Studies On The Relationship Between The Changes Of Tissue Factor Pathway And Cerebrocardiac Thrombotic Diseases

Many studies showed that the blood coagulation process plays an important role in the evolution of acute arterial thrombotic events. A two-stage theory of coagulation is widely accepted.Tissue factor pathway (TFP) is responsible for the initiation of coagulation. However, the contribution of coagulation factors especially in TFP to the development of ischemic arterial disease is still not clearly established. Recent experiments and clinical observations demonstrated that the disturbance of normal balance of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) is closely associated with the evolution of thrombotic episodes. TF is a transmembrane glycoprotein.TF binds factorⅦ(FⅦ) and leads to the activation of factorⅦ(FⅦa) and the formation of TF-FⅦa complex. In turn, TF-FⅦa complex activates factorsⅩandⅨvia limited proteolysis. Therefore, it initiates a cascade of enzymatic reactions resulting ultimately in fibrin clot formation. Therefore, it becomes apparent that the balance of TF-FⅦa and TFPI is a determinant of the initiation of coagulation.Several recent studies suggested that the changes of TFP are closely associated with thrombosis during the onset of acute cerebrocardiac diseases.The cerebrocardiac thrombotic diseases are one of the most common and serious clinical problem. Two mechanisms are responsible for it: atherosclerosis and thrombosis,but the arterial thrombotic is the principal cause of acute myocardial infarction(AMI) and acute ischemic stroke(AIS). TF initiates the TFP and plays an important role in the pathogenesis of atherosclerosis and coronary artery disease. TFPI is the unique physiological inhibitor of the TF-induced initation of blood coagulation and a key factor to prevent thrombosis.On the basis of the integrity of the structure,TFPI has two types: full-length TFPI (fl-TFPI) and truncated TFPI(tr-TFPI).The latter is truncated at the c-terminal end. According to whether bound to lipoproteins or not,TFPI is divided into two forms:free-TFPI and bound-TFPI. Compared with bound-TFPI, free-TFPI possesses much more anticoagulant activity. TFPI is mainly produced by endothelial cells and alternative mRNA splicing generates two forms: TFPIαand TFPIβ.Based on catalytic activity, FactorⅦcontains two styles: Zymogen and enzyme. However, up to now, the relationship between the changes of ratios among the different styles(types,forms) of each coagulation parameter and the evaluation of cerebrocardiac thrombotic diseases has been still not reported. Therefore, it is a novel research direction to investigate the association of TFP with the pathogenesis of cerebrocardiac thrombotic diseases and other thrombotic disease. On the other hand, high reactivity of peripheral blood monocyte as well as monocyte recruitment to the intima can strongly affect the development of atherosclerosis.In rest state, monocytes and endothelial cells do not express tissue factor;but under pathological conditions, lypopolysaccharide(LPS),interleukin-1(IL-1),tumor necrosis factor-α(TNF-α) and other proinflammatory agents can induce them to express tissue factor.It has been reported that angiotensinⅡ(AngⅡ) induced monocytes to express TF and its mechanism was involved NF-κB activation. However, no paper has reported the changes of TFmRNA in monocytes isolated from peripheral blood in patients with cardiovascular and cerebrovascular diseases. The Chinese nation consists of many nationalities and Xinjiang is regional autonomy of minority nationalities.Up to now, the changes of TFP during the onset of acute cerebrocardiac thrombotic diseases have not yet been observed in XinJiang.Purpose:The aim of the present study is 1) to select one kind of kits used to detect TFPI, which can offer more efficient and convenient clinical informations about thrombotic diseases; 2) to compare the levels of plasma TFP components in the healthy volunteers of Han nationality and Uygur nationality in Urumqi; 3) to observe the involvement of TFP and other blood coagulation factors during the onset of AIS and AMI, and furthermore, to demonstrate whether there are some differences in the changes of coagulation- anticoagulation system between AMI and AIS; 4) to observe the expression of TF mRNA in monocytes during the onset of AMI and AIS in patients.Methods:1) to compare the concentration of TFPI, Assaypro and American Diagnostica Inc (ADI) kits were used to determine the antigen levels.2) Plasma recalcification time was detected by manual operation.3)Plasma TF activity was assayed with the chromogenic method.4) Plasma TF?TFPI and FⅦantigen (FⅦAg) and activated FⅦ(FⅦa) and D-dimer were measured with enzyme linked immunosorbent assay (ELISA).5) FⅦcoagulation activity (FⅦ∶C) was observed in the one-stage clotting assay.6) Reverse transcriptase-polymerase chain reaction(RT-PCR) was used to determine the levels of TFmRNA in monocytes.Results:1) Comparison of two kinds of TFPI assay kits.