Binding of EGF1 Domain Peptide in Coagulation Factor Ⅶ with Tissue Factor and Its Implications for the Triggering of Coagulation

The binding function of EGF1 domain peptide with tissue factor(TF)and its ability of triggering coagulation were explored.The TF expression model in vitro was established by lipopolysaccha-ride induction.The affinity of EGFP-EGF1 and TF expressing cells was analyzed by fluorescence microscopy and flow cytometry(FCM).The affinity of EGFP-EGF1 and rat soluble TF was quantitated by surface plasmon resonance(SPR).The ability of EGFP-EGF1 in triggering coagulation was tested by prothrombin time assay.The FCM res...

High-yield expression of recombinant mouse coagulation factor Ⅶ in methylotrophic yeast Pichia pastoris

Objective: To explore high-yield secretory expression of recombinant mouse coagulation factor Ⅶ (rmF Ⅶ ) protein in Pichia pastoris (P. pastoris). Methods: The fragment of mF Ⅶ cDNA was amplified by PCR from a pcDNA3-mFⅦ plasmid. Then the cDNA fragment was subcloned into α-factor secretion signal open reading frame of pPIC9K secretory expression vector. The mutagenesis of mF Ⅶ was performed by Site-Direct Mutation and then verified by DNA sequencing. The yeast expression vector of rmF Ⅶ, named as pPIC9K-rmFⅦ, was linearized with Sac I and transferred into GS115 strains(his-Mut+)by electroporation. The recombinants were identified by direct PCR and selection on MM and MD plates. rmF Ⅶ was expressed in recombinant strains (his+Mut+) for 4 d. The expression level and activation of rmF Ⅶ in the BMMY medium were detected by SDS-PAGE and Western blot respectively. Results:pPIC9K-rmFⅦ was constructed and transferred to GS115 strains successfully. 48-hour post induction by methanol rmFⅦ protein was secreted into the culture supernatant. The molecular weight of the expressed products was shown to be about 46 kD by SDS-PAGE analysis. Western blot showed that the expressed rmF Ⅶ exhibited specificity and antigenicity. Conclusion: Since mFⅦ is considered as a tumor-targeting molecule , this study may provide a basis for further anti-tumor strategy on rmFⅦ.

Role of Coagulation Factor Ⅶ in Pathogenesis of Ischemic Heart Disease

To study the variation and significance of plasma coagulation factor Ⅶ （FⅦ） in different kinds of ischemia heart disease （IHD） and examine its relation with plasma lipid and gene polymorphism. FⅦa was determined with one stage clotting assay by using a recombinant soluble tissue factor （rsTF）. FⅦc was measured with one stage clotting assay. FⅦag was quantified with an enzyme-linked immunosorbent assay （ELISA）. Polymorphism was analyzed with PCR-urea-polyacrylamide gel electrophoresis. Our results showed that FⅦa in stable angina （SA）, unstable angina （UA）, obsolete and acute myocardial infraction （OMI, AMI） patients was higher than those of normal group with the differences being significant within any two groups. FⅦag in UA, OMI and AMI was higher than those in SA and normal groups. There were positive correlations between FⅦa and serum triglycerides, FⅦa and FⅦc, FⅦc and FⅦag. FⅦ-323 0/10 bp polymorphism analysis was performed in 60 patients and 0/10 bp polymorphism was found in 5 cases. FⅦc and FⅦag were much lower in cases of 0/10 bp groups than those in cases of 0/0 bp groups. It is concluded that there was activation of extrinsic coagulation pathway in every kind of IHD to different extent. FⅦa was the risk factor in the development of IHD, and more sensitive in reflecting the severity of cardiovacutar disease than FⅦc or FⅦag. FⅦa was higher in OMI, which may be one of the risk factors of re-infraction. Serum triglyceride may indirectly lead to the development of IHD by increasing the level of FⅦa, FⅦ-323 0/10 bp polymorphism was present in Chinese patients with IHD and it was correlated with the level of FⅦc, FⅦag in plasma. 10 bp allelomorphic gene was a protective factor against thrombogenesis.

