The Molecular Basis Of Venous Thromboembolism Caused By Hereditary Anticoagulant Protein Deficiency

Part One The incidence of hereditary anticoagulant protein deficiency in people with VTEObjective To explore the rate of anticoagulant protein deficiency and the incidence of hereditary anticoagulant protein deficiency in people with VTE.Methods Collect the peripheral blood of 277 clinical patients with acute or chronic VTE and 1000 healthy subjects(control group).Automatic coagulation analyzer and chromogenic substrate method were used to Detect the activities of AT?PC and PS(Antithrombin activity,AT:A;Protein S activity,PS:A;Protein C activity,PC:A).DNA sequencing was used to detect special gene mutation among those with lower activity of anticoagulant protein.Using statistical method to calculate the rate of anticoagulant protein deficiency and the incidence of hereditary anticoagulant protein deficiency in people with VTE.Results The rate of AT?PC?PS deficiency were 16%, 17.45%, 17.09%,separately.And the incidence of hereditary anticoagulant protein deficiency were as follows: ATD 1.5%?PCD 6.2%?PSD 0.7%.Conclusions Compared with normal subjects, the rate of anticoagulant protein deficiency and the incidence of hereditary anticoagulant protein deficiency in people with VTE were higher.It indicates that hereditary anticoagulant protein deficiency is a risk factor for VTE.Part two Molecular mechanisms of protein C deficiency caused by a novel missense mutationObjective To explore the molecular mechanisms of protein C deficiency caused by a novel missense mutation.Methods To analyse a patient with deep vein thrombosis,then extract his DNA from peripheral blood for PCR amplification and DNA sequencing analysis. Wild-type and mutant PROC c DNA expression plasmids(PC-wt and PC-c. 1157 T>C) were transfected into HEK293 T cell.RT-PCR was Carried out to detect the m RNA level of both plasmids transfected cells.Western blot was utilized to observe the PC expression in cell lysates and extracellular fluid. Laser confocal assay was carried out to detect the distribution of PC in The endoplasmic reticulum.Results Cell immunofluorescence assays prove that Overexpression plasmids were successfully transfected into HEK-293T(human embryonic kidney 293 T cells) cells. RT-PCR results indicate that the m RNA level in celles transfected with mutant plasmids were significantly higher than the wild ones. Western blotting assays also show no significant difference of protein C expressed in both cells,but the mutant type one have an upward trend;Comparing with mutant type,Protein C excreted into the extracellular statistically higher in the wild type.Laser confocal assay further showed that pc expressed in HEK-293 T cells transfected by PC-c. 1157 T>C obviously aggregated in The endoplasmic reticulum,comparing with the wild-types.Conclusions The main moleculor mechanisms of protein C deficiency in this pedigree is the declined expression and disturbed secretion caused by the mutation of PC-c. 1157 T>C.Part three Molecular mechanisms of protein S deficiency caused by a novel nonsense mutationObjective To explore the molecular mechanisms of protein S deficiency caused by a novel nonsense mutation.Methods To analyse a patient with deep vein thrombosis,then extract his DNA from peripheral blood for PCR amplification and DNA sequencing analysis.Family tree was also done according to the result.Wild-type and mutant PROS1 c DNA expression plasmids(ps-wt and PS-c.1771 del A) were transfected into HEK293 T cell. RT-PCR was executed to detect the m RNA level in both plasmids transfected cells.Western blot was utilized to observe the protein S expression in cell lysates.Elisa test was also used to detect protein S excreted into the extracellular.Laser confocal assay was carried out to detect the distribution of PS in The golgi apparatus.Results A new nonsense mutation PS-c.1771 del A was found in the PROS1 gene,family analysis showed this is hereditary anticoagulant protein S deficiency. Compared to the wild-types, the m RNA level and ps expression of HEK293 T cells sharperly decreased when transfected by PS-c.1771 del A.Elisa assay found protein S excreted into the extracellular also reduced in the mutant groups.Laser confocal assay further showed that ps expressed in HEK-293 T cells transfected by PS-c.1771 del A obviously decreased and rarely aggregated in The golgi apparatus,comparing with the wild-types.Conclusions The main moleculor mechanisms of protein S deficiency in this pedigree is the declined expression and disturbed secretion caused by the mutation of PS-c.1771 del A.