五步蛇毒蛋白C激活剂的纯化与活性分析

目的 研究五步蛇毒蛋白C激活剂(PCA)组分的分离纯化及其抗凝活性.方法 借助DEAE-Cellulose、CM-Sephadex C-50和Sephadex C-75柱层析,以离子交换和凝胶过滤法从皖南产五步蛇粗毒中分离纯化PCA组分;以SDS-PAGE凝胶电泳检测其纯度和分子量,发色底物法(Chromogenic Substrate Assay)测定PCA组分的生色反应能力;测定PCA对正常血浆KPTT和PT的影响.结果 从五步蛇粗毒中纯化的PCA组分经SDS-PAGE测定为单一区带,相对分子质量约为18.5 kD,等电点pH 4.9;发色底物法显示其具有生色反应能力;该PCA 1 mg/L在体外明显延长KPTT,但PT不受影响.结论 采用离子交换和凝胶过滤法,可从皖南产五步蛇毒中分离纯化出高纯度的PCA组分,该组分可抑制内凝途径影响血液凝固过程.     

玉米芯木聚糖硫酸酯抗凝血活性及其机制的研究

采用活化部分凝血活酶时间(APTT)、凝血酶时间(TT)和凝血酶原时间(PT)检测了玉米芯木聚糖硫酸酯(wisX-SB)的抗凝活性,结果表明wisX-SB能明显延长APTT和TT,而不影响PT,提示wisX-SB是通过内源性和/或共同途径发挥抗凝血作用的。采用发色底物法及纤维蛋白原转化实验分别考察了wisX-SB对凝血酶及纤维蛋白原的作用,结果提示wisX-SB的抗凝机制包括:直接抑制纤维蛋白原的转化;通过增强抗凝血酶III(AT-III)的活性,抑制凝血酶活性,从而达到抗凝目的。较低浓度时以前者为主,较高浓度下两者皆起作用。     

硇洲马尾藻(S.naozhouense)褐藻多酚的抗凝血活性研究

本文采用溶剂萃取法提取硇洲马尾藻(S.naozhouense)褐藻多酚,并通过超滤进行分子量分级得NW1和NW2两部分,检测其总多酚的含量.通过体内和体外实验检测NW1和NW2体内对凝血时间(CT)、出血时间(BT)、凝血酶原时间(PT)和血浆凝血酶时间(TT)的影响.结果表明,硇洲马尾藻多酚NW1在10～40 mg/kg.d剂量范围能显著延长CT和BT,在5～15 mg/mL浓度范围显著增加PT和TT,表现出良好的全面抗凝血活性;NW2能显著延长CT和BT,对TT有一定的延长作用,但对PT无效果,且在高浓度(15 mg/mL)时表现出显著的缩短PT作用.     

不同批号蕲蛇酶在家兔血液凝固系统上的生物活性检测

目的：研究不同批号蕲蛇酶静脉给予家兔后,对血液凝固系统各项指标的影响,以判定其药效的稳定性。方法：取家兔,纯白色,体重1．5－2．5kg,雌雄兼用,每日静脉注入蕲蛇酶1．0u/kg.d^-1,连用3－5天,药前、药后从心脏取血6ml,取全血1ml作血栓形成;余血以3．0％枸橼酸钠按1：9抗凝,分离富血小板血浆（PRP）,测定其血小板数目和血小板聚集率,以贫血小板血浆（PPP）测定凝血酶时间（TT）,凝血酶原时间（PT）,部分凝血活酶时间（KPTT）以及纤维蛋白原（Fg)含量。结果：5个批号的蕲蛇酶药后3天或5天均可明显使血栓形成的重量减轻,长度缩短,Fg含量减少（P＜0．01）,TT,PT,KPTT均延长（P＜0．05－0．01）,血小板数轻度减少,聚集率抑制达20％－50％,结论：不同批号的蕲蛇酶静脉给予家兔,具有显著的作用于血液凝固系统,导致血栓形成减少,表明对心脑血管内血栓形成药学相同,说明用家 兔作为鉴定药品质量是可行而必须。     

