinical value. Materials and methods. tPA was measured by a solid-phase enzyme immunoassay in tumor cytosol samples obtained from 800 patients with breast cancer. The median follow-up period was of 49.2 months. Results. Cytosolic tPA levels ranged widely in breast carcinomas (median: 3.9; range: 0.1– 315.3 ng/mg protein). tPA levels were significantly lower in large tumors, as well as in those showing poor differentiation, estrogen (ER) or PgR-negativity, aneuploidy, or a high S-phase fraction. In addition, low tPA intratumoral levels were associated with a high probability of both shortened relapse-free and overall survival in all patients and in the subgroup with node-negative tumors. However, our results did not show any significant relationship between intratumoral tPA levels and prognosis in the different subgroups of patients, stratified according to the type of systemic adjuvant therapy received (chemotherapy, tamoxifen or chemotherapy plus sequential tamoxifen). Conclusion. The results of the present investigation indicate that low intratumoral tPA levels are associated with aggressiveness and poor prognosis in breast cancer patients. However, the study suggests that tPA levels do not predict response to systemic adjuvant therapy.     

Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults

Introduction

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis, respectively. Plasma coagulative and fibrinolytic potentials in normal children and adults, and in representative pathologically altered hemostatic states, were evaluated via simultaneous assessment of thrombin and plasmin generation.

Materials and Methods

An assay of Simultaneous Thrombin and Plasmin generation (STP) was developed to measure thrombin and plasmin in plasma using individual fluorometric substrates. Coagulation is initiated with dilute tissue factor, phospholipid, and calcium in platelet-poor plasma; fibrinolysis is accelerated via tissue plasminogen activator (tPA). Abnormal states of hemostasis were investigated.

Results

STP assay reproducibility and normal adult and pediatric values for measured and calculated parameters have been established. Onset of both thrombin and plasmin generation was significantly delayed in children relative to adults (p < 0.001) and the maximum amplitudes of thrombin and plasmin generation were less in children than adults (p < 0.01). No significant differences were measured among pediatric age groups. The most profound impairments in thrombin generation were observed for extrinsic and common pathway factor deficiencies, with the exception of afibrinogenemia. Plasmin generation was severely impaired in deficiencies of fibrinogen and plasminogen as well as with decreased tPA reagent concentration and addition of aminocaproic acid. Plasmin generation was greatly enhanced by alpha-2-antiplasmin deficiency and excess tPA reagent.

Conclusion

Simultaneous assessment of thrombin and plasmin generation in plasma shows promise for affording an enhanced understanding of overall coagulative and fibrinolytic functions in physiological and pathologically altered states of hemostasis in children and adults.     

Asymmetric dimethylarginine impairs fibrinolytic activity in human umbilical vein endothelial cells via p38 MAPK and NF-κB pathways

Introduction

Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of nitric oxide (NO) synthase. An increased synthesis and/or a reduced catabolism of ADMA might contribute to the onset and progression of thrombosis. The present study was designed to evaluate the effect of ADMA on fibrinolytic factors in endothelial cells, and to investigate the cellular mechanisms.

Materials and Methods

Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of ADMA for various periods; Then HUVECs were preincubated with NO precursor (L-arginine), MAPK inhibitors, or NF-κB inhibitor (PDTC) before ADMA treatment to repeat the experiment. Protein levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and NF-κB activity were measured by ELISA; mRNA levels of tPA and PAI-1 were assayed by qRT-PCR; The activation of MAPK was characterized by western blot analysis.

Results

(1) ADMA decreased tPA antigen levels in time- and concentration-dependent manners, with the maximum effect of 30 μmol/L ADMA for 48 h (control 109.01 ± 4.15 ng/ml vs ADMA 86.76 ± 5.95 ng/ml, p < 0.01); (2) 30 μmol/L ADMA elevated antigen levels of PAI-1 in a time-dependent manner, with the maximum effect of 30 μmol/L ADMA for 48 h (control 2721.12 ± 278.02 ng/ml vs ADMA 3435.78 ± 22.33 ng/ml, p < 0.05); (3) ADMA reduced tPA mRNA levels and increased PAI-1 mRNA levels; (4) L-arginine, SB203580 (p38 MAPK inhibitor) and PDTC attenuated the effects of ADMA on tPA and PAI-1 significantly. (5) ADMA induced a rapid phosphorylation of p38 MAPK, and stimulated NF-κB activity greatly.

Conclusions

ADMA may accelerate thrombosis development by impairing fibrinolytic activity in vascular via inhibiting nitric oxide production and then activating its downstream p38 MAPK and NF-κB pathways.     

Relationships among tumor burden, tumor size, and the changing concentrations of fibrin degradation products and fibrinolytic factors in the pleural effusions of rabbits with VX2 lung tumors

The VX2 tumor is derived from a papilloma virus-induced rabbit epithelial cell line. If VX2 tumor cells (trapped in a plasma clot) are introduced intravenously into NZW rabbits, the cells lodge in the lung capillary bed and produce tumors. Independently of the tumor burden (ie, the total tumor weight per rabbit), approximately 15% of rabbits with VX2 lung tumors accumulate an effusion in the interpleural space and this pleural effusion contains products of hemostasis. We hypothesized that these products were of intra-tumoral origin and that they changed in concentration as tumor burden increased. Interrelationships among lung-, tumor-weights, and pleural effusion volumes, and the concentrations of fibrinolytic factors, their catabolic products, and other proteins of pleural effusions were measured in rabbits with a wide range of tumor burdens. Positive correlations between tumor burden and total lung weight and between pleural effusion volume and net lung weight suggested that interstitial fluid from the stroma of tumors passed directly into the extravascular space of the lung(s) and into the interpleural space(s). Analyses of pleural effusions indicated that plasminogen-, alpha(2)-antiplasmin-, and plasminogen activator inhibitor-1-related proteins, urokinase-like- and tissue-plasminogen activator activities, and vascular endothelial growth factor increased in concentration up to a tumor burden of approximately 20-25 g. Plasmin activity and intact fibrinogen were absent. The concentration of fibrin(ogen) degradation products did not change significantly up to a tumor burden of approximately 25 g but increased substantially as tumor burdens exceeded 25 g. In conclusion, interstitial fluid from tumors enters the extravascular space of the host and may accumulate with fluid from non-tumor sources as a pleural effusion. The concentrations of fibrinolytic factors and their products in pleural effusions reflect the tumor burden of the rabbit. Conceivably, the components of a malignant effusion contain much information about the extent of tumor growth.