Role of L-Arginine on the Expression of Coagulation Factor VIII Gene.

Objective: To explore the key sites in which L-arginine affects the expression of human coagulation factor VIII gene, and to create new drug targets for the treatment of hemophilia. Methods: A total of 5 human FVIII genes (A1, A2, A3, C1 and C2) with B domain deletion were transfected into human umbilical vein endothelial cells (HUVECs) as promoters. Run-on assay and ELISA analysis were performed to observe the driving effect of each domain gene on chloramphenicol acetyl transferase (CAT) gene transcription and expression, and the effect of L-arginine on each promoter. Results: In co-culture with L-arginine, transcriptional expression of the CAT gene was not detected in the PCAT3-Basic group (negative control without promoters), PA3-CAT3-Enhancer group or PC1-CAT3-Enhancer group. The transcriptional expression of CAT gene in the PCAT3-Control group (positive control with promoters) and PA1-CAT3-Enhancer group was unchanged compared with the non-L-arginine intervention, while the transcriptional expression of CAT gene in the PA2-CAT3-Enhancer group was significantly enhanced. Conclusions: A1 and A2 domain genes had promoter function and could initiate the transcription and expression of CAT gene, but A3, C1 and C2 domain genes could not. Moreover, L-arginine can significantly enhance transcription and expression of human coagulation factor VIII via A2 domain.

Investigation on abnormal gene loci of a Chinese pedigree with hereditary combined deficiency of blood coagulation factor XI, XII, and protein S.

OBJECTIVE: In order to clarify the interaction mechanism, the phenotype and abnormal gene loci of FXI, FXII, and PS were investigated in this study. METHODS: Chinese pedigree with hereditary combined deficiency of coagulation factor (F) XI, FXII, and PS was enrolled in our study. Activated partial thromboplastin time (APTT), partial thromboplastin time (PT), FXI:C, FXII:C, and protein S (PS):C were determined using the one-stage coagulation method. FXI:antigen (Ag), FXII:Ag, and PS:Ag were detected using enzyme-linked immunosorbent assay (ELISA). Exons and introns of the FXI, FXII, and PS genes were amplified by polymerase chain reaction (PCR), and gene sequencing results were analyzed using Chromas software. RESULTS: A deletion of two bases located in introns A-149 and-150 within the FXI gene of the proband, his father, wife, and both sons. A missense variant in exon 14 (GGT â†’ AGT, Gly542Ser) within FXII of the proband, his parents, and both sons. Four variants in exon 4 within the PS gene of all members of the pedigree: GTT â†’ GTG (Val46Val), CGC â†’ CTC (Arg49Leu), CGT â†’ CAT (Arg60His), and CAG â†’ TAG (Gln61stop). CONCLUSIONS: None of the pedigree members showed a tendency for bleeding or thrombosis. Therefore, we speculated that the lack of coagulation factors counteracted the lack of PS, restoring the balance between the coagulation and anticoagulation systems. Another possible explanation is that these defects individually have only partial penetrance.

Role of Ca2+, Calnexin and Calreticulin in Platelet from Adult Patients with Chronic Immune Thrombocytopenic Purpura.

BACKGROUND: Adult chronic immune thrombocytopenia (chronic ITP) is a common autoimmune hemorrhagic disease characterized by decreased platelet production and increased platelet destruction, leading to thrombocytopenia. In this study, Ca2+, calnexin (CNX) and calreticulin (CRT) within platelets from adult patients with chronic ITP were investigated. METHODS: Platelets were isolated from blood specimen collected from 20 adult patients with chronic ITP and 20 healthy volunteers. Ca2+, CNX and CRT were determined by flow cytometry, and the results were analyzed with EXPO32 ADC software. RESULTS: Flow cytometry showed the expressions of Ca2+ (74.19±19.40% vs 22.79±10.47%) was elevated (P<0.05). However, CNX (15.10±7.32% vs 41.79±14.45%) and CRT (25.11±12.66% vs 38.58±12.02%) were decreased markedly in platelets from adult patients with chronic ITP (P<0.05 compared with healthy volunteers). CONCLUSION: Based on enhanced expression of Ca2+ and attenuated expression of CNX and CRT in patients with chronic ITP, Ca2+ concentration and its associated down-regulated proteins may be important regulatory signals in the pathogenesis of chronic ITP.

Investigation of Lymphocyte Subsets in Peripheral Blood of Patients with Dyslipidemia.

OBJECTIVE: In order to evaluate the effect of dyslipidemia on cellular or humoral immunity in patients, changes in the absolute number of lymphocyte subsets were detected. METHODS: Flow cytometry was applied to determine the absolute value of lymphocyte subsets: B cell, NK cell, CD4+ T cell including the functional subset (CD4+CD28+), native subset (CD4+CD45RA+CD62L+), memory T cell subset (CD4+CD45RA-), CD8+ T cell including the functional subset (CD8+CD28+) and activated subsets (CD8+CD38+ and CD8+DR+). The relationship between lymphocyte subsets and hypercholesterolemia and hypertriglyceridemia was analyzed. RESULTS: The absolute values of CD19+ B cell, CD3+ T cell, CD4+ Th cell, CD4+CD28+ cell, naive CD4+ T cell and memory CD4+ T cell in patients with dyslipidemia were markedly higher than those in healthy controls (P<0.05). There was no significant difference between healthy controls and dyslipidemia patients in other lymphocyte subsets (P>0.05). The absolute values of CD3+ T cell and naive CD4+ T cell were significantly positively correlated with hypercholesterolemia in peripheral blood (r=0.291 and 0.306, respectively, all P<0.05). There was no significant correlation between hypertriglyceridemia and lymphocyte subsets (P>0.05). CONCLUSION: Dyslipidemia has potential effects on immune profiles in lymphocytes subsets, and changes in lymphocyte subsets in dyslipidemia patients may lead to immune dysfunction.