Coagulation factor Ⅶ gene polymorphisms are not associated with the occurrence or the survival of hepatocellular carcinoma:a report of 37 cases

Objective: Coagulation factor VII(FVII) triggers the extrinsic pathway of blood coagulation. In our previous study, we showed that FVII plays an important role in tumorigenesis of hepatocellular carcinoma(HCC). However, the role of FVII polymorphism in HCC is still unknown. The present study aimed to investigate the relationship between HCC carcinogenesis and single nucleotide polymorphism of FVII.Methods: Thirty-seven HCC patients and 30 healthy donors were recruited in this study. Four common FVII gene polymorphisms– a decanucleotide insertion at position –323(–323 ins10-bp), a G to T substitution at position –401(–401 G/T), a G to A substitution at position –402(–402 G/A), and a T to C substitution at position –122(–122 T/C) – were analyzed by sequencing or commercialized assays using genomic DNA isolated from blood samples. Clinicopathological parameters between control and HCC subjects were compared according to the specific genotypes.Results: The most common nucleotide variation was –402 G/A. However, no statistically significant difference was observed between healthy controls and HCC subjects for all four polymorphisms in terms of genotype distribution and allele frequencies,indicating that these polymorphisms may not affect HCC tumorigenesis. Furthermore, no association was found between–402 G/A polymorphisms and tumor stage, recurrence, and overall survival.Conclusions: Our results indicate that FVII polymorphisms may not be a key factor that clinically impact tumorigenesis and outcomes of HCC, although further investigations should be conducted to confirm our findings.

Polymorphisms of the coagulation factor Ⅶ gene and its plasma levels in relation to acute cerebral infarction differences in allelic frequencies between Chinese Han and European populations

Background Coagulation factor Ⅶ (F Ⅶ) levels in plasma are usually related to ischemic heart disease (IHD) and cerebral infarction shares many of the risk factors related to IHD. Is there any relationship between factor Ⅶ and cerebral infarction? We investigated the relationship between F Ⅶ and acute cerebral infarction and reported genotype frequencies and allelic frequencies of FⅦ gene polymorphisms in the Chinese Han population. Methods We recruited 62 patients with acute cerebral infarction confirmed by magnetic resonance imaging (MRI) from Ruijin Hospital,and 149 age-matched patients clinically free of vascular disease to act as controls. All of them were unrelated,and were from the Chinese Han population. FⅦ coagulant activity (FⅦc) was determined using an clotting assay,activated FⅦ (FⅦa) and FⅦ Ag were assayed using enzyme immunoassay kits. The FⅦ gene polymorphisms to be detected included-401G/T,-402G/A,5’F7A1/A2,IVS7 and R353Q. 5’F7 and IVS7 were revealed by means of a PCR and direct agarose gel electrophoresis. The rest were examined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results The results showed that FⅦc,FⅦAg and FⅦa were higher in the acute cerebral infarction group than in the control group ( P <0.01, P <0.05, P <0.05,respectively). There were no significant differences in the genotype frequencies of FⅦ gene polymorphisms between the two groups. The allelic frequencies in the Chinese Han population were as follows: -401G/T (96.64/3.36), -402G/A (52.01/47.99),5’F7A1/A2(96.64/3.36),IVS7 H5/H6/H7/H8 (0.34/52.35/46.98/0.34) and R353Q (95.64/4.36). There were significant differences ( P <0.01, P <0.001, P <0.001, P <0.001, P <0.001,respectively) in these allelic frequencies between the Chinese Han and European populations. Conclusions The results indicate that increased plasma FⅦ levels may contribute to thrombosis in cerebral infarction. And there was no significant difference in genotype frequencies of these five FⅦ gene polymorphisms between the acute cerebral infarction and control groups. Moreover,these results showed that the frequencies of protective allele, including -401T,5’F7 A2 and 353Q were lower, but that -402A, which was previously found to be associated with increased plasma FⅦ levels,is higher in Chinese Han population.

