ETA and ETB receptor antagonists synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures

Astrocytes produce and bind endothelins (ETs), suggesting that these cells have ET autoregulatory and eliminatory functions. To further investigate these functions in primary rat astrocytes, ET-1 levels in the cell culture media (RIA/HPLC) and intracellular content of ET-1 mRNA (RT PCR) were measured under basal and stimulated (thrombin, 2.2 U/ml) conditions in the presence and absence of ET_A and ET_B selective antagonists (BQ123 or LU135252, and BQ788, respectively). Neither basal nor stimulated ET-1 levels in astrocyte media were influenced by ET_A or ET_B antagonists alone, but were significantly increased by a combination of both. ir ET-3 levels were not affected by antagonist treatment. Exogenous ET-1, added to the cultures, was rapidly cleared from the supernatant; this clearance was markedly inhibited by a combination of BQ123 and BQ788. ET-1 mRNA levels were not altered by any treatment. To conclude, in primary rat astrocyte cultures, extracellular ET-1 is cleared by binding to ET-receptors, apparently involving both, ET_A and ET_B sites. Thus, a blockade of the astrocytic ET eliminatory function as a consequence of the in vivo application of non-selective ET receptor antagonists may lead to increased extracellular ET levels in the brain.     

Anticoagulant 1,2,3,4,6-pentagalloyl-β-d-glucopyranose isolated from geranium (Pelargonium inquinans Ait)

Geranium (Pelargonium inquinans Ait) leaves were extracted with 80% MeOH, and partitioned into n-hexane, ethyl acetate, BuOH and H2O to isolate the anticoagulant principles. The EtOAc fraction was found to be the most active, and was further purified using silica and octadecylsilane column chromatography employing a bioassay-guided fractionation method. The active compound was isolated and identified as 1,2,3,4,6-pentagalloyl-beta-D-glucopyranose (PGG) (compound I). The isolated anticoagulant significantly prolonged the activated partial thrombin time (APTT) and thrombin time (TT) using normal human plasma. One microgram of 1,2,3,4,6-pentagalloyl-beta-D-glucopyranose showed 0.063 heparin units in the APTT and 2.73 heparin units in the TT for anti-thrombosis. This is the first report of the isolation of PGG from geranium plants.     

Thrombin generation in chronic obstructive pulmonary disease: dependence on plasma factor composition

Background

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for thromboembolic events. We investigated thrombin generation profiles in COPD patients and their dependence on plasma factor/inhibitor composition.

Methods

Factors (f) (fII, fV, fVII, fVIII, fIX, fX), antithrombin, protein C (PC) and free tissue factor pathway inhibitor (fTFPI) from 60 COPD patients (aged 64.2 ± 10.1 years; a mean forced expiratory volume in 1 second [FEV₁], 55.6 ± 15.8% of predicted values) were compared with those for 43 controls matched for age, sex, weight and smoking. Patients receiving anticoagulation were excluded. Using each individual's plasma coagulation protein composition, tissue factor-initiated thrombin generation was assessed computationally.

Results

COPD patients had higher fII (115 ± 16 vs 102 ± 10%, p < 0.0001), fV (114 ± 19 vs 102 ± 12%, p = 0.0002), fVII (111 ± 15 vs 102 ± 17%, p = 0.002), fVIII (170 ± 34 vs 115 ± 27%, p < 0.0001), and fIX (119 ± 21 vs 107 ± 17%, p = 0.003), and lower fTFPI (17.7 ± 3.2 vs 18.9 ± 3.2 ng/ml, p = 0.047) compared with controls, while fX, antithrombin, and PC were similar in both groups. Computational thrombin generation profiles showed that compared with controls, COPD patients had higher maximum thrombin levels (+ 28.3%, p < 0.0001), rates of thrombin generation (+ 46.1%, p < 0.0001) and total thrombin formation (+ 14.4%, p < 0.001), together with shorter initiation phase of thrombin generation (p < 0.0001) and the time to maximum thrombin levels (p < 0.0001). Thrombin generation profiles in COPD patients can be normalized via correction of fII, fVIII , fIX and TFPI. The severity of COPD and inflammatory markers were not associated with thrombin generation profiles.

Conclusions

Prothrombotic phenotype in COPD patients is largely driven by increased prothrombin, fVIII, fIX, and lower fTFPI.     

Coagulation parameters in patients receiving dabigatran etexilate or rivaroxaban: two observational studies in patients undergoing total hip or total knee replacement

Introduction

Dabigatran and rivaroxaban have recently been added to the armamentarium for thromboprophylaxis in orthopedic surgery. Although this is their first licensed indication, others will soon follow. Owing to their claimed predictable anticoagulant response that dispenses with the need for monitoring coagulation, their effects are poorly described in routine cases. However, interpreting blood coagulation results and evaluating whether a treatment is properly targeted in the case of untoward incidents will become a common concern for clinicians.

Methods

Eighty patients undergoing total hip or knee replacement were included in two studies. Forty of them received dabigatran (study 1) and 40 rivaroxaban (study 2). Blood samples (n = 176 and 166) were taken preoperatively and twice a week from the first postoperative day.

Results

Dabigatran increased aPTTr about two-fold and PT about 1.2-fold, and it was mostly an initiation-phase modulator of thrombin generation. Mean circulating concentrations as measured by a diluted thrombin time were 105±85 ng/mL at T_max in samples from patients receiving the full dosing. They depended significantly on renal function, body weight and gender.Rivaroxaban increased aPTTr and PTr around 1.5 fold and modified the initiation and amplification phases of thrombin generation, with a lowered and prolonged thrombin burst. Mean circulating concentrations as measured by an antiXa test were 117 ± 78 ng/mL at T_max.With both drugs, routine coagulation tests, thrombin generation curves and functionally determined concentrations exhibited high interindividual variability.