适配子纤维蛋白靶向性验证及其抗凝活性测定

目的:验证适配子G81的纤维蛋白靶向性,评估适配子对凝血系统的影响。方法:以复钙法制备鼠源、人源体外纤维蛋白,将不同浓度Cy5.5标记的适配子溶液与之孵育,置于激光共聚焦显微镜下以固定的参数成像,用ImageJ软件进行相对荧光强度分析;将适配子G81溶液加入血浆中,通过倍比稀释法得到含浓度梯度适配子的血浆,采用SYSMEX CS-5100全自动血凝仪检测PT、APTT、TT,评估适配子G81对凝血功能的影响。结果:激光共聚焦显微镜显示适配子能与纤维蛋白结合,随着加入适配子量的增加其相对荧光强度逐渐增强,表明适配子可与纤维蛋白结合,统计分析提示荧光强度与适配子存在量效关系;人源、鼠源纤维蛋白结合的荧光强度无统计学差异(P0.05)。在抗凝活性检测中,血浆中适配子G81浓度达到200 pmoL/mL时,各浓度统计分析结果均显示P0.05,表明适配子对PT、APTT、TT的测量均没有统计学差异上的影响。结论:适配子G81具有纤维蛋白靶向性,且当加入的适配子剂量低于200 pmol/mL时对内、外源性凝血功能、凝血酶时间均无明显影响。     

金属离子对氧化硫硫杆菌生长及产酸的影响

目的:在前期研究中选育出的氧化硫硫杆菌TT03,为了解其对各种金属离子的适应能力而进行了试验.方法:在史塔克培养基中添加规定量的各种金属离子,接种TT03后进行摇动培养,测定培养液中的产酸以代表其实际生长状况.结果:在Co<'3+>、Zn2+为100mg/L而其他微量元素为1000mg/L时没有产生抑制作用,其中有些微量元素还表现出促进作用;除pb2+、Cd2+以外,重金属在1000mg/L下普遍表现出轻微的抑制作用,对产酸没有明显影响;当pb2+、Cd2+的含量分别在10mg/L、1mg/L时,对TT03产酸没有影响.在试验的金属离子中Cd2+对TT03产酸的抑制作用最强,在浓度30mg/L时,生长产酸被完全抑制.结论:在正常土壤环境中的金属离子含量,不会对氧化硫硫杆菌TT03的生长和产酸造成致命影响.     

HB提取条件的优选及其凝血实验研究

目的：探讨HB（一种促凝血草药的简称）提取条件的优选,及其氯仿萃取纯化后水相提取物的体内、外促凝血作用。方法：以家兔、大鼠为研究对象,分为空白组、云南白药对照组和HB氯仿萃取水相提取物组。观察家兔体外凝血时间;及给药10d后大鼠的血生化指标,如凝血酶原（PT）时间、活化部分凝血活酶（APTT）时间、纤维蛋白原（FIB）含量、凝血酶（TT）时间、血小板计数（PLT）。结果：HB水提最优工艺条件是：提取水量10倍,提取温度90℃。提取时间1.5h。体外促凝效果的观察：家兔的四种方法体外凝血时间明显短于空白及云南白药对照组（P〈0.01）,且凝血时间缩短70%以上。体内促凝作用观察：各用药组血小板计数均显著高于空白及云南白药组（p〈0.01）;高（5,10,20mg/kg）剂量组PT时间均显著短于空白组（p〈0.01）,与云南白药组相比无显著性差异（p〉0.05）;各剂量组APTT、FIB、TT较空白及云南白药组无显著性差异。结论：HB氯仿萃取纯化后水相提取物具有良好的体内、外促凝血作用,而其能使血小板计数显著升高是其主要止血机理之一。     

