In 2011, we reported that fibrinogen, rather than fibrin, was a major thrombus protein in acute PE [1]. The remaining proteins included serum albumin and cytoskeletal proteins.Fibrinogen makes embolus fragile, which theoretically explains the reason for wide thrombolytic time window: thrombolytic therapy can be extended to several days, 2 weeks, or even longer. It also explains why interventional fragmentation is effective for patients with acute PE.

The main component of red thrombus in acute VTE is fibrinogen. What is the relationship between fibrinogen and lymphocytes, leucocytes, platelets and erythrocytes? It is involved in the molecular mechanisms underlying the pathogenesis of acute VTE. Our pilot studies on the genomics, functional proteomics and informatics of acute VTE have demonstrated that the subunits of integrin β1, β2 and β3 are core proteins in the red thrombus [2]. The core proteins in thrombus bind to fibrinogen to generate unstable grid structure. When the grid structure is filled with erythrocytes,the soluble and fragile red thrombus forms. The subunits of integrin β1, β2 and β3 are distributed on the membranes of lymphocytes, leucocytes and platelets, respectively.As the configurations and expressions of these proteins change in VTE patients, the integrin subunits β1, β2 and β3 may serve as the early diagnostic markers of VTE. In the present study, 120 VTE patients diagnosed by imaging examinations and 120 sex and age matched non-VTE patients and healthy controls were recruited, and the expressions of integrin β1, β2 and β3 were detected in the peripheral blood cells.

A total of 120 inpatients with acute VTE were recruited from Apr 2011 to Dec 2012,including 47 males and 73 females, with an average age of 67.84±16.09 years (range:24-90 years). All acute DVT patients were diagnosed by ultrasonography or selective intravenous angiography. Acute PE patients were diagnosed by CT pulmonary angiography (CTPA) or selective pneumoangiography. Patients with malignancies,pregnancy, autoimmune diseases or on immunosuppressants were excluded.

During the same period, 120 sex and age matched non-VTE patients and healthy controls were also enrolled as controls. Non-VTE patients had no clinical symptoms of VTE and VTE was excluded by ultrasonography or CTPA. The study protocol was approved by the Ethics Committee of Tongji Hospital, and an informed consent was obtained from all patients in accordance with the declaration of Helsinki.

Detailed clinical history was collected immediately after patients were admitted to the hospital. A total of 2 ml of fasting peripheral blood samples were colleted from subjects in three groups and anti-coagulated with EDTA. Detection was carried out within two hours.

Monoclonal antibodies were employed in the detection of integrin β1(CD29),β2(CD18) and β3 (CD61) expression. Fluorescent antibodies against CD29, CD18 and CD61 were purchased from BD (USA). Briefly, 100 µl EDTA-anticoagulant peripheral blood was added into the test tubes and homotype control tubes were introduced simultaneously. According to different fluorescent markers, 20 µl of mouse IgG1-PC5, IgG1-FITC or IgG1-PE (mouse IgG2-PE substituted for IgG1-PE in the detection of CD29) was added. Subsequently, 20 µl of corresponding fluorescent antibodies were added. After mixing thoroughly, the mixture was kept in dark for 30 min at room temperature. Then, 500 µl of haemolysin (BECKMAN-COULTER, USA) was added into the tubes, followed by incubation at 37℃ for 30 min. After washing, 500µl of sheath reagent was added to each tube, followed by flow cytometry (EPICS XL-4, BECKMAN-COULTER). A total of 10000 cells were analyzed in each detection. The percentage of positive cells was calculated, and results were analyzed by the built-in SYSTEM-II software.

Statistical analysis was performed with SPSS 18.0 software. According to the Kolmogorov-Smirnov analysis, the blood levels of integrins showed a skewed distribution. Thus, these variables were presented as medians (1st, 3rd quartiles). The medians and interquartile ranges were plotted in the figures as a box and whisker plot.In addition, differences in the variables between patients and controls were examined with Student's t-test or two-tailed Mann-Whitney U-test. The Chi-square test and Fisher's exact probabilities were employed for the comparison between observed and expected frequencies. Furthermore, the receiver operating characteristic (ROC)curves for predicting survival were plotted and analyzed to compare the diagnostic performance. Youden's index [3] was calculated, and the optimum diagnostic cutoff levels, sensitivity, specificity, positive and negative predictive values were analyzed according to the maximum of Youden's index. A value of P<0.05 was considered statistically significant.