Polymorphisms in the coagulation factor Ⅶ gene and the risk of myocardial infarction in patients undergoing coronary angiography

Objective To investigate whether coagulation factor Ⅶ (FⅦ) polymorphisms play a role in the pathogenesis of coronary artery disease (CAD) and/or myocardial infarction (MI) in a series of Hans.Methods The Arg 353 Gln and HVR4 polymorphisms of FⅦ gene were determined in 374 patients undergoing selective coronary angiography by PCR and restriction fragment length polymorphism assay.Results The FⅦ genotype distribution was in accordance with Hardy-Weinberg equilibrium. The frequencies of FⅦ genotypes or alleles did not show significant differences between the CAD group and the controls or between the males and the females. The frequencies of carriers of the Gln 353 allele and (Arg/Gln+Gln/Gln) genotypes were significantly higher in the CAD patients without MI than in those with MI ( P =0.031,odds ratio 0.37,95% CI: 0.15-0.94). However,HVR4 polymorphisms were not significantly different between the two groups ( P >0.05).Conclusion Carrying the F Ⅶ Gln 353 gene may be a protective factor against MI in the Chinese Hans.

Association of coagulation factor Ⅶ with the risk of myocardial infarction in the Chinese

To elucidate the association of plasma factor Ⅶ coagulant activity (FⅦc) with the risk of myocardial infarction (MI) and to assess the influence of factor Ⅶ gene MspI polymorphism and lipid metabolism on FⅦc in the Chinese Methods A total of 137 patients with angiographically confirmed MI and 125 healthy individuals were evaluated retrospectively Plasma FⅦc was measured by one stage prothrombin time, and FⅦ genotype was determined after MspI digestion of polymerase chain reaction amplified genomic DNA Serum lipid levels were assessed by routine methods Results MI patients had significantly higher levels of FⅦc (119 5%±22 7% vs 99 9%±21 8%, P <0 01) and total serum cholesterol (5 80±1 06?mmol/L vs 5 53±1 08?mmol/L, P <0 05) than controls, but only FⅦc independently correlated with the risk of MI (OR=1 04, P <0 01) There were no significant differences in FⅦ genotype or allele frequency between patients and controls ( P >0 05) Subjects with the Gln353 allele were associated with significantly lower FⅦc levels than Arg353 homozygotes (99 7%±19 3% vs 111 4%±24 6%, P <0 05) Serum triglyceride was positively correlated with plasma FⅦc in both control ( r =0 25, P <0 01) and case ( r =0 87, P <0 01) groups, but this correlation was restricted to Arg/Arg genotype ( r =0 68, P <0 01) A significant correlation of total serum cholesterol with FⅦc only appeared in Arg/Arg homozygotes ( r =0 17, P <0 01) Conclusions Our findings support the role of plasma FⅦc as a risk factor for MI in Chinese Plasma triglyceride and FⅦ gene MspI polymorphism are two independent determinants of FⅦc Assay of this polymorphism will be helpful in determining who will benefit most from lipid lowing therapy

Expression and Purification of Human Coagulation Factor X in Mammalian CHO-DG44 Cells

[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.

Extrahepatic synthesis of coagulation factor Ⅶ by colorectal cancer cells promotes tumor invasion and metastasis

Background Blood coagulation factor Ⅶ （FⅦ） is physiologically synthesized in the liver and released into the blood. Binding of FⅦ to tissue factor （TF） is related to the metastatic potential of tumor cells, also a significant risk factor in the development of hepatic metastasis in patients with colorectal cancer （CRC）. It has been found that some cancer cells can produce FⅦ extrahepatically. However, litte is known about FⅦ and CRC. We therefore hypothesized that CRC cells may synthese FⅦ, leading to tumor invasion and metastasis.Methods We detected the expression of FⅦ protein in 55 CRC specimens by immunohistochemical staining. The FⅦ mRNA in 45 of 55 CRC cases, 6 colon cancer cell lines and one hepatoma cell line was measured by real-time reverse transcription-PCR （RT-PCR）. Transwell invasion assays were performed to evaluate the changes of cell migration and invasion of LoVo cancer cells in vitro. We further observed the likely effectors regulated by the TF/FⅦa complex Western blotting assay.Results Extrahepatic synthesis of FⅦ was detected in the cytoplasm of 32 （58.2%） CRC specimens byimmunohistochemistry, but not in normal mucosa. Liver metastasis （P=0.003） and TNM staging （P=0.005） were significantly correlated with FⅦ antigen expression. The positive ratios in stages Ⅰ, Ⅱ, Ⅲ and Ⅳ were 33.3%, 40.0%,52.4% and 87.5%, respectively. The expression of FⅦ mRNA in CRC with hepatic metastasis was significantly higher than CRC without hepatic metastasis （5.33±2.88 vs. 1.47±0.51, P=0.03）. Ectopic FⅦa induced a slight increase （1.34-fold） in the number of migrating cells, which was inhibited by the specific TF antibody. The formation of TF/FⅦacomplex resulted in a marked increase in the expression of matrix metalloproteinases （MMP）-2 （3.5-fold） and MMP-9（4.7-fold） in a time-dependent and dose-dependent manner.Conclusions Extrahepatic synthesis of FⅦ by CRC cells may promote tumor invasion and metastasis. MMPs, as downstream effectors of TF/FⅦa signaling, facilitate the development of metastasis in colon cancer.

