Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as γ-carboxylation, N- and O-glycosylation, β-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC–ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn₃₂₂ compared to Asn₁₄₅. Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser₆₀ and Ser₅₂ which are modified by oligosaccharide structures such as fucose and Glc(Xyl)_0–1–2, respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.     

冻干加热处理凝血因子类制剂的病毒灭活验证

在病毒灭活验证过程中,比较了冻干后不同加热方法对凝血因子类制剂中伪狂犬病毒(PRV)的灭活效果,包括80℃干烤72h、100℃水浴加热30min以及100℃蒸汽加热30min方法。结果表明80℃干烤72h对制品中PRV灭活较彻底,另外两种方法对制品中PRV的灭活效果均不理想。     

Flocculation of algae using chitosan

Flocculation of three freshwater algae, Spirulina,Oscillatoria and Chlorella, and onebrackish alga, Synechocystis, using chitosan was studiedinthe pH range 4 to 9, and chlorophyll-a concentrations inthe range 80 to 800 mg m^–3, which produces aturbidity of 10 to 100 nephelometric turbidity units (NTU) in water. Chitosanreduced the algal content effectively by flocculation and settling. Theflocculation efficiency is very sensitive to pH, and reached a maximum at pH7.0for the freshwater species, but lower for the marine species. The optimalchitosan concentration that is required to effect maximum flocculation dependedon the concentration of alga. Flocculation and settling were faster whenconcentrations of chitosan higher than optimal are used. The settled algalcellsare intact and live, but will not be redispersed by mechanical agitation. Thede-algated water may be reused to produce fresh cultures of algae.     

Conscientious metabolic monitoring on a patient with hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome undergoing anaesthesia

Summary. Currently we know not more than 50 patients who show an interesting combination of increased plasma ornithine concentrations, postprandial hyperammonemia, and homocitrullinuria (HHH-syndrome). Since exact knowledge of this severe, although rare syndrome is important for any perioperative or intensive medical treatment concerning therapy and progression of the disease, we report a comprehensive study on a 32-year old woman with this rare multifaceted disorder who had to undergo general anaesthesia. For the first time amino acid status in plasma, urine, cerebrospinal fluid and especially polymorphonuclear leucocytes, which in the investigation showed to be valuable tool for evaluating amino acid metabolism in nucleated cells in HHH-syndrome, and further important pathophysiologic indicators of cellular and metabolic function have been conscientiously investigated and compared. The pathophysiological repercussions of our results as well as the recommendations for conscientious therapeutical management are discussed. Received September 9, 1999 Accepted July 1, 2000     

高剂量生长抑素、奥美拉唑联合止血芳酸治疗急性上消化道出血合并凝血功能障碍患者的临床效果

目的:探讨高剂量生长抑素、奥美拉唑联合止血芳酸治疗急性上消化道出血合并凝血功能障碍患者的临床效果及安全性。方法:选择我院2014年1月~2017年12月收治的92例急性上消化出血合并凝血功能障碍的患者,并按随机数表法将其分为对照组和研究组。对照组予以常规剂量生长抑素、奥美拉唑联合止血芳酸治疗,研究组予以高剂量生长抑素治疗,其余奥美拉唑及止血芳酸用法同对照组。治疗后,比较两组的临床疗效、止血情况、住院时间,治疗前后血常规指标、凝血功能的变化及并发症的发生情况。结果:治疗后,研究组总有效率明显高于对照组[91.30%vs.74.42%](P0.05),而平均止血时间、再止血率及住院时间均明显短于对照组(P0.05);两组白细胞计数(WBC)、部分活化凝血酶原时间(APTT)及凝血酶原时间(PT)均较治疗前明显下降,血红蛋白(Hb)、红细胞计数(RBC)、红细胞压积(Hct)及血小板计数(PLT)均较治疗前明显上升,且研究组以上指标变化较对照组更明显(P0.05)。两组并发症的发生率比较差异均无统计学意义(P0.05)。结论:高剂量生长抑素、奥美拉唑联合止血芳酸治疗急性上消化道出血合并凝血功能障碍的效果明显优于常规剂量生长抑素、奥美拉唑联合止血芳酸治疗,其能够更有效缩短止血时间,避免再出血,且未增加药物不良反应,安全性高。     

A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonah_m) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonah_m reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.     