A total of 120 VTE patients and 120 non-VTE patients and healthy controls matched in age and sex were enrolled into this study. Among the 120 VTE patients, 72 (60.00%) were diagnosed with DVT and 48 (40.00%) with PE. There were 8 (6.67%) patients suffering from both DVT and PE. Patients' demographics, type of episodes, disease history and plasma integrin levels are shown in Table 4-1-1.

Blood Integrin levels were quantified by flow cytometry. The median levels of integrin β1, β2 and β3 were all significantly higher in VTE patients when comapred with non-VTE patients (P=0.000, 0.000 and 0.000, respectively) and healthy controls (P=0.000, 0.000 and 0.000, respectively). Between non-VTE patients and healthy controls, there was no statistical significance in the blood levels of integrin β1, β2 and β3 (P=0.572, 0.544 and 0.547, respectively). (Figure 4-1-1)

ROC curve analysis was utilized to assess diagnostic performance of these proteins.When a comparison was made between VTE patients and non-VTE patients, the AUC of integrin β1, integrin β2 and integrin β3 was 0.869 (P=0.000, 95%CI: 0.821-0.916), 0.809(P=0.000, 95%CI: 0.752-0.867) and 0.742 (P=0.000, 95%CI: 0.676-0.809), respectively, and that of combined three integrins and D-Dimer was 0.917 (P=0.000, 95%CI: 0.878-0.956),and 0.811 (P=0.000, 95%CI: 0.754-0.868), respectively (Figure 4-1-2).

When a comparison was made between VTE patients and healthy controls, the AUC of integrin β1 integrin β2 and integrin β3 was 0.875 (P=0.000, 95%CI: 0.829-0.922), 0.828 (P=0.000, 95%CI: 0.774-0.882), and 0.721 (P=0.000, 95%CI: 0.655-0.786),respectively, and that of combined three integrins was 0.915 (P=0.000, 95%CI: 0.876-0.954) (Figure 4-1-3).

When a comparison was made between VTE patients and non-VTE patients plus healthy controls, the AUC of integrin β1, integrin β2 and integrin β3 was 0.870 (P=0.000,95%CI: 0.825-0.915), 0.821 (P=0.000, 95%CI: 0.771-0.871) and 0.731 (P=0.000, 95%CI: 0.671-0.792), respectively, and that of combined three integrins was 0.916 (P=0.000, 95%CI:0.878-0.953) (Figure 4-1-4).

Ages are shown as mean (SD), integrins as median (1st, 3rd quartiles), and categorical data as the number and percentage to the sample group. Age was compared with Student's t test. Gender was compared with chi-square test. Integrin level was compared with Mann-Whitney U test.

Abbreviations:DVT: deep venous thrombosis, PE: pulmonary embolism, COPD:chronic obstructive pulmonary disease, CAD: coronary artery disease, CI: cerebral infarction.

VTE patients usually have nonspecific clinical presentations, which are difficult to diagnose [4]. Recently a number of biomarkers have been evaluated with regard to their potential for predicting VTE, but results are currently contradictory among different labs[5-8]. In this study, we evaluated whether three integrins (β1, β2 and β3) could serve as promising biomarkers for VTE.

Integrins are transmembrane receptors that mediate the adhesion between cells and cells, and between cells and extracellular matrix (ECM). As signaling receptors,integrins play important roles in regulating the signaling pathways involved in the growth, proliferation, differentiation, and migration of cells. Integrins are heterodimeric glycoproteins. In humans, there are 20 different integrins with special ligands formed by specific non-covalent binding of 18 α and 9 β subunits [9].

The subunit of β1 integrin is mainly expressed on the membranes of lymphocytes and platelets. The corresponding ligands are laminin, collagen, thrombospondin,fibronetin and vascular cell adhesion molecule 1 (VCAM-1) [10]. The subunit of β2 integrin is mainly expressed on the membranes of neutrophils and monocytes, and the corresponding ligands are fibrinogen, intercellular adhesion molecule (ICAM), factor X,and ic3b [11]. The β3 subunit of integrin is expressed on the membrane of platelet and the corresponding ligands are fibrinogen, fibronetin, vitronectin, vWF, thrombospondin and so on [12].