①Total-TFPI (t-TFPI) in plasma isolated from the control group was measured by Assaypro and ADI kits(﹟850) separatively. The data were presented as mean±S.D.i.e. Assayprro: 61.65±19.32μg/L; ADI:87.25±24.5μg/L. The confidence interval tested by ADI kit was more close to the widely accepted reference range of TFPI than that obtained from Assaypro kit in the normal control group.②The level of total-TFPI measured in patients with AMI by using Assaypro kit was 77.32±32.5μg/L and the upper limit of the confidence interval was below that of the reference range of normal people. However, the level of total-TFPI measured by ADI kit(﹟850) was 115.79±52μg/L, the upper limit of the confidence interval was significantly above that of the reference range of normal people.③In contrast to assaypro TFPI kit only used to detect total-TFPI,total(t)-TFPI as well as full length(fl)-TFPI were simultaneously measured by using ADI TFPI kit(﹟850) and the difference between t-TFPI and fl-TFPI is truncated(tr)-TFPI.Thus,three parameters about TFPI can be obtained by using ADI TFPI kit(﹟850).④The differences of the coefficient of variation of total-TFPI measurements obtained from AMI and the normal control groups by two kinds kits were not statistically significant.2) A preliminary observation of the plasma tissue factor pathway in the healthy volunteers of Han and Urygur nationalities,in Urumqi.①There were no significant differences in the antigen levels of TF,t-TFPI,fl-TFPI and in the ratios of TF/t-TFPI,TF/tr-TFPI,tr-TFPI/fl-TFPI between the healthy population of Han and Uygur nationalities. The levels of plasma FⅦAg,FⅦa and FⅦ∶C,D-dimer and the ratios among them were not significantly different.②The concentration of plasma tr-TFPI and the ratios of TF/fl-TFPI and tr-TFPI/t-TFPI in the Uygur nationality group were markedly lower than those in the Han nationality group.3) Observation on TFP and other coagulation factors during the onset of acute cerebrocardiac thrombotic diseases.①Plasma recalcification time in AMI and AIS groups was significantly shorter than that in the control group.②The levels of TF activity and the concentration of TF and TFPI antigen in plasma were significantly higher in the AMI and AIS groups than those in the control group. But the increament degree of TF was remarkably higher than that of TFPI. Therefore, the TF/TFPI ratio was markedly increasead in the AMI and AIS groups.In contrast to the AMI group,the increament degree of TF activity in plasma was markedly higher in the AIS group.As to TFPI, t-TFPI and fl-TFPI were significantly higher; on the contrary, tr-TFPI was markedly lower than that in the control group.Due to the changes of TF and TFPI, the TF/t-TFPI?TF/tr-TFPI and fl-TFPI/t-TFPI ratios were increased and the TF/fl-TFPI, tr-TFPI/t-TFPI and tr-TFPI/fl-TFPI ratios decreased simultaneously in the AMI and AIS groups compared with the control group.Although the tendency of the changes in most parameters of TFP was consistant in the AMI group and the AIS group,the TF activity in the AIS group was remarkably higher than that in the AMI group.③Compared with the control group,the levels of plasma FⅦ∶C were remarkably increased in AMI and AIS groups. There was no significant difference in FⅦantigen between the AMI and control groups. The concentration of FⅦa and the ratio of FⅦa/FⅦAg were more remarkably higher in the AMI group, whereas the ratio differences of FⅦa/FⅦ∶C or FⅦ∶C/FⅦAg between the control and AMI groups was not significant. On the other hand, there were no significant differences between AIS and control groups in FⅦa, FⅦAg and FⅦa/FⅦAg ratio. But the FⅦ∶C/FⅦAg ratio was remarkably higher and FⅦa/FⅦ∶C ratio was significantly lower than those in the AIS group.In contrast to the AMI group, FⅦa and the ratio of FⅦa/FⅦ∶C were decreased in the AIS group.④The levels of plasma D-dimer were remarkably increased in the AMI and AIS groups compared with the control group. 4) Observation on the expression and variation of peripheral monocytes TF-mRNA in patients with AMI and AIS:①The expression of TF mRNA in human peripheral monocytes was significantly higher in the AMI and AIS groups compared with the control group.②The levels of TF mRNA expression were positively related to the concentration of plasma TF in the three groups.Conclusions:1) The ADI kit used for assay of TFPI concentrations can offer more informations about its types than the Assaypro kit.2) Compared with the age-matched healthy subjects of Han nationality,there was no significant difference in the ingredients of plasma TFP,but the level of tr-TFPI was remarkably lower in the Uygur nationality.3) During the onset of AMI and AIS, the initiation of TFP is associated with the thrombotic events.4) In contrast to the control group,the levels of plasma FⅦ∶C were increased in the AMI and AIS groups;but plasma FⅦa and the ratio of FⅦa/FⅦAg were only elevated in the AMI group.Except TF activity, the tendency of the changes of TFP is consistent in both (AMI and AIS)groups.It may be more sensitive and reliable to use the ratios(such as TF/tr-TFPI,tr-TFPI/fl-TFPI)to evaluate the initiation status of coagulation.5) The level of D-dimer is considered to be a parameter for the secondary activation of fibrinolysis in patients with AIS and AMI.6) The expression of TFmRNA in monocytes was upregulated during the onset of AMI and AIS.