Experimental study on anticoagulant and antiplatelet aggregation activity of a chemically sulfated marine polysaccharide YCP

A polysaccharide YCP was prepared from a marine filamentous fungus Keissleriella sp. YS4108, which exhibited as a molecular weight (Mw) of 2.4x10(3) kDa and its three sulfated derivatives (YCP-SL, YCP-SM and YCP-SH) were synthesized, the degree of substitution (DS) of which were determined to be 0.13, 0.99 and 1.3, with the average molecular weight 0.64x10(3), 0.57x10(3) and 0.45x10(3) kDa, respectively. Anticoagulant activity and antiplatelet aggregation activity of these sulfated derivates were evaluated by activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and platelet aggregation assay. The results showed that YCP sulfates significantly prolonged APTT, TT and PT. The derivates showed no effects on thrombin in the presence or in the absence of antithrombin III (AT III) or heparin cofactor II (HC II), while the derivates effectively inhibited factor Xa in the presence of AT III. At the same time, YCP-SH also possessed potent antiplatelet aggregation activity in vitro compared with aspirin. YCP sulfates specifically interfered with different stages of the coagulation cascade, and the anticoagulant activity improved with the increasing DS and decreased Mw.     

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。     

Thermal depolymerization of alginate in the solid state

A new method of introduction carboxyl groups to chitosan sulfate by the acylation reaction between hydroxyethyl chitosan sulfates and butane dioic anhydride in homogeneous solution was used to obtain carboxybutyrylated hydroxyethyl chitosan sulfates. The structures of the derivatives were characterized by element analysis, FT-IR, ¹³C-NMR, and gel permeation chromatography. The content and position of the carboxyl groups could be controlled favorably. Their anticoagulant activity was determined for human plasma with respect to activated partial thromboplastin time (APTT), thrombin time (TT), and prothombin time (PT). The introducing of carboxyl groups to amino groups greatly prolonged the APTT and TT. The best result occurred when the degree of substitution of the carboxyl groups was about 0.4/unit that prolonged APTT and TT with about 5 and 1.5 times compared to that of the uncarboxylated hydroxyethyl chitosan sulfates; another conclusion is that introducing of carboxyl groups into N,O-position gave better results than that just into N-positions. Low S% chitosan sulfate and 6-O-desulfated chitosan sulfate showed little anticoagulant activity but their N,O-carboxybutyrylated derivatives (0.6/unit ds) showed increased APTT or TT, while their N-carboxybutyrylated derivatives (0.6/unit ds) gave no improvement. Generally, the introducing of carboxyl groups could not increase PT in spite of the position introduced.     

Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae

A fibrinolytic metalloprotease with in vitro fibrinolytic effects was purified from the edible mushroom Pleurotus ferulae using several chromatography steps including anion and ion exchange, gel filtration, and fast protein liquid chromatography columns. The molecular mass of the enzyme was estimated to be 20.0?kDa, as determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin zymography. The protease was active at 50°C, and pH 4.0, 5.0, and 8.0. The fibrinolytic activity of the enzyme was inhibited by ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′ tetraacetic acid and strongly inhibited by two metal ions, Cu and Mg. In vitro assays evaluating fibrinolytic activity on a fibrin plate, fibrin turbidity, and thrombolytic activity on fibrin clots using human fibrinogen and human thrombin revealed that the enzyme could hydrolyze fibrin polymers directly and inhibit the formation of fibrin clots. In activated partial thromboplastin time (APTT) and prothrombin time assays, the enzyme strongly prolonged the APTT, which detects an activity of intrinsic and common pathways. The enzyme showed strong in vivo protective effect against mortality/paralysis from epinephrine plus collagen-induced acute thromboembolism in in vivo model. Our findings suggest that the enzyme may have a potential for treatment and prevention of thrombosis-relative diseases.     