Polymorphisms in the genes for coagulation factor Ⅱ,Ⅴ,Ⅶ in patients undergoing coronary angiography

Objective: To determine whether polymorphisms in the genes for coagulation factor Ⅱ,Ⅴ,Ⅶ could predispose an individual to increase risk for coronary artery disease (CAD) and/or myocardial infarction (MI) in Chinese. Methods: We screened coagulation factor Ⅱ( G20210A),Ⅴ( G1691A),Ⅶ( R353Q and HVR4) genotype in 374 patients undergoing coronary angiography by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay. Results: The R353Q and HVR4 genotype of the factor Ⅶ distribution was in accordance with Hardy-Weinberg equilibrium. The frequencies of FVⅡ genotype or allele did not show statistically significant differences between CAD group and controls or between male and female.The frequencies of the Q allele and ( RQ + QQ) genotype were significantly higher among the CAD patients without myocardial infarction (MI) history than among those with MI history (P < 0.05). However, HVR4 polymorphism was not significantly different within groups. We only find one normal control of factorⅡ(G20210A) mutation. No coagulation factor Ⅴ(G1691A) mutation was found in the CAD patients and con-trois. Conclusion: The factor Ⅱ(G20210A),Ⅴ(G1691A) mutation is absent and may not be a major genetic factor for CAD and/or MI; the Q allele of the R353Q polymorphism of the factor Ⅶ gene may be a protective genetic factor against myocardial infarction in Chinese.

Diagnostic and Prognostic Value of Plasma Factor V Activity and Parameters in Thrombin Generation for Disseminated Intravascular Coagulation in Patients with Hematological Malignancies

In this study,we used plasma factor V activity and parameters of the thrombin generation test to discuss their diagnostic and prognostic value for disseminated intravascular coagulation (DIC) in patients with hematological malignancies.A total of 164 patients who were diagnosed with hematological malignancies in the Department of Hematology,Union Hospital,between Apr 2014 and Dec.2014 were enrolled in this study.There were 131 patients in the study group and 33 patients in the control group in terms of the laboratory results for DIC.The patients in the study group were divided into a DIC subgroup (n=59) and a non-DIC subgroup (n=72) based on the International Society of Thrombosis and Hemostasis (ISTH) Integral System,and they were divided into four subgroups [score ≤3 (n=35),score=4 (n=37),score=5 (n=47),and score >6 (n=12)] according to ISTH scores.Using 28-day mortality as the endpoint,the patients in the study group were divided into a survival subgroup (n=111) and a non-survival subgroup (n=20).The results showed that the plasma factor V activity was significantly weaker,and lag time and time to peak were significantly shorter in the study group than in the control group (P<0.01).The factor V activity,peak and endogenous thrombin potential (ETP) were significantly decreased in the DIC subgroup as compared with those in the non-DIC subgroup (P<0.01).Among factor V activity,lag time,peak,ETP,and ttPeak,only the factor V activity was significantly decreased in the nonsurvival subgroup compared with the survival subgroup (P<0.01).With the increase in ISTH score,the ETP and peak decreased gradually.The binary logistic regression analysis revealed that PLT,D-dimer,factor V activity and ETP had linear relationship with DIC diagnosed by ISTH Integral System.Using DIC diagnosed by ISTH Integral System as the endpoint,the area under curve (AUC) of factor V activity was found to be similar to that of blood platelet count (PLT) and prothrombin time (PT).In conclusion,factor V activity,ETP and peak had diagnostic value for DIC in patients with hematological malignancies,and only factor V activity had limited prognostic value.

