蚯蚓纤溶酶的分离纯化及临床作用研究进展

蚯蚓纤溶酶是从蚯蚓中分离的一种丝氨酸蛋白水解酶,有抗凝溶血栓、抗癌抗肿瘤、抗炎、神经修复等作用,在临床上是预防和治疗血栓疾病的有效药物。本文对蚯蚓纤溶酶的分离纯化与药理作用加以概述,为临床蚯蚓溶栓药物的研究提供依据。     

蚓激酶的最新研究进展

蚓激酶是一种蚯蚓溶纤蛋白酶,具有很强的纤维蛋白溶解活性,有溶解血栓和防止血栓形成的作用。本文对蚓激酶的结构、提取工艺及其功能进行了综述,为其进一步的开发利用提供理论依据。     

低分子量尿激酶与Annexin V的融合基因在家蚕杆状病毒表达系统中的表达

将低分子量尿激酶与膜联蛋白 (AnnexinV)的融合基因重组到家蚕杆状病毒转移载体pBacPAK8中 ,获得重组转移载体pBacPAK UKAV ,并与线性化的病毒Bm PAK6DNA共转染家蚕细胞 ,获得重组病毒BacPAK UKAV。将重组病毒BacPAK UKAV感染家蚕培养细胞 (MOI=10 )和 5龄幼虫 (10 5pfu/头 ) ,Western印迹方法表明表达产物大小约为 6 9kD。用纤维蛋白平板溶圈法测定表达产物的纤溶活性 ,其融合蛋白具有明显的纤溶活性 ,约为 5× 10 -5U/个细胞。用APTT法测定表达产物的抗凝活性 ,比野生病毒Bm PAK6感染表达产物的APTT时间延长了 1倍以上 ,表现出明显的抗凝活性。体外实验表明 ,家蚕培养细胞和幼虫表达的重组低分子量尿激酶与AnnexinV融合蛋白具有溶栓与抗栓双功能。研究结果为今后进一步探索具有溶栓抗栓功能的血栓药物提供了新的方向。     

中华圆田螺纤溶活性蛋白的 发现及活性研究

纤溶活性蛋白研究是当前研究之一,该蛋白具有良好的溶栓效果。中华圆田螺富含活性蛋白。本实验以中华圆田螺为研究对象,采用硫酸铵分段盐析分离和提取得到了活性粗蛋白。纤溶平板法证明其具有纤溶活性,并研究了温度、pH值、金属离子对其纤溶活性的影响。实验结果表明中华圆田螺活性蛋白最适作用温度为58℃;pH值为7～10范围内影响较小,活性较强;Na~+、K~+对其纤溶活性无影响,Ca~(2+)、Fe~(3+)对其有抑制作用。     

蚯蚓纤溶酶活性研究

蚯蚓是优质的蛋白质饲料,但蚯蚓体内含有纤溶酶等生物活性物质,大量使用会造成动物机体的损害。为了保证蚯蚓作为蛋白饲料在畜牧业生产中的应用安全,本研究采用琼脂糖-牛血纤维蛋白原平板测定了蚯蚓水解液在不同时间、不同pH值和不同温度下的纤溶酶活性。结果表明,随着pH值降低和温度的升高蚯蚓纤溶酶的活性逐渐降低,但直至pH值为1时或温度达到100℃时纤溶酶仍具有较强的活性。因此认为,蚯蚓纤溶酶在胃液的酸性条件下或通常的环境温度下难以完全灭活,把蚯蚓作为蛋白饲料或饲料添加剂使用时应严格控制动物的摄入量,以保证养殖业的安全生产。     

纳豆及纳豆激酶的研究进展

纳豆具有丰富的营养价值，并富含维生素K2等，可提高蛋白质的消化吸收率，在发酵过程中，可以产生纳豆激酶，降解纤维蛋白原，并可以将纤维酶原激活为纤溶酶，从而刺激产生纤溶酶原激活剂。纳豆激酶以安全、高效、稳定，且成本较低、易吸收、副作用小、作用时间长等优点，成为较为理想的保健及溶栓药物之一。本文对纳豆及纳豆激酶做了综述性研究．为更好的研究开发纳豆及纳豆激酶产品奠定基础。     

大黄注射液对犬血管搭桥术后纤溶系统的影响

为研究大黄注射液对犬血管搭桥术后纤溶系统的影响,将20只犬随机分为高(0.8 g.kg-1)、中(0.4g.kg-1)、低剂量组(0.08 g.kg-1)及对照组,同侧自体颈外静脉重建颈总动脉,头静脉滴注大黄注射液,ELISA定量分析测定术前及术后1、2、3、5、7、14、21 d纤溶系统各指标的变化。结果显示,用药后血小板α颗粒膜蛋白、D-二聚体、纤溶酶原激活剂的抑制物-1显著降低,组织纤溶酶原激活剂显著升高,且成剂量依赖关系;血清纤维蛋白(原)降解产物无显著变化。结果表明,大黄注射液能纠正搭桥术后血液高凝状态,恢复抗凝系统活性,预防血栓形成。     

