Factor Xa Promotes Differentiation of Meg-01 Cell Line / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect and mechanism of Factor Xa on the differentiation of Meg-01 cells into platelet-like particles.</p><p><b>METHODS</b>The Meg-01 cells were used as experimental object, Factor Xa was used as agonist. Cell proliferation was detected by CCK-8 assay. The viability of platelet-like particles was analyzed by AlamaBlue kit. MAPK/ERK pathway and PI3K/AKT pathway were assayed by Western blot. The expression of CD41b was analyzed by Western blot and flow cytometry. Cell cycle and apoptosis were detected by flow cytometry.</p><p><b>RESULTS</b>The Factor Xa (1 µg/ml) inhibited cell viability, induced apoptosis. Factor Xa triggered cell arrest at the G(2)/M stage and down-regulated the expression of SKP2. After Meg-01 cells were stimulated by Factor Xa, the expression of CD41b was up-regulated and the MAPK/ERK pathway and PI3K/AKT pathway were activated. The platelets-like particles stimulated by FXa activation were viable.</p><p><b>CONCLUSION</b>The Factor Xa maybe display some effect on the differentiation of megakaryocytes into platelets.</p>

Evaluation of clinical efficacy of BAY 38-4766 for cytomegalovirus rentinitis / 中华实验眼科杂志

Background At present,researches about retinitis and acquired immunodeficiency syndrome (AIDS) mainly focus on observation and analysis of the cytomegalovirus (CMV)-induced ocular complication.To screen the effective drugs is very important for the treatment of AIDS-related CMV-induced retinitis.Objective This study was to describe the clinical features,diagnosis and treatment outcome of CMV-induced retinitis with BAY 38-4766 and evaluate the relationship between CMV retinitis and AIDS.Methods This was a case observational study.A total of 154 eyes of 84 patients with CMV retinitis and AIDS were enrolled in this study.Before the definitive diagnosis of CMV retinitis,the AIDS course of these patients were 4-26 months.In the initial examination,the visual acuity ranged from finger counting to 0.4,and the number of CD4+ T-lymphocyte was 0-30 cells/μl.The survival time in the patients ranged from 3 weeks to 18 months.BAY 38-4766 was used in 117 eyes of 62 patients,and 102 eyes of 53 patients showed the srinked of retinal lesion and improvement of vision (0.1-0.7).BAY 38-4766 was used to treat the CMV retinitis in 117 eyes of 62 patients with the initial intravenous dose of 5 mg/kg,twice per day for consecutive 2 weeks and followed by oral dose 1 gram per day.The follow-up duration was 2 weeks to 18 months.The fundus feature,visual acuity and CD4+ T-lymphocyte counts were analyzed.This study proposal was approved by Ethic Committee of the 474th Hospital of PLA,and written informed consent was obtained from each patient prior to entering this study.Results The numbers of CD4+T-lymphocytes increased to 12-402 eells/μl after administration of BAY 38-4766.The CMV retinitis aggravated and the vision decreased in the untreated 22 patients with the CD4+T-lymphocytes 0-30 cells/μl.Conclusions CMV retinitis is the most common intraocular complication in patients with AIDS.Diagnosis of CMV retinitis is based on the characteristics of necrotizing retinitis,which is typically associated with retinal hemorrhage and vasculitis.BAY 38-4766 is an effective drug for the treatment of CMV retinitis.

Research progress in hirudin fusion protein--review / 中国实验血液学杂志

Natural hirudin extracted from the secretion of medical leech salivary gland is a single-chain peptide containing 65 aminoacid residues with molecular weight of 7000 D, and exists in three isomers of HV1, HV2 and HV3. Hirudin possesses three disulfide bridges forming the structure of core cyclic peptides, which binds to the catalytic site of thrombin so as to inhibit the catalysis of thrombin. Its c-terminus rich in acidic aminoacid residues possesses hydrophilicity, and is free on the molecular surface, and can bind with fibrin recognition site of hirudin. The minimal segment of 12 - 16 C-terminal acidic residues keeps the minimal activity of anti-thrombosis. Thus, hirudin, as a potent and specific inhibitor of thrombin, can be used to protect from and to treat clinically thrombosis. As it has some disadvantages such as short half-life, bleeding side-effect and mono-function, and so on, hirudin has been fused with some other functional proteins in recent years. The obtained fusion proteins can prolong the half life of hirudin, or relieve it bleeding side effect, or bring new functions, such as thrombolysis, inhibiting the platelet aggregation, targeting specifically. The research progress in hirudin fusion protein was summarized in this review.

Purification of Recombinant Fusion Protein Staphylokinase-Hirudin Expressed by Escherichia coli and Analysis of its Dimer / 中国生物工程杂志

The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from the high density-fermented engineered E.coli by means of ion-exchange chromatography (IEC) and gel filtration (GF). The purity of rSFH reached to more than 98% determined by RP-HPLC and SDS-PAGE, and the yield was up to 0.7g per liter of fermentation broth. The analysis of homologous dimmer of rSFH appeared during the purification and calculation of the surface hydrophobic area had been carried out by means of hydrophobic chromatography and MALD-TOF. The influence of sodium chloride and temperature on the behavior of rSFH reversible dimerization was analyzed by high performance sized- exclusive chromatography(HPSEC). It is concluded that the hydrophobic interaction played an important role in the reversible dimerization of rSFH.

Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris / 生物工程学报

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.