超高龄患者填充骨水泥型人工股骨头置换术后的凝血功能 变化及意义

目的:观察骨水泥填充对人工股骨头置换术术后超高龄老年患者凝血系统的影响。方法:选择80岁以上骨水泥型人工股骨头置换术患者29例,于术前、术后当天及术后第3天空腹抽取静脉血,测定凝血功能相关指标,包括凝血酶原时间(PT)、部分凝血活酶时间(APTT)、凝血酶时间(TT)、凝血酶原活动度(PTA)、国际标准化比值(INR)、纤维蛋白原(FIB)、D二聚体(DD)、抗凝血酶Ⅲ(ATⅢ)、血小板(PLT)水平,并对结果进行比较分析。结果:患者术后当天FIB、DD、显著升高(P<0.05),ATⅢ降低(P<0.05),提示高凝状态,且纤溶亢进,此时段TT、PT延长(P<0.05),血小板明显降低,提示存在出血风险;术后第3天TT、PT显著延长(P<0.05),ATⅢ恢复到术前水平,FIB,DD水平较手术后当天下降,提示术后第3天有明显的出血倾向,凝血与纤溶系统逐渐恢复平衡。结论:骨水泥型人工股骨头置换术对80岁以上超高龄患者凝血功能有显著影响,术后当天高凝状态、纤溶亢进,存在潜在出血风险,术后第3天有明显出血倾向,提示高龄患者术后应适当补充凝血因子且须谨慎使用抗凝药物。     

一种新的钙依赖性磷脂结合蛋白——Annexin32对血凝及血栓形成的影响

通过探讨一种新的钙依赖性磷脂结合蛋白Annexin32对家兔凝血时间和兔、大鼠血栓形成的影响, 对其主要药效学进行了研究, 并与普通肝素进行了比较. 兔、大鼠静脉给药, 检测用药后不同时间家兔白陶土部分凝血活酶时间(KPTT)、体外血栓形成和大鼠体内血栓形成的变化. 结果显示, 按1 mg/kg静脉注射Annexin32后, KPTT明显延长(P < 0.01), 但小于肝素延长幅度, 体外血栓形成仪测定显示血栓长度、重量均明显减少(P < 0.01), 大鼠体内血栓重量明显降低(P < 0.01), 提示Annexin32具有抗血栓形成的功能, 并有对凝血系统影响小的特征.     

聚氨酯表面偶联重组水蛭素及其抗凝血酶活性的评价

在PU和Chro表面上涂覆含羧基硬段的PU1溶液并用次甲基蓝吸附法测定表面羧基含量;用EDC接枝牛血清白蛋白并用FT-ATR-IR谱的差减测定表面BSA接枝量;用EDC偶联rHir并用放射性同位素测定表面rHir偶联量,最后用白陶土部分凝血酶时间测定(KPTT)、凝血酶原测定(PT)、凝血酶时间测定(TT)试验评价样品的抗凝血酶活性.CPU和C-Chro表面羧基含量分别为3.77和3.03 mmol/m²,CPU-BSA和C-Chro-BSA表面BSA含量分别为426和262 mg/m²,CPU-BSA-¹²⁵I-rHir和C-Chro-BSA-¹²⁵I-rHir表面rHir含量分别为716.7和691.1 μg/m²,比对照样品都有很大的提高.抗凝血酶试验评价表明,rHir偶联样品的KPTT、PT、TT时间都明显延长,它们的抗凝血酶活性确有提高,抗血栓性能得到有效的改善.     

Antimicrobial peptide buforin I inhibits tissue factor-initiated coagulation

The enhanced extrinsic blood coagulation following septic shock often manifests cardiovascular complications. The upregulated monocytic tissue factor (mTF) was shown to be a primary contributor to the extrinsic hypercoagulation following acute bacterial endotoxin (LPS) infection. A single-stage clotting assay monitors TF-initiated coagulation. We herein demonstrate a novel anticoagulant activity of antimicrobial peptide Buforin I (BF I) in offsetting LPS-induced mTF hypercoagulation in THP-1 cells, which was confirmed in a cell-free in vitro model, showing that BF I effectively blocked rabbit brain thromboplastin (rbTF) procoagulant activity. Upon inclusion of 25 microM BF I into human plasma, the prolonged prothrombin time (PT) was consistent with the depressed TF-initiated coagulation. In a two-stage chromogenic assay monitoring S-2288 hydrolysis, BF I significantly inhibited not only mTF- but also rbTF-catalyzed FVII activation accompanied by the diminished FVIIa formation. The inhibition by BF I of FVII activation accounted for its novel anticoagulant activity in offsetting mTF-initiated hypercoagulation.     