动物凝血和抗凝血功能的检查及临诊意义

1止血和凝血机制 在生理状态下，血液中有少量纤维蛋白生成，并覆盖在血管内膜上，保护血管内皮，以维持血管的正常通透性，同时生成的纤维蛋白又被纤维蛋白溶酶所溶解，凝血与纤溶处于动态平衡状态，保持机体不出血，也无血栓形成，血液是流体状态而循环于全身。当这种平衡失调时，便可导致出血不止或形成血栓，即止血与凝血障碍。止、凝血障碍的机制十分复杂，其涉及微血管、血小板、各凝血因子、抗凝因子及纤维蛋白溶解系统等诸多因素。在正常时止、凝血系统与抗凝血和纤维蛋白溶解系统处于动态平衡状态，遂能既无出血也无血栓形成。若止、凝血活性减弱或抗凝血及纤溶活性增强则会引起低凝状态发生出血症状。相反会引起高凝状态或导致血栓形成。前者临床上统称为出血性疾病，后者统称为血栓性疾病。     

牛乳乳酶的研究进展

哺乳动物的乳汁已经分离出60多种固有酶。乳酶是牛乳的重要组成部分,乳酶对泌乳形成和乳汁分泌过程起着重要的调节作用,乳酶还是乳中抗氧化过程和先天性免疫系统的重要组成部分。乳汁形成、泌乳过程、泌乳中止过程以及乳腺退化过程与机体诸多生理过程相互影响,与机体的消化系统、纤溶酶系统、纤溶酶激活物-纤溶酶原-纤溶酶为基础的负反馈体系等多种机制相互调节。本文对牛乳中的纤溶酶、N-乙酰基-1-β-D-氨基葡萄糖苷酶(NAGase)、黄嘌呤氧化还原酶(XO)、乳酸过氧化物酶和组织蛋白酶D等几种重要固有酶的研究进展进行了综述。     

溶栓药替奈替普酶研究进展

替奈替普酶(Tenecteplase)是在阿替普酶(Alteplase)的基础上,用Asn、Glu和Ala-Ala-Ala-Ala代替其分子中的Th103、Asn117和Lys296-His-Arg-Arg2993个位点而得到的生物技术制品,属于新型的第3代溶栓药,具有体内半衰期长,溶栓效果好,溶栓时间短,纤维蛋白特异识别性强,使用方便及危险性低等特点.替奈替普酶主要用于可以引起心肌梗死等较为严重的血栓性疾病的溶栓治疗,是目前有效的溶栓药物之一.文章对溶栓药的研究概况及第3代溶栓药替奈替普酶的基础研究和临床应用进行了综述.     

Effect of sample preparation on basophilic stippling in bovine blood smears

The effect of sample preparation on the amount of basophilic stippling of erythrocytes (BSE) was studied using blood from a calf with chronic experimental lead poisoning. The combination of EDTA anticoagulation and rapid drying of the blood smear resulted in the most BSE. Alcohol prefixation reduced BSE. Wright-Leishman stain was better than Wright stain in demonstrating BSE.     

基于蛋白组学体外分析益母草水煎液对补体和凝血级联通路相关活性因子的作用

旨在通过蛋白组学探索益母草水煎液对人真皮微血管内皮细胞(human dermal microvascular endothelial cells, HDMECs)凝血与抗凝血相关因子表达的调节作用。MTT法筛选益母草水煎液对HDMECs的安全浓度；使用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification, iTRAQ)技术分析50和100 μg·mL^-1益母草水煎液作用24 h后HDMECs蛋白表达谱的变化，通过对比各组蛋白谱的变化筛选出差异显著的相关通路，分析该通路中筛选到的可信差异表达蛋白(differentially expressed proteins, DEPs)，并选取可信DEPs中与凝血与抗凝血相关的拮抗因子进行RT-PCR和ELISA验证。结果表明：1 mg·mL^-1以下益母草水煎液对HDMECs无毒副作用；益母草水煎液能够同时调节与凝血相关的血小板活化等过程和与抗凝血相关的肝素结合过程。对筛选出的补体和凝血级联通路中的5种可信DEPs分析显示，与空白组相比，50 μg·mL^-1益母草水煎液组凝血酶原(F2)、抗凝血酶-Ⅲ(AT-Ⅲ)、组织型纤溶酶原激活剂(t-PA)、凝血因子Ⅴ(F5)和激肽原(KNG)同时显著下调，100 μg·mL^-1益母草组F2、t-PA、AT-Ⅲ、KNG显著下调；与50 μg·mL^-1益母草水煎液组相比，100 μg·mL^-1益母草水煎液组F2、AT-Ⅲ、t-PA、F5和KNG等凝血级联相关因子均显著升高。结果提示，益母草水煎液可显著改变HDMECs蛋白表达谱，并可能通过调控补体和凝血级联通路相关因子F2、AT-Ⅲ、t-PA、F5、KNG蛋白的表达对凝血与抗凝血相关拮抗因子发挥双向调控作用。     

