Exploratory Study On Thrombosis Markers For Acute Thrombotic Diseases

As a main category of illness,thrombotic diseases have close relationship with eachclinical department.Atherosclerosis and its thromboembolic complication has become thefirst top of causes for death and disability both in western countries and China.Theincidence is increasing year by year.Process of thrombogenesis is a long period of duration.When a patient visits his doctor,a thrombus has already formed leading to vascularembolism.Vital organs,including the heart and brain which are very sensitive to ischemia,have bad response to therapy after an event of embolism.Therefore,thrombotic diseasesseverely do harm to human well-being.If a diagnosis of thrombosis during the early stageof thrombogenesis is performed and interventional measures are taken in time,the harmfuleffect caused by thromboembolic diseases can be weakened to a great extent.At present,accurate diagnosis for thrombotic diseases mainly depends on imageological evidence at the regional location of thrombosis.In clinical practice,detection of plasma d-dimer,activated partial thromboplastin time (APTT),prothrombintime (PT),and level of fibrinogen is considered to be an initial screening proceedingwithout a positive conclusion on the sensitivity and specificity.The pathogenesis ofthrombotic diseases is very complicated.At the early stage of thrombosis or prior to theformation of a thrombus,negative results might be shown about the above-mentionedscreening markers,ultimately leading to delay in precise diagnosis.When positive findingsare indicated,the illness usually develops to be at an intermediate or advanced stage duringwhen the most effective treatment couldn't be performed.Therefore,among high-riskgroup with thrombotic diseases,the popular research today in the field of thromboticdiseases is concentrated on allround assessment of risk and performance of diagnosis ofthrombosis at an early stage.For studies on proteomics,time-of-flight mass spectrometry (TOF-MS) is an extensiveand high-throughout research technique at molecular level.Application of TOF-MScoupled with bioinformatics and experiments on gene function are playing more and moreimportant roles in the field of bringing molecular mechanisms of a disease to light.Usingsurface enhanced laser desorption / ionization time of flight mass spectrometry(SELDI-TOF-MS) and protein biochips,exploratory studies were performed to drawplasma protein fingerprints for arterial and venous thrombosis in order to discover specificprotein biomarkers which could discriminate between patients with thrombotic diseasesand control subjects with high sensitivity and specificity and to provide experimentalevidence for developing new specific biomarkers used for diagnosis of thrombosis in thefuture.Meanwhile,coupled with detection of correlated biomarkers and application of aseries of platforms for liquid-phase gene chips related with coagulation,anticoagulation,and fibrinolytic systems,a fast,precise,and comprehensive system for early diagnosisshould be set up to find the presence of a thrombus based on clinical investigation. PartⅠRetrospective Study on Changes of Coagulation Function andEvaluation on Risk Factors for Acute Cerebral InfarctionObjective Several studies have indicated an association between changes ofcoagulation,anticoagulation,and fibrinolytic proteins and development of cerebrovasculardisease,but few reports referred to their roles together.In this report,we systematicallyinvestigated the relationship between sixteen coagulation markers of them and acutecerebral infarction,as well as considering the contribution of blood lipids and otherpossible risk factors.Methods Inpatients diagnosed for acute cerebral infarction (ACI) (n=50) andhospital controls (n=54) were recruited from Union Hospital.All blood samples werecollected from ulnar vein on the mornings of 2^nd day of hospitalization.Plasmaconcentrations of protein C (PC),free proteinS (FPS),total protein S (TPS),thrombomodulin (TM),activated FⅦ(FⅦa),FⅦantigen (FⅦAg),p-selectin (p-sel),tissue-type plasminogen activator (t-PA),and plasminogen activator inhibitor-1 (PAI-1)were assayed by ELISA kits;Activity of tissue factor (aTF) was analyzed by chromogenicactivity assay kits;Activated protein C (APC) ratio,activated partial thromboplastin time(APTT),prothrombin time (PT),thrombin time (TI),fibrinogen (Fbg),and D-dimer weredetected in an automated coagulation analyzer (Sysmex CA-7000,Japan).Blood lipidswere detected using an automatic biochemical analyzer (Hitachi 7170A,Tokyo,Japan) at Department of Laboratory Medicine.Results Plasma levels of thrombomodulin,fibrinogen,and activity of tissue factorwere significantly higher in cases than in control subjects (P＜0.001,P＜0.01,and P＜0.05,respectively);Concentration of high-density lipoprotein cholesterol (HDL-C) wassignificantly lower in cases than in controls (P＜0.01).Multivariate logistic regressionanalysis showed that hypertension and high plasma levels of thrombomodulin,fibrinogen,and aTF were significantly associated with presence of ACI (odds ratio [OR],143.74,P＜0.001;OR,2.05,P＜0.05;OR,2.09,P＜0.05;OR,1.02,P＜0.05,respectively).Conclusion Our findings indicate that hypertension and elevation of plasmathrombomodulin,fibrinogen,and aTF are independent risk factors for ACI. PartⅡAssessment of Risk Factors for Acute Deep Venous ThrombosisObjective To observe the changes of coagulation function in patients with acutelower-limb deep venous thrombosis (DVT) and evaluate the risk factors for DVT.Methods Plasma APTT,PT,TT,fibrinogen (Fbg),and D-dimer were detected by anautomated coagulation analyzer in 62 acute lower-limb DVT patients and 70 controlsubjects;And retrospective studies on the clinical data of all patients were done by binarylogistic regression analysis.Results (1) In DVT group,plasma APTT,PT,TT,D-dimer,and fibrinogen andD-dimer / fibrinogen ratio (D/F ratio) were higher when compared with control group;allof the differences were significant (all P＜0.01);(2) There were positive correlationsbetween D-dimer and fibrinogen both in DVT and control groups (r= 0.475,P＜0.01;r=0.564,P＜0.01,respectively);(3) Logistic analysis indicated that acute lower-limb DVTwas associated with the presence of hypertension and increased plasma level of fibrinogen(odds ratio [OR],24.99,P＜0.01;OR,4.346,P＜0.01,respectively).Conclusions Hypertension and elevated plasma level of fibrinogen are independentrisk factors for acute lower-limb DVT. PartⅢStudy on Plasma Protein Fingerprints for Acute Thrombotic DiseasesExperiment 1 Proteomic study on plasma potentialbiomarkers for acute cerebral infarctionObjective Previous researches indicated that no single biologic marker was used inthe routine diagnosis of acute cerebral infarction (ACI).In this experiment,surfaceenhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS)technology was used to screen potential specific biomarkers in plasma samples frompatients with ACI.Methods Plasma samples were drawn from 32 cases with ACI and from 60 healthycontrol subjects.Plasma proteomic profiling was detected by the SELDI-TOF-MStechnology coupled with weak cation exchange arrays (CM10 ProteinChip,CiphergenBiosystems).A diagnostic pattern was introduced with the help of a bioinformatics tool(nonlinear support vector machine) and the method of leave-one-out cross validation wasused to estimate the discriminating power of this pattern.Results A differential pattern consisting of 13 biomarkers was selected based ontheir collective contribution to the optimal separation between patients with ACI andcontrol subjects with a sensitivity of 84.4% and specificity of 95.0%,respectively.Conclusion Plasma proteomic profiling with SELDI-TOF-MS and ProteinChiptechnologies shows potential in discriminating patients with acute cerebral infarction andcontrol subjects.Diagnosis of acute cerebral infarction should probably depend on the useof a panel of biomarkers. Experiment 2 Proteomic study on plasma potential biomarkers foracute myocardial infarction and deep vein thrombosisObjective The present study is concerned about searching for specific biomarkersfor thromboembolic (arterial and venous) diseases by the use of Surface-Enhanced LaserDesorption / Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS).Methods We screened for potential biomarkers in 69 plasma samples,includingsamples from 20 patients with idiopathetic deep vein thrombosis (DVT),20 patients withacute myocardial infarction (AMI),and 29 healthy controls without a history ofthromboembolism.Pretreated plasma samples were analyzed on the Protein BiologySystem IIc plus SELDI-TOF-MS (Ciphergen Biosystems,Fremont,CA).Proteomic spectraof mass to charge ratio (m/z) were generated by the application of plasma to immobilizedmetal affinity capture (IMAC-3) ProteinChip arrays activated with copper.Results A pattern of three biomarkers (m/z:2 667,5 914,and 6 890 Da,respectively)with a total accuracy of 100% was selected based on their collective contribution to theoptimal separation between patients with AMI and healthy controls.Another patternconsisting of only one biomarker (m/z:5 914 Da) could totally discriminate patients withDVT and control subjects.For further analysis between patients with AMI and those withDVT,a pattern of four biomarkers (m/z:3 418,5 271,33 378,and 68 125 Da,respectively)was selected with a total accuracy of 82.5%.Conclusions Plasma proteomic profiling with SELDI-TOF-MS and ProteinChiptechnologies provides high sensitivity and specificity in discriminating patients withthrombosis and healthy subjects.The discovered biomarkers might show great potential forearly diagnosis of thromboembolic diseases.