Factors V and VII anticoagulant activities in the salivary glands of feeding Dermacentor andersoni ticks

The salivary glands of Dermacentor andersoni ticks possess anticoagulant activities that can alter the clotting time of rabbit whole blood. Salivary gland extracts from female ticks inhibit both the intrinsic and extrinsic coagulation systems, and maximal activities against both pathways occur when the ticks attain about 250 mg feeding weight. These anticoagulants are directed against both coagulation factors V and VII, but they do not affect factors II or X. Despite this salivary anticoagulant activity, heavily tick-infested rabbits suffer no visible alteration of their peripheral blood coagulability and have no detectable circulating fibrin degradation products, suggesting that the ticks do not secrete a factor with fibrinolytic activity.     

Effects of high-molecular-weight cryoprotectants on platelets and the coagulation system

The objective of this study is to examine the effects of the most widely used high-molecular-weight cryoprotectants on the coagulation system. Dextran, hydryoxyethyl starch (HES), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and albumin were added at different concentrations in the range between 0.01-1% (w/v) to solvent/detergent-treated plasma. Using a STA/STA Compact coagulation analyzer the following clotting tests were performed: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Factor V, and Factor VIII percentage of activity. PVP and PEG caused a significant increase in APTT, a decrease in Factor VIII percentage of activity, and a slight decrease in TT, while PT and Factor V percentage of activity remained unchanged. Dextran, HES, and albumin did not effect the clotting tests. The effect of high-molecular-weight cryoprotectants on platelets was assessed by platelet-induced clot retraction (PICR) and aggregation with thrombin and agglutination with ristocetin. Platelet aggregation and agglutination were unaffected by all cryoprotectants tested; however, PICR was significantly reduced in the presence of PVP or PEG. Possible mechanisms by which PVP and PEG interfere with the coagulation system are discussed. We also raise issues concerning the development of one-step blood cryopreservation techniques which do not require cryoprotectant removal prior to transfusion.     

Anticoagulant activity of sulfated polysaccharide isolated from fermented brown seaweed Sargassum fulvellum

A sulfated polysaccharide with anticoagulant properties was isolated from the fermented brown seaweed Sargassum fulvellum. Freeze-dried S. fulvellum was fermented in an incubator for 10^th week at 25°C to convert seaweed macromolecules into anticoagulant sulfated polysaccharides (ASP). Anticoagulant activity was determined by an activated partial thromboplastin time (APTT) test using citrated human blood plasma. The 8^th week S. fulvellum crude seaweed extract (SWE) exhibited the highest blood anticoagulant activity. Therefore, 8^th week crude SWE was used for purification of ASP by two steps; DEAE cellulose anion-exchange followed by Sepharose 4B chromatography. The isolated ASP showed a single spot on agarose gel electrophoresis, which confirmed the purification status of our ASP. Polyacrylamide gel electrophoresis (PAGE) analysis showed that the molecular mass of the purified ASP was between 8 and 20 kDa. Polysaccharide and sulfate concentrations of the purified ASP were 180 and 29.70 μg mL⁻¹ respectively. ASP recovery was 1.32% (w/w) from the crude polysaccharide applied to the DEAE column. Purified ASP had a pH of 3.86 and was considered an acidic polysaccharide. Moreover, both ASP and heparin showed a relative clotting factor of 27.47 at the concentrations of 180 and 60 μg mL⁻¹ respectively. Therefore, S. fulvellum ASP can be considered a weaker anticoagulant than heparin. Results of the APTT, PT, and TT clotting assays showed that ASP was able to inhibit both intrinsic and extrinsic blood coagulation pathways. Finally, this study established a feasible and simple experimental protocol to isolate anticoagulant from fermented seaweeds leading to potential further development of anticoagulant agent for the pharmaceutical industry.     