Preparation and Structure of a New Coagulation Factor XI Catalytic Domain for Drug Discovery

Human blood coagulation factor XI （FXI） is a key enzyme in the amplification phase of blood coagulation cascade, and is recognized as an important target for anti-coagulant development in recent years. We designed a new mutant form of FXIa catalytic domain rhFXI370-607 （N73Q-N113Q-C123S）, and report here the facile preparation, protein crystallization, and crystal structure of this protein. We highlight a few unique structural features of FXIa after comparison with the trypsin family serine proteases at sequence and structural levels. This work provides a foundation to develop new small molecular FXIa inhibitors with increased potency and specificity.

Clinical association between coagulation indicators and bone metastasis in patients with gastric cancer

BACKGROUND Bones are one of the most common target organs for cancer metastasis.Early evaluation of bone metastasis(BM)status is clinically significant.Cancer patients often experience a hypercoagulable state.AIM To evaluate the correlation between coagulation indicators and the burden of BM in gastric cancer(GC).METHODS We conducted a single-center retrospective study and enrolled 454 patients.Clinical information including routine blood examination and coagulation markers were collected before any treatment.Patients were grouped according to the status of BM.Receiver operating characteristic curves were used to assess diagnostic performance and determine the optimal cutoff values of the above indicators.Cutoff values,sensitivity and specificity were based on the maximum Youden index.Univariate and multivariate logistic regression analyses were used to evaluate the relationships between biomarkers and BM.RESULTS Of the 454 enrolled patients,191 patients were diagnosed with BM.The receiver operating characteristic curve analysis suggested that prothrombin time(PT)Wang X et al.Coagulation indicators predict bone metastasis WJGO https://www.wjgnet.com 1254 July 15,2023 Volume 15 Issue 7[cutoff:13.25;sensitivity:0.651;specificity:0.709;area under receiver operating characteristic curve(AUC)=0.738],activated partial thromboplastin time(aPTT)(cutoff:35.15;sensitivity:0.640;specificity:0.640;AUC=0.678)and fibrin degradation products(FDP)(cutoff:2.75;sensitivity:0.668;specificity:0.801;AUC=0.768)act as novel predictors for BM.Based on multivariate logistic regression analysis,the results showed the independent correlation between PT[odds ratio(OR):3.16;95%confidence interval(CI):1.612-6.194;P=0.001],aPTT(OR:2.234;95%CI:1.157-4.313;P=0.017)and FDP(OR:3.17;95%CI:1.637-6.139;P=0.001)and BM in patients with GC.Moreover,age,carcinoembryonic antigen,erythrocyte and globulin were found to be significantly associated with BM.CONCLUSION Coagulation markers,namely PT,aPTT and FDP,might be potential predictors for screening BM in patients with GC.

Expression of tissue factor in pancreatic adenocarcinoma is associated with activation of coagulation

瞄准：在 thromboembolism 的开发在胰腺的癌症和它的角色学习织物因素(TF ) 的表示。方法：TF 表示被北污点和间接免疫荧光在八根人的胰腺的癌房间线学习。或者拼接的 TF (asTF ) 的表示被 RT-PCR 估计。另外， TF 表示被免疫荧光与胰腺的腺癌(PCa ) 在 19 个病人的胰腺的纸巾决定，有慢性胰炎(CP ) 的 9 个病人和 20 正常控制。血浆样品(30 PCa 病人， 13 CP 病人和 20 控制) 为可溶的 TF 层次和凝结激活标记被调查[thrombin-antithrombin III 建筑群(梭织) ，凝血素碎片 1 + 2 (F1 + 2 )] 。结果：所有胰腺的癌房间线表示了 TF (8/8 ) ，他们中的大多数表示了 asTF (6/8 ) 。在蛋白质水平的 TF 表示没与癌房间线的区别相关。几乎，二件胰腺的癌症组织样品染色了为 TF (17/19 ) 积极。在 CP 的所有样品，弱染色被限制为胰腺的管房间，而一些仅仅内皮下房间在正常控制的 9/20 是积极的。TF 并且在 PCa 病人梭织层次而 2 铺平的提高的 F1 + 没与控制相比到达统计意义，显著地与控制相比被提高。在病人们梭织的 CP 和 F1 + ， 2 个层次证明了显著地与控制相比被提高，尽管梭织举起少些比在 PCa 病人被读。结论：我们结束那除了起来房间膜上的 TF 的调整表示，可溶的 TF 可能在胰腺的癌症贡献凝结系统的激活。