慢性乙型肝炎患者血清白介素17A和胆碱酯酶的表达及临床意义

目的:检测慢性乙型肝炎(CHB)患者血清白介素17A(IL-17A)、胆碱酯酶(CHE)水平,并分析其临床意义。方法:选取2018年1月到2019年3月期间在重庆三峡中心医院接受治疗的CHB患者84例,根据病情严重程度将所有患者分为轻度组30例、中度组28例、重度组26例,另选取同期在重庆三峡中心医院进行体检的健康志愿者50例作为对照组。比较各组的凝血四项指标[纤维蛋白原(FIB)、凝血酶原时间(PT)、凝血活酶时间(APTT)、凝血酶时间(TT)]、肝功能指标[谷丙转氨酶(ALT)、谷草转氨酶(AST)]、IL-17A、CHE水平,采用Pearson相关分析CHB患者血清IL-17A、CHE与凝血四项、ALT、AST的相关性。结果:重度组、中度组、轻度组、对照组的FIB、CHE水平逐渐升高,PT、APTT、TT、ALT、AST、IL-17A水平逐渐降低,两两比较均有统计学差异(P0.05),IL-17A与FIB、CHE呈负相关,与PT、APTT、TT、ALT、AST呈正相关(P0.05);CHE与FIB呈正相关,与PT、APTT、TT、ALT、AST呈负相关(P0.05)。结论:CHB患者血清中IL-17A、CHE水平与患者的肝功能和凝血功能密切相关,联合检测IL-17A和CHE有助于患者的病情评估以及预后判断。     

蛇伤凉血合剂对尖吻蝮蛇咬伤患者凝血四项的影响

目的观察蛇伤凉血合剂对尖吻蝮蛇咬伤的临床疗效。方法将60例尖吻蝮蛇咬伤患者随机分为观察组和对照组各30例,两组患者均给予常规治疗,治疗组加服蛇伤凉血合剂,观察比较两组患者就诊时(咬伤2h内)、咬伤后72h凝血酶时间(TT)、凝血酶原时间(PT)、活化部分凝血酶原时间(APTT)、纤维蛋白原(Fg)的变化。结果两组患者就诊时TT、PT、APTT、Fg均在正常范围,两组比较差异无统计学意义。伤后72h,两组患者的TT、PT、APTT均升高,Fg均降低,而且组间比较差异均有统计学意义(P0.05)。结论蛇伤凉血合剂对尖吻蝮蛇咬伤所致凝血功能障碍有防治作用。     

Influence of functional groups on the in vitro anticoagulant activity of chitosan sulfate

A new method for the chemical modification of chitosan sulfate was used to prepare N-propanoyl-, N-hexanoyl- and N,O-quaternary substituted chitosan sulfate. Structural analysis by elemental analysis, FTIR, 13C NMR, and 1H NMR spectroscopy, and gel-permeation chromatography showed that these methods could conveniently be used for the introduction of functional groups. The influences of the acyl or quaternary groups on the anticoagulant activity of the polysaccharides were studied with respect to activated partial thromboplastin time (APTT) thrombin time (TT), and prothrombin time (PT). The propanoyl and hexanoyl groups increased the APTT activity, and the propanoyl groups also increased the TT anticoagulant activity slightly, while the N,O-quaternary chitosan sulfate showed only a slight TT coagulant activity.     

术中保温联合早期系统化康复训练对老年全髋关节置换术患者凝血功能、康复效果和生活质量的影响

摘要 目的：观察术中保温联合早期系统化康复训练对老年全髋关节置换术（THA）患者凝血功能、康复效果和生活质量的影响。方法：选择我院2018年8月～2021年8月期间收治的80例老年THA患者,按随机数字表法将其分为对照组（40例）和观察组（40例）。对照组患者接受早期系统化康复训练,观察组患者接受术中保温联合早期系统化康复训练,对比两组术中低体温发生率、凝血功能、康复效果和生活质量。结果：观察组的术中低体温发生率明显低于对照组（P<0.05）。观察组的下床活动时间、住院时间短于对照组（P<0.05）。两组术后8周Harris髋关节功能评分均升高,且观察组高于对照组（P<0.05）。对照组术后7 d凝血酶原时间(PT) 、活化部分凝血活酶时间( APTT) 及凝血酶时间( TT) 、纤维蛋白原（FIB）均升高（P<0.05）,观察组术前、术后7 d PT、APTT、TT、FIB组内对比无统计学差异（P>0.05）,观察组术后7 d PT、APTT、TT、FIB低于对照组（P<0.05）。对照组术后6个月躯体职能、角色职能、情绪职能、认知职能、社会职能评分升高,且观察组高于对照组（P<0.05）。结论：术中保温联合早期系统化康复训练可促进老年THA患者康复,有效保护机体凝血功能,促进患者生活质量提高,效果显著。     

藕节止血作用研究

本实验通过测定毛细管凝血时间(CT)、剪尾法出血时间(BT)、活化部分凝血酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)、优球蛋白溶解时间(ELT)等指标研究藕节醇提物及其不同极性段的体内止血作用.结果表明,藕节醇提物显著缩短小鼠CT、BT,显示很好的止血作用,可能是主要通过缩短APTT、PT,延长ELT...     