Effect of clopidogrel on tissue-plasminogen activator-induced in vitro thrombolysis of feline whole blood thrombi

OBJECTIVE: To determine if clopidogrel enhanced the thrombolytic rate of tissue-plasminogen activator (t-PA) on an in vitro feline whole blood thrombosis model. ANIMALS: 9 purpose-bred cats. PROCEDURE: Blood obtained from cats before (baseline) and after treatment with clopidogrel (75 mg, p.o., q 24 h for 3 days) was anticoagulated with sodium citrate (9:1 volume-to-volume ratio) to which 1 microCi of I125-fibrinogen was added. Thrombi were formed by the addition of calcium chloride and bovine thrombin. Thrombi were placed into autologous plasma to which 0.1 mg of t-PA was added. Plasma samples were collected at different time points to determine the amount of released I125-fibrin split products. Thrombolytic rates were calculated by determining the time to 25%, 50%, and 75% thrombolysis (t25, t50, and t75, respectively). Confidence intervals for t25, t50, and t75 at baseline were compared with those after treatment. RESULTS: There were no significant differences in thrombolytic rates between values obtained at baseline and after clopidogrel treatment (t25, 18.0 vs 18.5 minutes; t50, 63.3 vs 65.6 minutes; and t75, 163.0 vs 170.1 minutes, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Clopidogrel did not have an effect on the rate of thrombolysis of feline whole blood thrombi induced by t-PA in this in vitro model.     

Regional Citrate Anticoagulation for Intermittent Hemodialysis in Dogs

Background

The traditional systemic heparinization used for anticoagulation in extracorporeal therapies may cause fatal complications in animals at risk of bleeding.

Hypothesis/Objectives

To develop and validate a protocol of regional citrate anticoagulation (RCA) for intermittent hemodialysis in dogs.

Animals

A total of 172 dogs treated with hemodialysis for acute kidney injury.

Methods

In vitro titration was performed, adding trisodium citrate and calcium chloride to heparinized canine blood. A tentative protocol was used first in 66 treatments with additional heparinization and subsequently in 518 heparin‐free treatments. Safety and adequacy of RCA were assessed based on clinical and laboratory monitoring, dialyzer pressure gradient, treatment completion, and visual scoring of the extracorporeal circuit.

Results

Addition of 1 mmol/L citrate to heparinized blood decreased the ionized calcium concentration by 0.23 mmol/L (95% confidence interval [CI], 0.16–0.30) and 1 mmol/L calcium increased it by 0.62 mmol/L (95% CI, 0.45–0.79). Heparin‐free treatments were initiated with infusion of trisodium citrate (102 mmol/L) at 2.55 mmol/L blood and calcium chloride (340 mmol/L) at 0.85 mmol/L. Citrate and calcium administrations were adjusted in 27 and 34% of the treatments, respectively. Overall, anticoagulation was satisfactory in 92% of the treatments, with expected azotemia reduction in 95% (urea) and 86% (creatinine), stable dialyzer pressure gradient in 82%, and clean extracorporeal circuits in 92% of the treatments. Eighteen treatments (3.5%) were discontinued prematurely, 9 because of clotting and 9 for reasons unrelated to the RCA procedure.

Conclusions and Clinical Importance

Regional citrate anticoagulation allows safe and efficient heparin‐free hemodialysis in dogs at risk of bleeding.     

Hemostatic abnormalities in nephrotic syndrome

Nephrotic syndrome is often associated with a hypercoagulable state and thrombotic complications. Thrombosis may be due to a number of abnormalities in blood, including AT III deficiency, increased concentrations of fibrinogen, factors V and VIII, and platelet hyperaggregability. The therapeutic approach to thrombosis in nephrotic syndrome is the use of anticoagulants as a preventive measure or an attempt at thrombolysis with streptokinase, urokinase, or stanozolol.     

