Preclinical Research Of Inherited Anti-coagulation Factor And Acquired Coagulation Factor Deficiency

Coagulation factors and anti-coagulation factors are the important elements on maintaining normal haemostasis and preventing thrombosis of human being. According to the causes, anticoagulation factors'deficiency can be divided into two groups: hereditary and secondary. Inherited anti-coagulation factor deficiency is single gene inherited disease. Though the prevalence rate of these disorders is low, patients with these diseases may not only suffer the pains in their life, but also may pass it down to their offspring. The discovery of related gene mutation from proband will be beneficial both to the offspring and to a better understanding of the relationship between the protein structure and function of related anti-coagulation factor. Study on the diversity of clinical phenotype and the inconsistency with the coagulant theory in existence caused by different gene mutations, also will help us to further understand the mechanism of blood coagulation and haemostasis. In this paper, molecular mechanisms causing deficiencies of antithrombin(AT) and protein S(PS) in plasma were studied in three pedigrees with inherited AT deficiency and two pedigrees with inherited protein S deficiency, respectively.Inherited antithrombin deficiency is inherited as an autosomal dominant trail, with the prevalence of 2/10000⁵/10000 in population and 1%⁸% in venous thromboembolism (VTE) patient. AT deficiency is the major inherited risk factor of VTE. Proband 1, 2, 3 in this paper was a 72-year-old , a 23-year-old, a 32-year-old female, respectively. Phenotype analysis revealed that the AT antigen (AT:Ag) and AT activity (AT:A) of proband 1 was 121mg/L and 57.2%, respectively, whereas protein C activity (PC:A) and protein S activity (PS:A) were all in normal range. After direct genomic DNA sequencing of AT gene including 7 exons and their boundaries'sequence, a heterozygous A9 850G in exon 5 of AT gene was identified in the propositus 1. A9 850G missense mutation caused His369Arg substitution of AT protein. Phenotype analysis revealed that the AT:Ag and AT:A of proband 2 was 119mg/L and 54.6%, respectively, whereas PC:A and PS:A were all in normal range. A heterozygous CT deletion in exon 3A of AT gene in 5 384-5 or 5 386-7 was identified in the propositus 2. The mutation would introduce a stop code at 162aa, causing the deletion of 230 aa in C-terminal from synthesized AT protein. Phenotype analysis revealed that the AT:Ag and AT:A of proband 3 was 111mg/L and 40.0%, respectively, whereas PC:A and PS:A were all in normal range. A heterozygous 15bp deletion in exon 4 of AT gene in 7 421-35 was identified in the propositus 3. The missense mutation caused Glu 229Ala substitution and deletion of 5 aa of AT protein. Three novel mutations lead to type I inherited AT deficiency.Transient expression experiments were performed using COS-7 and CHO cells transfected with the expression vectors containing the wild-type or the mutated recombinant cDNA (Atwt, pcDNA/AT H369R, pcDNA/AT 5 386-7delCT, pcDNA/AT 7 421-35del15bp) and analyzed using ELISA, Western-blot and Immunofluorescence staining. The study on in vitro transient expression of pcDNA/AT 5 386-7delCT transfected to COS-7 cell revealed that AT:Ag could not be detected in culture supernatant. Compared to AT wt, the AT:Ag of pcDNA/AT 5 386-7delCT was only 16.2% in cell lysate. Cell immunofluorescence assay results indicated that the intensity of fluorescence was significantly reduced in pcDNA/AT 5386-7delCT cell compared with AT wt cell. Expression studies in vitro were also performed with two AT gene mutations causing homozygous AT deficiency. The AT:Ag in culture supernatant and cell lysate after transient transfected with A9 850G mutated AT cDNA expression plasmid (pcDNA/AT H369R) to COS-7 cell, were 40.2% and 172.1% respectively compared to that of AT wt. However, the result that the AT:A in culture supernatant was 29.4%. The AT:Ag in culture supernatant and cell lysate after transient transfected with 7 421-35del15bp mutated AT cDNA expression plasmid (pcDNA/AT 7 421-35del15bp) to COS-7 cell, were 14.6% and 150.3% respectively compared to that of AT wt. However, the result that the AT:A in culture supernatant was 10.5%. Cell immunofluorescence assay results showed that the AT protein with A9 850G and 7 421-35del15bp substitution mutation had secretion defect. The intracellular AT molecules were dominantly located in ER or other pre-Golgi structure.Inherited PS deficiency is the inherited risk factor of VTE. Proband 1, 2 in this paper was a 27-year-old, a 24-year-old female, respectively. They presented with recurrent and multiple thrombosis. TPS:Ag is 136mg/L, FPS:Ag is 41mg/L, PS:A is 48.6%, novel heterozygous mutation c.C121T(cDNA PROS1, GenBank, NM₀00313.1) in exon 2 of PROS1 gene was identified for proband 1, c.C121T missense mutation caused Arg-1Cys substitution of PS protein. TPS:Ag is 83mg/L, FPS:Ag is 26mg/L and PS:A is 29.2%, heterozygous mutation Gln522Stop in exon 14 of PS gene was identified for proband 2, the mutation is first reported in China. Heterozygous Arg-1Cys (R-1C) in PROS1 gene is the cause of typeâ…¡inherited PS deficiency. Heterozygous Gln522Stop in PROS1 gene is the cause of typeâ… inherited PS deficiency.We tested the coagulation factor's activity, coagulation factors'antibody, anticardiolipin antibody (ACL), lupus anticoagulant (LA) and thrombin generation in sixty schizophrenic patients. Twenty five patients with schizophrenia had been taking chlorderazin, twenty patients had been taking clozapine, and twenty patients had been no taking antipsychotic drugs. Chlorpromazine and clozapine associated with LA and coagulation factors (Fâ…§, Fâ…¨and Fâ…ª) deficiency. ETP are quite good parameters for predicting the bleeding and thrombosis risk of patients.