海参多糖对急性不完全性脑缺血的保护及抗凝作用的研究

目的:研究海参多糖(PSU)对大鼠急性不完全性脑缺血(AICI)损伤的保护作用及抗凝血作用。方法:SD大鼠60只,随机分为5组(n=12):假手术组、模型组、海参多糖低(30 mg/(kg.d))、中(60mg/(kg.d))、高(120mg/(kg.d))剂量组。双侧颈总动脉永久性结扎法复制大鼠急性不完全性脑缺血模型,观察海参多糖对大鼠行为障碍、脑组织含水量、血清C-反应蛋白(CRP)水平的变化情况,以及对凝血酶原时间(PT)、活化的部分凝血酶时间(APTT)、凝血酶时间(TT)和纤维蛋白原含量(FIB)等血液凝血指标的影响。结果:海参多糖可改善行为学障碍,减少脑组织含水量,降低血清CRP水平;可显著延长APTT,TT时间,减少FIB含量(P<0.05)。结论:海参多糖能明显减轻大鼠急性不完全性脑缺血损伤,并具有显著的抗凝血作用。     

凝血及纤溶系统在腹腔镜子宫全切手术后的变化

目的:比较分析妇科腹腔镜子宫全切术与开腹子宫全切术对凝血及纤溶系统功能的影响。方法:选择75例接受子宫全切除术的患者,随机分为腹腔镜组38例和开腹组37例,分别于术前24h、术后24h及术后48h采用凝固法检测血浆凝血酶原时间(PT),活化部分凝血活酶时间(APTT),凝血酶时间(TT),纤维蛋白原(FIB)水平,采用酶联免疫吸附双抗体夹心法(ELISA)测定血浆D-二聚体(D-D)的含量。自动血细胞技术分析仪测定血小板(PLT)。结果:①腹腔镜组与开腹组术前24h及术后48h血浆PT、APTT、TT、FIB、PLT、D-D水平相比差异无统计学意义(P>0.05)。②两组病人术后24h血浆PT、APTT均缩短,组间相比差异无统计学意义(P>0.05),但与术前24h相比差异有统计学意义(P<0.05)。③TT及PLT在术前24h及术后24h水平相比差异无统计学意义(P>0.05)。④FIB、D-D两组术后24h均升高,与术前相比差异有统计学意义(P<0.05),组间比较差异无统计学意义(P>0.05)。结论:腹腔镜子宫全切除术特殊的二氧化碳气腹的建立,头低臀高的膀胱截石位,气管插管的全麻协同作用,使患者的血流动力学发生改变,血流速度减慢,同开腹手术一样,均可使患者术后血液呈现不同程度高凝状态,从而均可增加术后下肢静脉血栓形成的危险。因此,对有高危因素的患者应加强围手术期的管理,防止血栓性疾病的发生。     

弥漫性血管内凝血产妇围术期凝血与纤溶系统指标的检测及临床意义

目的:探讨弥漫性血管内凝血(DIC)产妇围术期凝血与纤溶系统指标检测的临床意义。方法:选择2017年1到2017年12月在我院接受治疗的DIC孕妇57例(DIC组)为研究对象,采取分层抽样的方法选择同期在我院进行产检的正常孕妇57例(健康孕妇组)及在我院体检的健康非孕妇57例(非孕妇组)作为对照,比较各组凝血酶原时间(PT)、凝血酶时间(TT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、D-二聚体(D-D)及血小板计数(PLT)变化,根据DIC组的治疗结果分为有效组和无效组,并比较两亚组治疗前PT、TT、APTT、FIB、D-D、PLT,采用Pearson相关分析法分析DIC组治疗前各检测指标间的相关性。结果:与健康孕妇组、非孕妇组比较,DIC组PT、TT、APTT延长(P0.05),D-D水平升高(P0.05),FIB、PLT水平降低(P0.05);与非孕妇组比较,健康孕妇组PT、TT、APTT缩短(P0.05),D-D水平降低(P0.05),FIB、PLT水平升高(P0.05)。DIC组患者治疗后有效组治疗前的PT、TT、APTT短于无效组(P0.05),D-D水平低于无效组(P0.05);FIB、PLT水平高于无效组(P0.05)。Pearson相关分析结果显示,除PT与APTT之间无明显相关性(P0.05)外,其他凝血、纤溶系统指标之间均存在一定的相关性(P0.05)。结论:DIC孕妇围术期凝血与纤溶系统指标异常改变,检测凝血与纤溶系统指标对DIC孕妇的诊疗具有重要意义。     