Homocysteine induces procoagulant activity of red blood cells via phosphatidylserine exposure and microparticles generation

Increased homocysteine (Hcy) levels in plasma correlate with the risk of thromboic events. Red blood cells (RBCs), the most abundant blood cells in circulation, also play an active role in the process of thrombus formation. However, the effect of Hcy on procoagulant activity (PCA) of RBCs is unclear. In the present study, RBCs from healthy adults were treated with Hcy (8, 20, 80, 200, 800 μmol/L) for 24 h. Phosphatidylserine (PS) exposure of RBCs and red blood cell-derived microparticles (RMPs) release were detected using Alexa Fluor 488-lactadherin. PCA was assessed by coagulation time and purified clotting complexes testes. We found that Hcy treatment dose dependently enhanced PS exposure and consequent PCA of RBCs. Hcy also elevated the formation of procoagulant RMPs, with statistical significance at 800 μmol/L of Hcy. Moreover, 128 nmol/L lactadherin inhibited about 90 % PCA of RBCs and RMPs. Our data suggest that PS exposure and RMPs shedding are key sources for Hcy-induced PCA of RBCs. Lactadherin could be used to modulate the anticoagulant and procoagulant balance in this process.     

Anticoagulant activity of M-LAO, l-amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is l-amino acid oxidase (LAO), which catalyzes the oxidative deamination of l-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.     

Anticoagulant activity of M-LAO,L-amino acid oxidase purified from Agkistrodon halys blomhoffii,through selective inhibition of factor IX

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.     

New Insights into the Biological Activity of Lichens: Bioavailable Secondary Metabolites of Umbilicaria decussata as Potential Anticoagulants

This study reports the in vitro anticoagulation activity of acetonic extract (AE) of 42 lichen species and the identification of potential bioavailable anticoagulant compounds from Umbilicaria decussata as a competent anticoagulant lichen species. Lichens’ AEs were evaluated for their anticoagulant activity by monitoring activated partial thromboplastin time (APTT) and prothrombin time (PT) assays. A strong, positive correlation was observed between total phenolics concentration (TPC) of species and blood coagulation parameters. U. decussata was the only species with the longest clotting time in both APTT and PT assays. The research was moved forward by performing in vivo assays using rats. The results corroborated the dose-dependent impact of U. decussata’s AE on rats’ clotting time. Major secondary metabolites of U. decussata and their plasma-related bioavailability were also investigated using LC-ESI-MS/MS. Atranol, orsellinic acid, D-mannitol, lecanoric acid, and evernic acid were detected as possible bioavailable anticoagulants of U. decussata. Our findings suggest that U. decussata might be a potential anticoagulant lichen species that can be used for the prevention or treatment of coagulation-related issues such as cardiovascular diseases (CVDs).     

Plasmin converts factor X from coagulation zymogen to fibrinolysis cofactor

Known anticoagulant pathways have been shown to exclusively inhibit blood coagulation cofactors and enzymes. In the current work, we first investigated the possibility of a novel anticoagulant mechanism that functions at the level of zymogen inactivation. Utilizing both clotting and chromogenic assays, the fibrinolysis protease plasmin was found to irreversibly inhibit the pivotal function of factor X (FX) in coagulation. This was due to cleavage at several sites, the location of which were altered by association of FX with procoagulant phospholipid (proPL). The final products were approximately 28 and approximately 47 kDa for proPL-bound and unbound FX, respectively, which did not have analogues when activated FX (FXa) was cleaved instead. We next investigated whether the FX derivatives could interact with the plasmin precursor plasminogen, and we found that plasmin exposed a binding site only on proPL-bound FX. The highest apparent affinity was for the 28-kDa fragment, which was identified as the light subunit disulfide linked to a small fragment of the heavy subunit (Met-296 to approximately Lys-330). After cleavage by plasmin, proPL-bound FX furthermore was observed to accelerate plasmin generation by tissue plasminogen activator. Thus, a feedback mechanism localized by proPL is suggested in which plasmin simultaneously inhibits FX clotting function and converts proPL-bound FX into a fibrinolysis cofactor. These data also provide the first evidence for an anticoagulant mechanism aimed directly at the zymogen FX.