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 ...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...展开更多
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). Howeve...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.展开更多
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 determine...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.展开更多
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 pcDN...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.展开更多
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...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展开更多
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 ...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.展开更多
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 35...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.展开更多
基金supported by grants from National Basic Research Program of China(973 Program,No.2007CB935803)National Natural Sciences Foundation of China(No.30825018)
文摘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...
文摘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.
文摘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.
基金Supported by the grants from Academician Foundation of Chongqing (2004BC5006)
文摘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.
文摘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
基金TheworkwassupportedbytheNationalNaturalScienceFoundationofChina (No 3 983 0 180 )andClydeWuFoundationofShanghaiInstituteofHematology
文摘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.
基金ThisprojectwassupportedbyagrantfromtheKeyProjectoftheZhejiangProvinceScienceandTechnologyCommission (No 0 2 110 3 166)
文摘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.