Thrombolytic,anticoagulant and antiplatelet activities of codiase,a bi-functional fibrinolytic enzyme from Codium fragile

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. In this study, codiase, a new bi-functional fibrinolytic serine protease having thrombolytic, anticoagulant, and antiplatelet activities was purified from marine green alga, Codium fragile. The molecular weight of the enzyme was estimated to be 48.9 kDa by SDS-PAGE, and mass spectrometry. Fibrin zymography analysis showed an active band with similar molecular weight. The N-terminal sequence was found to be APKASTDQTLPL, which is different from that of other known fibrinolytic enzymes. Codiase displayed maximum activity at 30 °C and pH 6.0, and the activity was inhibited by Zn²⁺ and Fe²⁺. Moreover, the enzyme activity was strongly inhibited by serine protease inhibitor such as PMSF. Codiase exhibited high specificity for the substrate S-2288, and the K_m and V_max values for this substrate were found to be 0.24 mM and 79 U/ml respectively. Fibrin plate assays revealed that it was able to hydrolyze fibrin clot either directly or by activation of plasminogen. Codiase effectively hydrolyzed fibrin and fibrinogen, preferentially degrading α- and Aα chains, followed by γ–γ, and γ-chains. However, it provoked slower degradation of Bβ and β-chains. The structural change of fibrin clot and fibrinogen by codiase was also detected by FTIR-ATR spectroscopy analysis. In vitro and in vivo studies revealed that codiase reduces thrombosis in concentration-dependent manner. Codiase was found to prolong activated partial thromboplastin time (APTT), and prothrombin time (PT). PFA-100 studies showed that codiase prolonged the closure time (CT) of citrated whole human blood. These favorable antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to mice and NIH-3T3 cells, make it a potential agent for thrombolytic therapy.     

Characterization of a proteolytic enzyme from Lachesis muta venom (Serpentes: Viperidae).

A proteolytic enzyme from L. muta stenophrys was isolated by gel filtration on Bio Gel P-100 followed by FPLC on MONO S column. The enzyme exhibited proteolytic activity toward casein, hemoglobin and fibrinogen with a pH optimum around 10. The activity was inhibited by EDTA while trypsin inhibitors were not inhibitory. It is a glycoprotein, Mr 14 kDa with a high content of Asp, Glu, and Leu residues and a low content of Cys and Trp. The protease is devoid of myotoxic, hemorrhagic, esterolytic and amidolytic activities. It lyses the alfa and beta chains of human fibrinogen and releases kinin from L.M.W. kininogen. No release of histamine was observed upon incubation with mast cells.     

Hemostasis system indices in the course of a 105-day hermetic chamber isolation experiment

Changes in hemostasis indices in the course of a 105-day hermetic chamber isolation experiment were studied. The following indices were measured: the activated partial thromboplastin time (APTT); the prothrombin time (PT); the prothrombin index (PI); the international normalized ratio (INR); the thrombin time (TT); the concentrations of fibrinogen, soluble fibrin-monomer complexes (SFMC), D-dimer, and plasminogen (PG); and the activities of antithrombin III, protein C, and α₂-antiplasmin. Isolation was accompanied by PT prolongation (with PI decreasing and INR increasing) observed until the seventh day of the aftereffect period, as well as a decrease in the PG concentration. The changes are likely to have been related to the specific motor activity mode, the effect of compensatory physical exercise, and changes in the characteristics of protein and lipid metabolisms.     

