Research On The Mechanisms Of Tumor Associated Hypercoagulability

Tumor patients often have the combination of acute and chronic arteriovenous thrombosis events,seriously affecting the quality of life of patients.As early as 1856,Trousseau's syndrome was first mentioned,and it is a description of mobile thrombophlebitis of cancer patients.As people learning more about this disease,the range of it covers all the arteriovenous thrombosis diseases with the conditions of Cancer Associated Hypercoagulability(CAH),which makes the patients have thrombophilia habitus and the complications are easy to occur,such as Venous Thromboem Bolism(VTE),Pulmonary Embolism(PE),Disseminated Intravascular Coagulation(DIC),and so on,which greatly affecte the survival rate of cancer patients.At the same time,this abnormal hyperglytic background further accelerates the growth and spread of tumor cells,which can make the tumor cells in circulation more likely to precipitate down,resulting in localized growth,and the activated platelets form platelets cover around tumor cells,making the tumor cells escape from NK cell immune attack.The patients' in vivo coagulation activation forms interrelated network that can promote each other with inflammation,thrombosis,tumor progression,metastasis and other.Therefore,the correct acknowledgement of hypercoagulable state of the tumor and timely and effective prevention and treatment,play a vital role in improving the quality of life of cancer patients.In the clinical observation of many tumor hypercoagulations,the scope mainly includes VTE,PE.For nervous system arterial embolism,it's paid less attention.An autopsy report showed that 15% dead patients due to cancer had a definite apoplexy of pathologic diagnosis,but only about half of the patients were found to have apoplexy of clinical symptoms during their lifetime.Compared with traditional apoplexy,patients with tumor merger and ischemic apoplexy were worse than prognosis and had longer hospital stay.So far,the early detection and early treatment of such patients is not very clear.Neuroticists often experience the cryptogenic infarction without clear causes,and the study shows that the incidence of cryptogenic infarction is about 20-46%.It is thought that the hypercoagulable state caused by unidentified tumors may be one of the causes of this type of cryptogenic infarction.If the pathogenesis hidden behind the cryptogenic infraction,part of the patients can get timely and effective treatment and secondary prevention,the quality of life of patients can be improved greatly.At present,the prevention for tumor hypercoagulability in the clinical often takes use of tumor venous thromboembolic event prevention,which is agreed by the control experts.Due to conditions and other reasons,it is not fully promoted.And because tumor patients' thrombosis involves multidisciplinary,the evidence-based medical information is too much and complex,and lacks of systematical summary;Secondly,despite the concept of thrombosis prevention has been generally accepted,but also it lacks of evidence-based medicine for the prevention and treatment of cancer patients,thus limiting the prevention and treatment work of tumor patients with thrombosis.In the current expert consensus,the prevention criteria of thrombosis evaluation is mainly from the risk assessment scale,lack of quantitative,standardized blood test indicators to guide clinical prevention and treatment,so we urgently need the indicators similar with blood measurement indicators in vascular disease risk factors(blood lipids,blood sugar,uric acid,etc.)to help us find the high-risk groups in thrombotic events for timely preventive treatment,thereby improving the quality of life and survival of patients.In the mechanism study of tumor hypercoagulability states,the following items are often mentioned: Tissue factor(TF),micro-particles(MP),cancer pro-coagulant(CP)and so on.Tissue factor is one of the important proteins.It can be the form combining with micro-particles,namely Tissue Factor-positive micro-particles(TF-MPs)can be released from tumor cells into circulation.This compound contains tissue factors,phospholipids Phosphatidylserine(PS).In the study of solid tumors,blood cancer patients,it's found that patients had high levels of tissue factor particles.Is it the cause of tumor hypercoagulable or the result of hypercoagulability? In the study of animal experiments,it was found that the TF-MPs produced by injecting human tumor cells into animal bodies could produce reactions similar to those of DICs.Because of the structural characteristics of TF-MPs,is it possible to think that it is one of the main mechanisms causing hypercoagulation of cancer patients? Can it be considered as a predictor of the occurrence of thrombotic events in cancer patients?In summary,we speculate that the hypercoagulable state of tumor is one of the pathogenesis of nervous system thromboembolic events,especially for the nervous system cryptogenic infarction,tissue factor particles is the main reason causing tumor hypercoagulable state.Whether TF-MPs can be used as a clinical indicator of hypercoagulability,it depends on whether it can effectively reflect the degree of hypercoagulability and whether it really can play a role in promoting