In silico evidence of Remdesivir action in blood coagulation cascade modulation in COVID-19 treatment

BACKGROUND Coronavirus disease 2019(COVID-19)has demonstrated several clinical manifestations which include not only respiratory system issues but also liver,kidney,and other organ injuries.One of these abnormalities is coagulopathies,including thrombosis and disseminated intravascular coagulation.Because of this,the administration of low molecular weight heparin is required for patients that need to be hospitalized.In addition,Remdesivir is an antiviral that was used against Middle East Acute Respiratory Syndrome,Ebola,Acute Respiratory Syndrome,and other diseases,showing satisfactory results on recovery.Besides,there is evidence suggesting that this medication can provide a better prognosis for patients with COVID-19.AIM To investigate in silico the interaction between Remdesivir and clotting factors,pursuing a possibility of using it as medicine.METHODS In this in silico study,the 3D structures of angiotensin-converting enzyme 2(ACE2),Factor I(fibrinogen),Factor II(prothrombin),Factor III(thromboplastin),Factor V(proaccelerin),Factor VII(proconvertin),Factor VIII(antihemophilic factor A),Factor IX(antihemophilic factor B),Factor X(Stuart-Prower factor),and Factor XI(precursor of thromboplastin(these structures are technically called receptors)were selected from the Protein Data Bank.The structures of the antivirals Remdesivir and Osetalmivir(these structures are called ligands)were selected from the PubChem database,while the structure of Atazanavir was selected from the ZINC database.The software AutoDock Tools(ADT)was used to prepare the receptors for molecular docking.Ions,peptides,water molecules,and other ones were removed from each ligand,and then,hydrogen atoms were added to the structures.The grid box was delimited and calculated using the same software ADT.A physiological environment with pH 7.4 is needed to make the ligands interact with the receptors,and still the software Marvin sketch®(ChemAxon®)was used to forecast the protonation state.To perform molecular docking,ADT and Vina software was connected.Using PyMol®software and Discovery studio®software from BIOVIA,it was possible to analyze the amino acid residues from receptors that were involved in the interactions with the ligands.Ligand tortions,atoms that participated in the interactions,and the type,strength,and duration of the interactions were also analyzed using those software.RESULTS Molecular docking analysis showed that Remdesivir and ACE2 had an affinity energy of-8.8 kcal/moL,forming a complex with eight hydrogen bonds involving seven atoms of Remdesivir and five amino acid residues of ACE2.Remdesivir and prothrombin had an interaction with six hydrogen bonds involving atoms of the drug and five amino acid residues of the clotting factor.Similar to that,Remdesivir and thromboplastin presented interactions via seven hydrogen bonds involving five atoms of the drug and four residues of the clotting factor.While Remdesivir and Factor V established a complex with seven hydrogen bonds between six antiviral atoms and six amino acid residues from the factor,and Factor VII connected with the drug by four hydrogen bonds,which involved three atoms of the drug and three residues of amino acids of the factor.The complex between Remdesivir and Factor IX formed an interaction via 11 hydrophilic bonds with seven atoms of the drug and seven residues of the clotting factor,plus one electrostatic bond and three hydrophobic interactions.Factor X and Remdesivir had an affinity energy of-9.6 kcal/moL,and the complex presented 10 hydrogen bonds and 14 different hydrophobic interactions which involved nine atoms of the drug and 16 amino acid residues of the clotting factor.The interaction between Remdesivir and Factor XI formed five hydrogen bonds involving five amino acid residues of the clotting factor and five of the antiviral atoms.CONCLUSION Because of the in silico significant affinity,Remdesivir possibly could act in the severe acute respiratory syndrome coronavirus 2 infection blockade by interacting with ACE2 and concomitantly act in the modulation of the coagulation cascade preventing the hypercoagulable state.