Conclusion

Routine coagulation tests are altered in patients receiving dabigatran or rivaroxaban, but their alterations poorly reflect the circulating concentrations as determined by functional approaches.     

Thrombin in ischemic neuronal death

Thrombin plays a role in cerebral ischemia as rats subjected to focal cerebral ischemia were protected by the intracerebral injection of hirudin, a selective thrombin inhibitor. To separate the roles of thrombin in cell death and in coagulation, we have used an in vitro approach to test the effect of hirudin and of protease nexin-1 (PN-1), a cerebral thrombin inhibitor, on neuronal ischemia. Rat organotypic hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation (OGD) during 30 min. Hirudin or PN-1 administered after OGD significantly prevented neuronal death in the CA1 region. After 24 h, there was a marked increase in thrombin immunoreactivity on Western blots. Thrombin therefore contributes to ischemic damage in neural tissue in vitro.     

Repair of rabbit segmental defects with the thrombin peptide, TP508.

The synthetic peptide, TP508 (Chrysalin), was delivered to rabbit segmental bone defects in biodegradable controlled-release PLGA microspheres to determine its potential efficacy for enhancing healing of non-critically and critically sized segmental defects. Non-critically sized radial defects were created in the forelimbs of New Zealand White rabbits, which were randomized into three treatment groups receiving 10, 50 and 100 microg doses of TP508 in the right radius and control microspheres (without TP508) in the left radius. Torsional testing of the radii at six weeks showed a significant increase in ultimate torque, failure torque, ultimate energy, failure energy, and stiffness when treated with TP508 compared to controls (p<0.01 for all measures). Thus, TP508 appeared to enhance or accelerate bone growth in these defects. In a second set of experiments, critically sized ulnar defects were created in the forelimbs of New Zealand White rabbits, which were randomized into two groups with each rabbit receiving microspheres with 100 or 200 microg of TP508 into the right ulnar defect and control microspheres (without TP508) alone into the left ulnar defect. Bone healing was evaluated with plain radiographs, synchrotron-based microtomography, and mechanical testing. Radiographs of the rabbit limbs scored by three blinded, independent reviewers demonstrated a significantly higher degree of healing when treated with TP508 than their untreated control limbs (p<0.05). Three-dimensional synchrotron tomography of a limited number of samples showed that the new bone in TP508-treated samples had a less porous surface appearance and open marrow spaces, suggesting progression of bone remodeling. Torsional testing of the ulnae at nine weeks showed a significant increase in maximum torque and failure energy when treated with TP508 compared to controls (p<0.01 for both measures). These results suggest that TP508 in a controlled release delivery vehicle has the potential to enhance healing of segmental defects in both critically and non-critically sized defects.     

星点设计-效应面法优化O-羧甲基壳聚糖复凝止血海绵的处方

目的：优化O-羧甲基壳聚糖（O-CMC）复凝止血海绵处方并考察其止血效果。方法：建立兔耳动静脉创伤出血模型,以止血时间及出血量为指标,以水溶性O-CMC为载体,戊二醛为交联剂,加入凝血酶作为复凝血剂,采用星点设计-效应面法优化O-CMC复凝止血海绵处方。结果：O-CMC复凝止血海绵的最优处方为：O-CMC 2.49 mg·ml~（-1）,凝血酶49.87 IU·ml~（-1）,戊二醛0.49 mg·ml~（-1）。其止血效果明显优于O-CMC单方止血海绵和市售明胶海绵。结论：O-CMC复凝止血海绵具有良好的止血效果。     

A cost effective endovascular approach for management of post-catheterization profunda femoris artery pseudoaneurysm using thrombin

Post-catheterization PSA is one of the most commonly encountered vascular complications of cardiac and peripheral angiographic procedures. We report the case of patient who developed deep-seated profunda femoris artery pseudoaneurysm (PSA) following cardiac catheterization. Despite, repeated ultrasound guided compressions the PSA failed to close and instead produced local site pressure ulcers. The secondary infection followed which precluded use of percutaneous thrombin injection. The PSA was finally closed via a total endovascular technique combining intravascular thrombin injection and coil embolization, thus obviating the need for expensive measures like cover stents or invasive surgical repairs.     

人凝血酶对扁桃体手术创面止血的安全性和有效性研究

目的:评价人凝血酶对扁桃体手术创面的止血作用和安全性。方法:采用前瞻、随机、对照的方法,80例行双侧扁桃体摘除手术的患者随机分为研究组和对照组,在研究组自身对照观察有效性,即右侧创面局部给予人,左侧0.9%氯化钠溶液,对比观察创面出血的止血时间、出血量和单位面积出血量。对照组和研究组间比较观察凝血相和肝肾功能等安全性指标。结果:使用人凝血酶创面止血时间为(62.8±24.6)s,创面出血量(4.9±6.7)g,创面单位面积出血量(0.79±0.98)g.cm-2;明显低于使用生理盐水创面的(94.7±36.9)s,(7.3±13.4)g,(1.14±1.95)g.cm-2;两者比较均差异有统计学意义。研究期间,所有患者未出现任何局部或全身不良事件;凝血指标和肝肾等主要脏器功能的变化也不明显,且2组结果相似。10例研究组患者观察HBsAg、抗HCV、HIV抗体和梅毒抗体等指标3个月无异常。结论:人凝血酶对扁桃腺手术创面毛细血管出血有较好的止血作用和安全性。