hypercoagulability.To verify these problems,we firstly analyzed the clinical characteristics of traditional nervous system cerebral infarction(CI)patients and tumor merge cerebral infarction with compare of them,and found the unique clinical features of this population.To verify the role of tumor hypercoagulation in cryptogenic cerebral infarction,the existing conditions of plasma TF-MPs in tumor patients' bodies was investigated,and the coagulation activity and procoagulant mechanism was explored and the feas ibility of taking plasma TF-MPs as the test indicators of evaluating hypercoagulation.The main research results are as follows: Part I Observation of the tumor hypercoagulable state in nervous system thrombosis eventObjective: To discuss the clinical characteristics of thromboembolic events of tumor patients secondary nervous system and the role of tumor hypercoagulability in cryptogenic infarction.1 According to cerebral infarction at acute stage accompanied by a medical history of tumor at active stage,these patients were divided into experimental group and control group,58 cases for each group.Comprehensive analyses of clinical characteristics,blood test indexes and imaging data were carried out for each group.2 Subgroup analys is: According to the classification criterion of the Trial of Org 10172 in Acute Stroke Treatment,the experimental group was further divided into two groups: Stroke of other undete-rmined etiology,(SUE)and conventional stroke etiology(CSE).Comprehensive analysis was carried out on the clinical data of these two groups to further explore the role of hypercoagulability in SUE.3 Plasma TF-MPs was measured for all patients in the subgroups and intergroup comparisons were carried out.The correlation of Plasma TF-MPs with D-dimer was analyzed.Results:1 The proportions of SUE and multi-zone infarction and D-dimer of the experimental group were higher than those of the single infarction group(P<0.01).2 Analyses showed that D-dimer(<0.3),APTT and imaging characteristics were independent factors affecting the diagnosis of infarction type.The higher the D-dimer level(<0.3),the higher the risk of tumor and infarction was(OR(95%CI)=10.474(2.588~42.390));the higher APTT,the lower the risk of tumor and infarction was(OR(95%CI)= 0.780(0.671~0.906));the imaging characteristics was multiple territorial infarctions(OR(95%CI)=11.055(2.390~51.130)).3 In subgroup analysis,the occurrence rate of distal metastasis,D-dimer,and multiple territorial infarctions were much higher than those of the combination group where traditional vascular diseases occurred(P<0.01).4 The median TF-MPs of the SUE group was 42.50(27.25~51.25)/ul,that of the traditional vascular disease group was 18.50(13.25~24.00)/ul,and that of the experimental group was higher than that of the control group,showing Methods: an statistically significant difference(Z=5.413,P<0.001).5 Correlation analysis was carried out on the levels of plasma TF-MPs and D-dimer of all patients in the experimental group(Spearman correlation coefficient r=0.477,P<0.001).The results indicated the positive correlation of TF-MPs and D-dimer.Conclusions:1 Different from those of traditional cerebral infarction,patients with tumor and cerebral infarction are characterized by multiple territorial infarctions,high D-dimer level and being accompanied by history of tumor at active stage.2 When tumor patients with a high D-dimer level are likely to suffer from embolism,prolonged APTT is a protective factor for thrombosis prevention.Imaging results suggest that multiple territorial infarctions are associated with tumor.3 Hypercoagulable state is the major pathogenic mechanism of SUE secondary to tumor.Plasm TF-MPs is associated with hypercoagulable state.Part II Discussion on the hypercoagulability and TF-MPs level in the patients with breast carcinomaObjective: To observe the role of the hypercoagulable state and TF-MPs level in the body of patients with breast carcinoma in promoting hypercoagulability.Methods: 87 patients who were hospitalized for the first time(10 stage I,16 stage II,and 32 stage IV according to TNM staging system;8 cases with carcinoma in situ,15 cases with ductal breast carcinoma and 64 with aggressive carcinoma)were divided into the experimental group,and 20 patients with benign breast disease were included as controls.1 The routine coagulation indicators(including PT,APTT,FIB and D-dimer)of these two groups were analyzed.2 Calibrated automated thrombogram(CAT)was carried out for all patients to observe the coagulation indicators of the experimental group and the control group(Peak,ETP,lag-time and tt Peak).3 The TF-MPs of all subjects were measured to compare the differences in TF-MPs level between the experimental group and the control group,between different breast cancer stages and between different tumor subtype classifications.4 The correlations of TF-MPs with coagulation indicators were analyzed.Results:1 In the experimental group,PT and APTT were shortened and FIB and D-dimer levels were decreased compared with the control group,showing statistically significant differences(P<0.05).2 The TF-MPS level of the experimental group(76.69±18.89)was obviously higher than that of the control group(57.58±12.97)(P<0.01).TF-MPS level was increased