Effects of Intravenous Thrombolytic Therapy with Alteplase on Neurological Function,Coagulation Function and Serum Inflammatory Factors in Patients with Acute Cerebral Infarction

Objective:To investigate the effects of intravenous thrombolysis therapy with alteplase on neurological function,coagulation function and serum inflammatory factors in patients with acute cerebral infarction.Methods:A total of 96 patients with acute cerebral infarction admitted to our hospital from September 2017 to October 2019 were randomly divided into two groups,with 48 patients in each group.The control group(n=48)received routine treatment,and the observation group received intravenous thrombolysis therapy with alteplase on the basis of routine treatment.The neurological deficit score,prothrombin time(PT),activated partial thromboplastin time(APTT),tumor necrosis factor-a level(TNF-α),and high-sensitivity C-reactive protein(hs-CRP)were compared between the two groups after 15 days of treatment.Results:After treatment,NIHSS scores in both groups were lower than those before treatment;PT levels were increased,while APTT,TNF-αand hs-CRP levels were all decreased in both groups,and the changes in the observation group were greater than those in the control group,with statistically significant difference(P<0.05).Conclusions:Intravenous thrombolysis therapy with alteplase can improve the neurological function,coagulation function and serum levels of inflammatory factors in patients with acute cerebral infarction,which is worthy of clinical application.

Effects on coagulation factor production following primary hepatomitogen-induced direct hyperplasia

AIM:To investigate the molecular mechanisms involved in coagulation factor expression and/or function during direct hyperplasia(DH) -mediated liver regeneration.METHODS:Direct hyperplasia-mediated liver regeneration was induced in female C57BL/6 mice by administering 1,4-bis[2-(3,5-dichloropyridyloxy) ] benzene(TCPOBOP) ,a representative hepatomitogen.Mice were weighed and sacrificed at various time points [Day 0(D0:prior to injection) ,3 h,D1,D2,D3,and D10] after TCPOBOP administration to obtain liver and blood samples.Using the RNA samples extracted from the liver,a comprehensive analysis was performed on the hepatic gene expression profiling of coagulation-related factors by real-time RT-PCR(fibrinogen,prothrombin,factors Ⅴ,Ⅶ,Ⅷ,Ⅸ,Ⅹ,Ⅺ,Ⅻ,XⅢβ,plasminogen,antithrombin,protein C,protein S,ADAMTS13,and VWF) .The corresponding plasma levels of coagulation factors(fibrinogen,prothrombin,factors Ⅴ,Ⅶ,Ⅷ,Ⅸ,Ⅹ,Ⅺ,Ⅻ,XⅢ,and VWF) were also analyzed and compared with their mRNA levels.RESULTS:Gavage administration of TCPOBOP(3 mg/kg body weight) resulted in a marked and gradual increase in the weight of the mouse livers relative to the total body weight to 220% by D10 relative to the D0(control) ratios.At the peak of liver regeneration(D1 and D2) ,the gene expression levels for most of the coagulationrelated factors(fibrinogen,prothrombin,factors Ⅴ,Ⅶ,Ⅷ,Ⅸ,Ⅺ,Ⅻ,XⅢβ,plasminogen,antithrombin,protein C,ADAMTS13,VWF) were found to be downregulated in a time-dependent manner,and gradually recovered by D10 to the basal levels.Only mRNA levels of factor Ⅹ and protein S failed to show any decrease during the regenerative phase.As for the plasma levels,5 clotting factors(prothrombin,factors Ⅷ,Ⅸ,Ⅺ,and Ⅻ) demonstrated a significant decrease(P < 0.05) during the regeneration phase compared with D0.Among these 5 factors,factor Ⅸ and factor Ⅺ showed the most dramatic decline in their activities by about 50% at D2 compared to the basal levels,and these reductions in plasma activity for both factors were consistent with our RT-PCR findings.In contrast,the plasma activities of the other coagulation factors(fibrinogen,factors Ⅴ,Ⅶ,XⅢ,and VWF) were not significantly reduced,despite the reduction in the liver mRNA levels.Unlike the other factors,FX showed a temporal increase in its plasma activity,with significant increases(P < 0.05) detected at D1.CONCLUSION:Investigating the coagulation cascade protein profiles during liver regeneration by DH may help to better understand the basic biology of the liver under normal and pathological conditions.