The results of our pilot study showed that the β1, β2 and β3 subunits of integrin were core proteins of red thrombus in VTE. Through binding of these core proteins to ligands, platelets aggregate to the coralloid structures, and then core proteins bind to fibrinogen to generate grid structures of the thrombus. It is basic mechanism underlying the pathogenesis of VTE.

The present study showed that the blood levels of integrins β1, β2, and β3 were all significantly higher in VTE patients than in non-VTE patients and healthy controls.The ROC curves showed that the AUC of integrin β1, β2 and β3 subunits in diagnosing acute VTE was 0.870, 0.821 and 0.731, respectively. When integrins β1, β2, and β3 were combinated as a diagnostic marker for VTE, the AUC was 0.916, and the sensitivity,specificity, positive and negative predictive value were 84.6%, 90.8%, 81.7% and 92.0%,respectively, which indicates the potential for using blood integrins β1, β2, and β3 as biomarkers for the diagnosis of VTE (Table 4-1-2).

D-Dimer is a degradation product of cross-linked fibrin and forms immediately when the thrombin-generated fibrin clots are degraded by the plasmin. Thus, it reflects the whole activation of blood coagulation and fibrinolysis. Being the best-recognized biomarker for initial assessment of suspected VTE, D-Dimer with a negative value may safely rule out both DVT and PE with a sensitivity of 83%-96% and a negative predictive value of nearly 100%. However, because of its poor specificity of about 43%-71% in diagnosing VTE [13-17], D-Dimer testing has to be included in comprehensive sequential diagnostic strategies that incorporate clinical probability assessment and imaging techniques. Our study showed that combined measurement of thrombus core proteins (different integrin subunits) had a moderate sensitivity (84.6%), but a high specificity (90.8%) in diagnosing VTE. In patients, especially those with elevated D-Dimer, measurement of integrins is useful in the diagnosis of VTE and can avoid excessive imaging examinations.

According to the treatment time, anticoagulant therapies in VTE can be grouped into three classes: 1 month, 3 months and lifelong anticoagulant therapy. Hemorrhage is an important complication of anticoagulation therapy. There is lack of objective indicators for determining the course of warfarin therapy and deciding the reduction or withdrawal of warfarin therapy [18-22]. In this study, during 6-12 month follow-up,no evidence of VTE recurrence was found in 10 VTE patients whose integrin subunits β1, β2, and β3 restored to the normal range after Warfarin withdrawal. These findings suggest that when the expressions and configurations of integrin subunits β1, β2, and β3 return to the normal levels, they no longer bind to the corresponding ligands. Thus, the thrombosis was destroyed gradually. These may be potential indicators to determining the duration of oral anticoagulant therapy.

(Published:Int J Clin Exp Med 2014; 7: 2578-2584.)

1.Wang L, Gong Z, Jiang J, et al. Confusion of wide thrombolytic time window for acute pulmonary embolism: mass spectrographic analysis for thrombus proteins. Am J Respir Crit Care Med 2011; 184:145-146.

2.Xie Y, Duan Q, Wang L, et al. Genomic characteristics of adhesion molecules in patients with symptomatic pulmonary embolism. Mol Med Rep 2012; 6:585-590.

3.Youden WJ (1950) Index for rating diagnostic tests. Cancer 3: 32-35.

4.Torbicki A, Perrier A, Konstantinides S, et al. Guidelines on the diagnosis and management of acute pulmonary embolism: the Task Force for the Diagnosis and Management of Acute Pulmonary Embolism of the European Society of Cardiology (ESC) Eur Heart J. 2008;29:2276-2315.

5.Righini M, Perrier A, De Moerloose P, Bounameaux H. D-Dimer for venous thromboembolism diagnosis: 20 years later. J Thromb Haemost. 2008;6:1059 -1071.

6.Di Nisio M, Squizzato A, Rutjes AW, Buller HR, Zwinderman AH, Bossuyt PM. Diagnostic accuracy of D-dimer test for exclusion of venous thromboembolism: a systematic review. J Thromb Haemost. 2007;5:296 -304.

7.Rectenwald JE, Myers DD Jr, Hawley AE, Longo C, Henke PK, Guire KE, Schmaier AH, Wakefield TW. D-dimer,P-selectin, and microparticles: novel markers to predict deep venous thrombosis. A pilot study. Thromb Haemost.2005; 94:1312-1317.

8.Ingrid Pabinger and Cihan Ay. Biomarkers and Venous Thromboembolism. Arterioscler Thromb Vasc Biol.2009;29:332-336.