with tumor stage(P<0.001)and invasiveness(P<0.05).3 The results of CAT suggested that the lag-time of the experimental group(2.352±1.274min)was shorten than that of the control group(3.582±2.330min),showing statistically significant differences(P<0.05);the Peak of the experimental group(434.97±159.28 nmol/L)was lower than that of the control group(354.10±144.11nmol/L),showing statistically significant differences(P<0.05);the ETP of the experimental group(2586.41± 964.44nmol/l.min)was lower than that of the control group(2036.00±1133.47 nmol/l.min),showing statistically significant differences(P<0.05);the tt Peak of the experimental group(3.383±1.699min)was lower than that of the control group(6.182±3.541min),showing statistically significant differences(P<0.05).4 In the experimental group,Peak and ETP was increased with TNM stage and invasiveness.5 Correlation analysis indicated that TF-MPs was negatively correlated with PT(r=-0.328,P<0.05),APTT(r=-0.393,P<0.05),lag-time(r=-0.426,P<0.05)and tt Peak(r=-0.411,P<0.05)and was positively correlated with FIB(r=0.421,P<0.05),D-dimer(r=0.473,P<0.05),Peak(r=0.389,P<0.05)and ETP(r=0.381,P<0.05).Conclusion:Coagulopathy is common in patients with malignant breast carcinoma,and TF-MPs plays an important role in promoting the hypercoagulability of tumor.Part III Study on the mechanism of TF-MPs from breast cancer cells in promoting hypercoagulabilityObjective: To identify the role of TF+MPs related to breast cancer cells in promoting hypercoagulabilityMethods: After TF expression of breast cancer cell MDA-MB-231,453,549 and MCF7 was determined by PCR and Western blot,2 strains with high TF expression(experimental group)and with low TF expression(control group)were cultured.1 TF+MPs from breast cancer cells were extracted by physical and mechanical method after successful culture.2 Flow cytometry was used to carry out quantitative and qualitative measurements under the preset conditions that anti-human TF antibody and Annexin-FTIC were double positive and defined as TF-MPs because their sizes were smaller than 1um.These TF-MPs were measure for five times to obtain the mean value an d compared between different groups.3 One-stage clotting was employed to measure the procoagulant activity(PCA)function of the TF-MPs suspensions from two types of cells in the same quantity,and PCA was compared between different groups.4 Monoclonal anti-human tissue factors(a TF)in different doses were used to block TF,Annexin-v and phosphatidylserine(PS)to compare the effects of different doses on PCA.At the same time,the second control group was prepared.PBS in the same dose gradient was added into the suspension and then compared with PCA after adding a TF and Annexin-v in gradient doses in order to verify the structural basis of TF-MPs PCA.Results:1 The TF expression level of MDA-MB-231 was the highest among the four cell types,followed by 453,549 and MCF7 in order.2 After the physical and mechanical stimulation of MDA-MB-231 and MCF-7,breast cancer cells-related double positive TF-MPs of less than 1um were successfully extracted through the verification of flow cytometry.The micro particles of MDA-MB-231 expressing high TF(55.80±1.64/ul)was obviously lower than that of MCF-7 expressing low TF(10.40±1.14)(P<0.01).3 The PCA results showed that the PCA of TF-MPs suspension of MDA-MB-231(220.84±2.20)was obviously stronger than that of MCF-7 group(36.13±0.85)(P<0.01).4 TF monoclonal antibodies in gradient doses from in ascending order were added into TF-MPs suspension,and PCA was decreased with the increase of TF antibody,showing a statistically significant difference compared with the stock solution(P<0.05).PCA had no obvious decline as TF antibody was increased to 50ug/ml,suggesting that PCA was totally inhibited when a TF reached 50ug/ml,and a further increase of a TF dose wo uld have no impact on PCA.5 The PCA of a TF in 0,12.5 and 25ug/ml was decreased in turn,and there were statistically significant differences between different groups(P<0.05),suggesting that TF monoclonal antibody inhibited PCA after blocking TF.6 The same TF-MPs suspension was added into PBS in the same gradient dose as control group.Compared with a TF group,the PCAs of a TF experimental groups were lower than those of PBS groups,further verifying the effect of TF monoclonal antibody on the PCA of TF-MPs.7 PCA declined with the increase of the Annexin-V added,showing statistically significant differences compared with the stock solution.PCA had no obvious decline when Annexin-V was increased to 50ug/ml,but the PCA was still higher than PBS(P<0.05),suggesting that when Annexin-V reached 50ug/ml,the PS on the surface was totally blocked,so any further increase would have no impact on PCA,but it did not totally inhibit PCA.8 Compared with the control groups with the same gradient dose of PBS,the PCAs with different doses of Annexin-V were higher than PBS(P<0.05).Conclusions:1 TF-MPs from breast cancer cells have significant procoagulant activity.2 TF-MPs induced PCA mainly relies on TF and PS on the microparticle surface,and TF plays a dominant role and PS plays a support role in procoagulation.3 The quantity of TF-MPs relies on the TF expressed on cell membrane.4 TF-MPs can be released automatically from tumor cell.