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。     

Direct acting anti-thrombotic serine protease from brown seaweed Costaria costata

A direct acting antithrombotic serine protease (CCP) was purified from brown seaweed Costaria costata. CCP was a monomeric protease with molecular mass of 60,547.598 daltons as determined by mass spectrometry. The N-terminal sequence of CCP was SCNSCLDKVDADGLN. Proteolytic activity was inhibited by PMSF and APMSF. CCP exhibited high amidolytic activity toward substrate S-2251 with apparent K_m and V_max values were 14.5 μM and 183.5 U/ml respectively. Fibrin plate and fibrin zymography results revealed that CCP was able to degrade fibrin clots directly. It specifically hydrolyzed Aα and α and Bβ and β chains followed by γ and γ–γ chains of human fibrinogen and fibrin respectively. Cleavage of fibrin clot and fibrinogen was emphasized by observing the alteration of secondary structure using FTIR spectroscopy. Morphological alteration of fibrin clot was also evidenced by fluorescent microscopic observation. CCP reduced thrombus effectively in vitro. In vivo observation showed that it prevented/decreased thrombus formation in carrageenan-induced mice tail model. CCP prolonged activated partial thromboplastin time (APTT) and had little effect on prothrombin time (PT). Platelet function analyzer (PFA-100) tests showed that CCP prolonged closure time (CT). These data suggest that CCP could have therapeutic potential for the treatment of thrombosis.     

Mannich bases of scutellarein as thrombin-inhibitors: Design,synthesis, biological activity and solubility

Two series of 8-aminomethylated derivatives were prepared by Mannich reaction of scutellarein (2) with appropriate aliphatic amines, alicyclic amines and formaldehyde. All the compounds were tested for their thrombin inhibition activity through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). The antioxidant activities of these target products were assessed by 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay method and the solubility were assessed by ultraviolet (UV). The results showed that morpholinyl aminomethylene substituent derivative (3d) demonstrated stronger anticoagulant activity, better water solubility and good antioxidant activity compared with scutellarein (2), which warrants further development as a agent for ischemic cerebrovascular disease treatment.     

Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom

By Q-sepharose column ion-exchange chromatography, alkyl-sepharose column hydrophobic chromatography the purified fibrinogenolytic enzyme was obtained from Agkistrodon halys halys venom. It is a single peptide-chain with molecular weight about 28 kDa. It was founded that this enzyme cleaved A alpha-chain of fibrinogen, pH-optimum was determined in the range of 7.5-8.0. Its fibrinogenolytic activity was estimated 15.6 mM fibrinogen/min per mg protein; caseinolytic activity was estimated 7.5 c.u., and amidolytic activity was 0.325 mM pNA/min/mg and 0.175 mM pNA/min/mg for S2238 and S2251 respectively; K(m) was 5.6 mM. The enzyme activity was inhibited by DFP and benzamidine. These results suggest that the enzyme is serine protease. It inhibited the platelet-aggregation.     

高凝状态对非小细胞肺癌患者深静脉血栓形成的影响

目的:探讨高凝状态对非小细胞肺癌患者深静脉血栓形成的影响,为临床治疗提供依据。方法:选择2010年5月至2013年6月在我院接受治疗的非小细胞肺癌患者195例,记录患者姓名、年龄、性别、病理类型、手术、临床分期、放化疗、伴随疾病、身体状况评分(eastern cooperative oncology group,ECOG评分)及是否发生血栓等指标。调查入选患者的高凝状态指标,包括血小板(platelet,PLT)、纤维蛋白原(fibrinogen,Fib)、血浆凝血酶原时间(plasma prothrombin time,PT)、凝血酶时间(thrombin time,TT)、活化部分凝血活酶时间(activated coagulation time of whole blood,APTT)和D-二聚体(D-dimer)。结果:195例非小细胞肺癌患者中,PLT高于正常上限的有42例(21.5%),FIB升高的有78例(40%),TT延长的有12例(6.2%),PT缩短的有45例(23.1%),D-D升高的有60例(30.8%),APTT缩短的有15例(7.7%)。两项指标异常的有62例(31.8%),FIB和D-D升高的有32例(16.4%);三项指标异常的有27例(13.8%),PLT、FIB及D-D升高的有15例(7.7%);四项指标异常的有5例(2.6%),PLT、FIB及D-D升高,而PT缩短的有5例(2.6%)。所有患者各凝血指标中位值PLT为219×109/L,FIB为3.6 g/L,TT为13.4 s,PT为10.8 s,D-D为0.341 mg/L,APTT为29.7 s。结论:高凝状态对非小细胞肺癌患者深静脉血栓形成具有重要的促进作用,应受到临床的重视。