9.Vyas SP, Vaidya B. Targeted delivery of thrombolytic agents: role of integrin receptors. Expert Opin Drug Deliv.2009; 6(5):499-508.

10.Lityńska A, Przybylo M, Ksiazek D, et al. Differences of alpha3beta1 integrin glycans from different human bladder cell lines. Acta Biochim Pol 2000; 47(2):427-34.

11.Solovjov DA, Pluskota E, Plow EF. Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2. J Biol Chem 2005; 280(2):1336-45. Epub 2004 Oct.

12.Gerber DJ, Pereira P, Huang SY, et al. Expression of alpha v and beta 3 -integrin chains on murine lymphocytes.ProcNatl Acad Sci USA. 1996; 93(25):14698-703.

13.Bounameaux H, Cirafici P, Schneider PA et al. Measurement of D-dimer in plasma as diagnostic aid insuspected pulmonary embolism. Lancet 337:196-200; 1991.

14.Bozic, M, Blinc A, Stegnar M. D-dimer, other markers of haemostasisactivation and soluble adhesion molecules in patients with different clinicalprobabilities of deep vein thrombosis. Thromb Res 108:107-114; 2002.

15.Declerck PJ, Mombaerts P, Holvoet P et al. Fibrinolyticresponse and fibrin fragment D-dimer levels in patients with deep veinthrombosis. Thromb. Haemostasis 58:1024-1029; 1987.

16.Di Nisio, M.; Squizzato, A.; Rutjes, A. W.;et al.Diagnostic accuracy of D-dimer test for exclusion of venousthromboembolism: a systematic review. J. Thromb. Haemostasis 5: 296-304; 2007.

17.Di Nisio M, Squizzato A, Rutjes AWS, et al. Diagnostic accuracy of D-dimer test for exclusion ofvenous thromboembolism: a systematic review. J Thromb Haemost 2007; 5(2): 296- 304.

18.Kahn SR, Lim W, Dunn AS, et al. Prevention of VTE in nonsurgical patients: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012; 141(2 Suppl):e195S-226S.

19.Le Gal G, Kovacs MJ, Carrier M, et al. Validation of a diagnostic approach to exclude recurrent venous thromboembolism. J Thromb Haemost 2009; 7: 752-59.

20.Palareti G, Cosmi B, Legnani C, et al. D-dimer testing to determine the duration of anticoagulation therapy. N Engl J Med 2006; 355: 1780-89.

21.Prandoni P, Lensing AW, Prins MH, et al. Residual venous thrombosis as a predictive factor of recurrent venous thromboembolism. Ann Intern Med 2002; 137: 955-60.

22.Bounameaux H, Righini M. Thrombosis: duration of anticoagulation after VTE: guided by ultrasound? Nat Rev Cardiol 2009; 6: 499-500.

American College of Chest Physicians, ACCP, has put forward various risk factors of acquired VTE, including surgery, trauma, infection, tumor, aging, pregnancy, longbedding and immobilization, etc [1]. Acute infection is commonly faced in clinical practice, and there is a 2-3 times increased incidence of VTE in patients with communityacquired or hospital-acquired infections [2-4].

Acute venous thrombosis is red thrombus, which is composed of red blood cells,platelets, white blood cells and plasma proteins. In 2011, we reported that the main component of red thrombus in acute PE patients was fibrinogen, rather than fibrin,with only a small quantity of cellular cytoskeletal and plasma proteins [5]. Fibrinogenic thrombus is dissolvable, which can explain why delayed thrombolytic therapy is effective for acute and subacute VTE and thrombi are autolytic in some VTE patients.However, the action mechanism of fibrinogen in thrombosis remains unclear. We hypothesized that, due to the binding of fibrinogens (ligands) and activated receptors on surfaces of various leukocytes, platelets and lymphocytes, the thrombus protein network is constructed and red thrombus forms, with erythrocytes and plasma components filled in the spaces. In our previous studies [6, 7], genomics analysis, proteomics analysis and bioinformatics analysis of acute venous thrombi of PE patients confirmed that integrins β1, β2 and β3 were the core proteins of acute venous thrombi. Integrin β1 is mainly localized on lymphocytes, integrin β2 is mainly localized on neutrophils and integrin β3 is mainly localized on platelets. Moreover, activated integrin β3 was involved in the accumulation of platelets, receptors of integrin β2 and β3 bound to fibrinogens to form the biofilter-like grid structure of thrombi filled with red blood cells, forming red thrombi.