凝血与纤溶指标与颅脑损伤后急性创伤性凝血病的关系及对脑心综合征的预测效能研究

摘要 目的：分析凝血与纤溶指标与颅脑损伤（TBI）后急性创伤性凝血病（ATC）的关系及对脑心综合征（CCS）的预测效能。方法：选取2020年1月～2022年12月本院收治的80例TBI患者作为研究对象,分别检测并计算非ATC患者与ATC患者、非CCS患者与CCS患者的凝血酶原时间（PT）、活化部分凝血酶原时间（APTT）、D-二聚体（D-D）、凝血酶-抗凝血酶复合物（TAT）、纤溶酶-α2纤溶酶抑制物复合物（PIC）水平和国际标准化比值（INR）、TAT/PIC比值;并采用双变量Spearman相关性检验凝血与纤溶指标的相关性,建立多因素Logistic模型分析TBI后ATC和TBI合并CCS的影响因素,同时分析凝血与纤溶指标对TBI合并CCS的预测效能。结果：80例TBI患者的ATC发生率为27.50%;与非ATC组比较,ATC组PT、APTT、D-D水平较高,TAT/PIC比值较低（P＜0.05）。PT、APTT、D-D与颅脑损伤后ATC呈正相关性,TAT/PIC与ATC呈负相关性。多因素logistic分析结果显示,TAT/PIC是颅脑损伤后ATC的独立危险因素（P＜0.05）。80例TBI患者的CCS发生率为20.00%;与非CCS组比较,CCS组PT、APTT、D-D水平较高,TAT/PIC比值较低（P＜0.05）。多因素logistic分析结果显示,PT、APTT、D-D、TAT/PIC是TBI合并CCS的独立危险因素（P＜0.05）。TBI合并CCS预测中,PT、APTT、D-D的ACU均＞0.70,TAT/PIC＞0.85。结论：TAT/PIC与TBI后ATC存在一定关联,同时还能预测CCS的发生,在TBI患者预后预测方面具有指导意义。     

凝血因子药物市场分析

凝血因子是一类特殊的药物,是血友病等血液疾病的治疗药物,目前已经成为血液制品的重要组成部分。国外已经有二十多种重组凝血因子药物上市,2015年全球重组凝血因子药物的市场规模已经达到78.54亿美元,未来还将持续增长,Baxalta公司的重组凝血因子产品销售额位居全球首位,达到28.40亿美元。国外有多种重组凝血因子处于研发阶段,其中长效重组凝血因子将成为新的市场增长点。国内各类凝血因子药物的批签发状况良好,且随着国家发展和改革委员会取消血液制品最高零售价,各类产品价格均有不同幅度增长,其中人纤原蛋白原增长幅度最高,达到189%。国产凝血因子市场空间巨大,但存在产品供给和研发力度不足等问题,发展受到限制,必须改革行业管理制度、提高血浆分离技术、加强重组产品研发。     

连续股神经阻滞与静脉自控镇痛对全膝关节置换术后患者凝血功能的影响

目的:比较连续股神经阻滞(CFNB)与静脉自控镇痛(PCIA)在全膝关节置换术中的应用效果及对患者凝血功能的影响。方法:选取2014年1月至2015年12月间我院行单侧全膝关节置换术的患者80例,按照随机数字表法分为CFNB组和PCIA组,每组各40例,两组患者分别接受CFNB和PCIA治疗。观察两组患者术后6 h、12 h、24 h、48 h视觉模拟疼痛评分(VAS),两组患者分别于麻醉前(T1)、术毕(T2)、术后1 d(T3)、术后2 d(T4)进行血栓弹力图检查,观察两组凝血功能变化。并于术后随访1年,比较两组患者膝关节功能。结果:术后6 h、12 h、24 h、48 h CFNB组患者VAS评分显著低于PCIA组患者(P0.05)。T2、T3、T4时点CFNB组患者凝血反应时间(R)、血凝块形成时间(K)较T1升高,血凝块聚合形成速率(α角)、血凝块最大振幅(MA)较T1降低,PCIA组患者R、K较T1降低,α角、MA较T1升高,T2、T3、T4时点CFNB组患者R、K高于PCIA组患者,α角、MA低于PCIA组患者,差异均有统计学意义(P0.05)。两组患者术后均完成1年的随访,两组患者KSS评分、膝关节最大屈曲度、膝关节最大伸直度比较无统计学差异(P0.05)。结论:CFNB对于全膝关节置换术术后患者镇痛效果优于PCIA,有利于改善患者凝血功能,不影响术后患者膝关节功能的恢复。     

氟比洛芬酯复合小剂量芬太尼对腹腔镜胆囊切除术患者镇痛效果及凝血功能的影响

目的:探讨氟比洛芬酯复合小剂量芬太尼在腹腔镜胆囊切除术后静脉自控镇痛中的应用及对患者凝血功能的影响。方法:选择2015年11月~2016年11月于我院行腹腔镜胆囊切除术的患者102例,随机分为对照组和研究组,每组51例。对照组患者术后采用小剂量芬太尼静脉自控镇痛,研究组患者术后采用氟比洛芬酯复合小剂量芬太尼静脉自控镇痛。观察并比较两组患者手术前后血清纤维蛋白原(Fg),活化部分凝血酶原时间(APTT),凝血酶原时间(PT),血小板计数(PLT),P物质,5-烃色胺(5-HT),白细胞介素-6、8(IL-6、IL-8)水平以及术后并发症的发生情况。结果:术前,比较两组Fg、APTT、PT、PLT、P物质、5-HT、IL-6、IL-8无差异(P0.05);术后,两组Fg、APTT、PT、PLT、P物质、5-HT、IL-6、IL-8均较术前上升,研究组低于对照组,差异均有统计学意义(P0.05)。研究组术后并发症率低于对照组(P0.05)。结论:氟比洛芬酯复合小剂量芬太尼能够提高腹腔镜胆囊切除术患者静脉自控镇痛的效果,改善凝血功能,降低炎症因子水平。