Research on variation and significance of immune cells, inflammatory factors and coagulation functions in patients with pulmonary tuberculosis

Objective:To investigate variation of T lymphocyte subsets, inflammatory factors and coagulation functional indexes at different stages for patients with pulmonary tuberculosis and significance of discussion and treatment on mechanism of pulmonary tuberculosis. Methods:48 cases of patients with pulmonary tuberculosis at progressive stage treated in our hospital were selected as the progression group, and 50 cases of patients with pulmonary tuberculosis at remission stage were selected as the remission group. Meanwhile, 48 cases of healthy population in our hospital were selected as the control group. Variations and significances of T lymphocyte subsets (CD3+, CD4+, CD8+ and CD4+/CD8+), inflammatory factors [interleukin-2 (IL-2), interleukin-10 (IL-10), interferon-γ (IFN-γ) and tumor necrosis factor-α(TNF-α)] and coagulation function [Fg (fibrinogen), TT (thrombin time), PLT (platelet) and D-D (D-dimer)] were analyzed.Results: Coagulation function (Fg, TT, PLT and D-D), T lymphocyte subsets CD8+ and inflammatory factors (IL-2, IL-10 and TNF-α) in patients with pulmonary tuberculosis in progression group were significantly higher than in healthy population of control group (P<0.05). T lymphocyte subsets (CD3+, CD4+ andCD4+/CD8+) and IFN-γ were significantly lower than in healthy population of control group (P<0.05). Coagulation function (Fg, TT, PLT and D-D), T lymphocyte subsets CD8+ and inflammatory factors (IL-2, IL-10 and TNF-α) in patients with pulmonary tuberculosis in remission group were significantly lower than in patients of progression group (P<0.05), but significantly higher than in healthy population of control group (P<0.05). T lymphocyte subsets (CD3+, CD4+ and CD4+/CD8+) and IFN-γ in patients with pulmonary tuberculosis in remission group were significantly higher than in patients of progression group (P<0.05), but significantly lower than in healthy population of control group (P<0.05).Conclusions:Significant variations appeared on T lymphocyte subsets, inflammatory factors and coagulation functional indexes at different stages for patients with pulmonary tuberculosis, which had important significance on discussion and treatment of pulmonary tuberculosis mechanism.

Change of Coagulation Factor Ⅷ and Antithrombin Ⅲ Activity in Bank-Stored Blood

Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd day, and 70 % of the activity at the 7th day. FⅧ:c showed no obvious change after 24 h, until the 3rd day. It lost 40 %-60 % of the activity after 36 h and was reduced to the 30 % of the original activity at the 5th day. Our results suggested that at the 3rd day coagulation factor Ⅷ of bank stored blood can be used to replenish antithrombin Ⅲ, while bank stored blood in one day can be used to replenish FⅧ.

Maltose-binding Protein Improving the Crystallizability of C2 Domain of Human Coagulation Factor V

Human coagulation Factor V（FV）, together with Factor Xa, assembles to prothrombinase complex on activated cell surface, which converts prothrombin into thrombin, leading to fibrin deposition. The C2 domain of FV is believed to be a primary anchor for the assembly of pro- thrombinase on the cell surface, and was proposed as a target to intervene with pathological thrombotic events. We report here the crystal structure of the C2 domain of FV fused to maltose-binding protein（MBP）. The fusion tag of MBP is critical to generate the crystal for this study. There is no strong interaction between MBP and FVC2. The overall structure of FVC2 is similar to the previous FVC2 structures, suggesting the MBP fusion does not perturb the molecular structure of FVC2. This crystal form of FVC2 can be used for future study of molecular interaction between FVC2 and its inhibitors.