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.

Apoptosis in platelets from adult patients with chronic idiopathic thrombocytopenic purpura.

Adult chronic idiopathic thrombocytopenic purpura (cITP) is a chronic and usually life-long haemorrhagic disorder in which enhanced platelet destruction and weakened platelet production lead to thrombocytopenia. Platelets were isolated from blood samples collected from 40 adult patients with cITP and 40 healthy volunteers. Mitochondrial membrane potential (ΔΨm) and plasma membrane phosphatidylserine externalization were determined by flow cytometry, and activation of caspase-3 and expressions of Bax, Bak and Bcl-xL were analysed by western blotting. Flow cytometry showed increased mitochondrial depolarization and lower ΔΨm in platelets from adult patients with cITP. In addition, plasma membrane phosphatidylserine externalization was observed on platelets from adult patients with cITP, but rarely from healthy volunteers. Western blot analysis of platelet proteins revealed that, in adult cITP patients, caspase-3 was activated, which cleaved gelsolin and to release a 47-kDa fragment. Moreover, the expressions of Bax and Bak were elevated, and Bcl-xL was decreased markedly in platelets from adult patients with cITP. Our findings reveal, based on loss of mitochondrial membrane potential (Δψm), phosphatidylserine exposure, caspase-3 activation, enhanced expression of Bax and Bak, and attenuated expression of Bcl-xL, that platelet death in the pathogenesis of thrombocytopenia in chronic ITP in adults is apoptotic.

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.

The diagnostic profile of rare acute myelomegakaryoblastic leukemia.

Acute myelomagakaryocytic leukemia is a diagnostic and therapeutic challenge owing to its heterogeneity and overlapping features with other types of acute leukemia. In order to build a diagnostic profile, we analyzed the biological, clinical and hematologic characteristics of acute myelomagakaryocytic leukemia. We found that, in three patients diagnosed with acute myelomagakaryocytic leukemia, there were two types of leukemia cells. One type was myeloblastic with positive peroxidase (POX) stainig and the expression of antigens CD13 and CD33. The other type was megakaryoblastic with negative POX staining and the expression of antigens CD36, CD41, CD42a and CD61. Three patients displayed the same cytogenetic abnormality, a (9: 22) translocation. Among the three patients with RT-PCR, two patients displayed BCR-ABL fusion gene amplification and one patient showed a previously undescribed OTT-MAL fusion gene amplification.

Investigation on glucocorticoid receptors within platelets from adult patients with immune thrombocytopenia.

Objective: The expression of glucocorticoid receptors within platelets from newly diagnosed Immune Thrombocytopenia (ITP) patients in the adult was investigated.Methods: GR expression in platelets from newly diagnosed ITP patients and healthy controls was measured using flow cytometry. Subsequently, platelets RNA and proteins were isolated and used for confirming the flow cytometry results by using RT-qPCR and ELISA.Results: Flow cytometry showed that the percentages of platelets expressing GRα and GRß from ITP patients were significantly higher than those from healthy controls (P < 0.05). qPCR and ELISA confirmed that GRα and GRß were increased at both RNA transcription and protein expression levels within platelets from ITP patients compared with healthy controls.Conclusion: We speculated that the up-regulation of glucocorticoid receptor within platelets may be an important biological feature of platelets in patients with ITP, and may also play an important role in the treatment of ITP, which is worthy of further study.

Study on the Role of Calreticulin Within Platelet from Adult Patients with Chronic Immune Thrombocytopenic Purpura.

To observe the differences in proteins between adult patients with chronic immune thrombocytopenic purpura (ITP) and healthy adults. 30 patients with chronic ITP and 30 healthy controls were enrolled into the study. The platelet total protein was extracted from peripheral venous blood of 10 chronic ITP patients and 10 healthy controls respectively, and subjected to two-dimensional electrophoresis (2-DE) to find the differential protein spot between chronic ITP patients and healthy controls, then the differential protein spots were identified by mass spectrometry. Subsequently, platelets RNA and proteins were isolated from the other 20 chronic ITP patients and 20 healthy controls respectively, and used for confirming the 2-DE and mass spectrometry results by using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA). 2-DE combined with mass spectrometry revealed that calreticulin (CRT) expressed normally within platelets from healthy controls, while it reduced within platelets from patients with chronic ITP. qPCR and ELISA confirmed that CRT was decreased at both RNA transcription and protein expression levels within platelets from chronic ITP patients compared with healthy controls. Decreased transcription and expression of CRT within platelets may play an important role in the pathogenesis of chronic ITP, which is worthy of further study.

Preliminary investigation about the expression of tubulin in platelets from patients with iron deficiency anemia and thrombocytosis.

OBJECTIVE: In order to inquire into the pathogenesis of increased platelet counts in peripheral blood of patients with iron deficiency anemia (IDA), the phenomenon of thrombocytosis was confirmed, and then the expression of tubulin within platelets from IDA patients was investigated. METHODS: Peripheral blood samples were collected from 79 patients with IDA and were divided into 2 groups, group of IDA with normal platelet counts (34 cases), and group of IDA with increased platelet counts (thrombocytosis) (45 cases). Additionally, 45 peripheral blood samples from healthy volunteers were enrolled as a group of healthy controls. Count of platelets in peripheral blood was detected by means of LH-780 hematology analyzer and hemocytometer under a microscope respectively, and analyzed statistically. RESULTS: There was no statistical difference between platelet counts detected by LH-780 hematology analyzer and hemocytometer under a microscope (P > .05). The mean fluorescence intensity (MFI) of both α-tubulin and ß-tubulin within platelets from IDA patients with thrombocytosis was significantly less than that from healthy volunteers and IDA patients with normal platelet counts (P < .01), and there was no statistical difference between the latter two groups (P > .05). CONCLUSION: Some patients with IDA are accompanied by thrombocytosis, from which the expression of α-tubulin and ß-tubulin within platelets reduced obviously compared with those with normal platelet counts and healthy controls respectively. It is implied that downregulation of tubulin probably is a part of the pathogenesis leading to increased platelet counts in IDA.