Antidotal effect of vitamin K1 against warfarin-induced anticoagulation in horses

Warfarin-induced anticoagulation and reversal of the induced anticoagulation by vitamin K1 were evaluated in 4 mature horses. Each horse was given warfarin IV until the prothrombin (PT) time was prolonged by approximately 1.5 times the predosing base-line value. In experiment 1, we evaluated the time required for PT to return to the predosing value (PT reversal time) after warfarin administration was discontinued. Between each experiment, a 1-week rest period was allowed. In experiment 2, two doses of vitamin K1 (100 mg/dose) were administered IM 6 hours apart, and the PT was monitored hourly for 24 hours. In experiments 3 and 4, the horses were dosed with warfarin as in experiment 1, and the PT reversal time was evaluated after administration of 300- and 500-mg doses of vitamin K1 IM, respectively. In experiment 5, one horse was eliminated from the study, 1 horse was given 300 mg of vitamin K1 IV, and 2 horses were given 300 mg of vitamin K1 subcutaneously (SC); the reversal times were evaluated in the 3 horses given vitamin K1. Therapeutic response time was designated as the time required for the mean PT time of treated horses to reach the midpoint between the longest mean PT time achieved during anticoagulation and the mean base-line PT time. The therapeutic response time, without supportive therapy, after discontinuation of warfarin administration was 30 hours, and there was a PT reversal time of approximately 5 days from the last dose of warfarin. The 100-mg dose of vitamin K1 shortened the therapeutic response time to 12 hours and the PT reversal time to 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of blood clotting with the total thrombus analysis system in healthy dogs

To date, coagulation tests are unable to reflect in vivo coagulation status in the same system, including platelet function, fibrin clot formation, and whole blood flow. The Total Thrombus Analysis System (T-TAS), which is a microfluidic assay that simulates conditions in vivo, measures whole blood flow at defined shear rates under conditions designed to assess platelet function (PL-chip) or coagulation and fibrin clot formation (AR-chip). The T-TAS records occlusion start time, occlusion time, and area under the curve. We evaluated this test in healthy control dogs. We also investigated the effect in vivo of acetylsalicylic acid (ASA), and the effect in vitro of an anticoagulation drug (dalteparin; low-molecular-weight heparin; LMWH). The CV of the AUC of both chips was good (CVs of 6.45% [PL] and 1.57% [AR]). The inhibition of platelet function by ASA was evident in the right-shift in the PL test pressure curve. The right-shift in the AR test pressure curves showed that the administration of LMWH inhibited both platelets and the coagulation cascade. The T-TAS may be useful in the evaluation of canine blood coagulation.     

蜱的功能分子研究及其应用前景

蜱既是常见的吸血外寄生虫 ,又是人和动物许多重要疾病的媒介。为了持续地吸血 ,蜱唾液腺可分泌大量生物活性分子 ,并在抗凝血、抗炎症、免疫抑制等方面发挥重要作用。现已分离鉴定出的蜱抗凝血多肽 (TAP)等新型抗凝血和其他多种具有免疫调节的功能分子。此外 ,抗蜱基因工程疫苗候选抗原分子、酶及酶的抑制剂分子、抗药性相关分子、抗菌多肽和毒素等也在被深入研究。这些功能分子不仅在蜱及蜱传染病防制中显示着重要作用 ,同时也在医药生物制剂开发研制上表现出潜在的重要价值 ,显示着它们多方面广阔的应用前景     

Use of recombinant tissue-plasminogen activator in a dog with chylothorax secondary to catheter-associated thrombosis of the cranial vena cava

A 4-year-old, castrated male Maltese developed cranial vena caval thrombosis and chylothorax following central venous catheterization for treatment of postoperative sepsis. Vena caval thrombolysis was attempted using recombinant human tissue-plasminogen activator (t-PA). Thrombolytic therapy led to an acute reduction in the size of the caval thrombus and was followed by prompt resolution of the chylothorax. Hemorrhage at the entry sites of a jugular catheter and esophagostomy tube placed at the time of treatment was a dose-limiting complication of t-PA therapy in this dog.     

Thrombogenicity of dietary milkfat, fish oil and hydrogenated coconut oil in a pig model

Abstract Extract Several indicators of thrombosis and thrombolysis were measured in four groups of 16 pigs fed for 10 weeks on either a low fat basal ration or rations containing 10% anhydrous milkfat (AMF), 10% fish oil (MaxEPA), or 10% hydrogenated coconut oil (HCO). At the end of the feeding period, pigs on the three test fat/oil rations were subjected to balloon angioplasty of both femoral arteries. Thrombus size at the site of injury was measured both morphometrically and using autologous blood platelets labelled with (99)Tc-HMPAO (technetium - "Deretec").