Acute infection is a risk factor of VTE, but why is acute infection prone to VTE?Is there any relevance between core proteins of acute venous thrombi-- integrin β1, β2 and β3 and acute infection? To answer the question, we catched a case-control study,the differential expression of integrin β1 and β2 and β3 was compared between acute infection group and non-infection group, the relative risk of increased expression of integrin β1 and β2 and β3 in acute infection was acquired, and their clinical importance was also investigated.

A total of 230 patients with acute infection diagnosed from April 2011 to April 2012 in the emergency unit were recruited into this study, including 118 males and 112 females,aged 23-93 years, with a mean age of 72.53 years old. The classification of acute infection was according to the previously reported criteria [8], and the patients included 197 cases of respiratory tract infections (pneumonia and bronchitis), 19 cases of urinary tract infection, 19 cases of skin and soft tissue infection, 7 cases of abdominal infection(liver and gallbladder and gastrointestinal tract) and 8 cases of sepsis without clear foci. Among them, 18 cases were complicated with two kinds of infections. All infected patients were diagnosed in our hospital. Meanwhile, 230 age and gender matched inpatients without infection served as the control group, including 114 males and 116 females, aged 21-98 years (mean 70.31 years). Patients with cancer, autoimmune disease or patients taking immunosuppressive drugs were excluded. Patients with clinical symptomatic thrombus were also excluded. This study was approved by the Ethics Committee of Affiliated Tongji Hospital of Tongji University, and informed consent was obtained before study.

Detailed clinical data were collected from each acute infection patient and control patient on admission. Blood routine test, hsCRP and d-dimer were detected. HsCRP was detected by immune scatter turbidimetry, using Siemens BNII specific protein and auxiliary reagent. D-dimer was detected by Latex enhanced immune turbidimetric turbidity method, using SYSMEX CA1500 automatic blood coagulation analyzer.Fasting venous blood (2 ml) was collected from the cubital vein in the morning and anticoagulated with EDTA. Two hours later, the anti-coagulated blood was processed as follows.

Monoclonal antibodies against integrin β1 (CD29), β2 (CD18) and β3 (CD61)(BD company) were used to detect the integrin β1, β2 and β3, respectively. Three tag monoclonal antibodies (BECKMAN-COULTER) were used for CD ₃, CD ₄ and CD ₈ detection. In brief, 100 µl of EDTA treated blood was added to each tube and control tube was also included. Then, 20 µl of mouse IgG1-PC5, IgG1-FITC or IgG1-PE was added (20 µl of IgG2-PE was mixed with CD29), followed by addition of corresponding fluorescence antibodies (20 µl). Following vortexing, incubation was done in dark for 30 min at room temperature. Then, 500 µl of hemolysin (BECKMAN-COULTER) was added, followed by incubation at 37°C for 30 min. Following washing, 500 µl of sheath fluid was added to each tube, followed by flow cytometry (EPICS XL-4; BECKMANCOULTER). The PMT voltage, fluorescence compensation and sensitivity of standard fluorescent microspheres (EPICS XL-4; BECKMAN-COULTER) were used to adjust the flow cytometer and a total of 10000 cells were counted for each tube. The corres ponding cell population in the scatterplot of isotype controls was used to set the gate, and the proportion of positive cells was determined in each quadrant (%). SYSTEM-II was used to process the data obtained after flow cytometry.

SPSS18.0 statistical software was used for statistical analysis. Normality test was performed for all measurement data using the Kolmogorov-Smirnov test, with P> 0.05 as normal distribution. Data of normal distribution were expressed as means ± SD and were compared with t test between groups. Corrected t-test was applied when heterogeneity of variance. Non-normal data were expressed as median P ₅₀ and interquartile range(P ₂₅-P ₇₅), and group comparison was analyzed using nonparametric test (Mann-Whitney U test). Measurement data were compared using chi-square test. The association degree between two categorical variables was analyzed by calculating the relative risk (Relative Risk, RR). P <0.05 was considered statistically significant for all tests.

A total of 230 patients with acute infection and 230 patients without acute infection matched in age and sex were enrolled into this study. Among the 230 patients with acute infection, 197(85.7%) were diagnosed with respiratory tract infections (RTI), 19(8.3%)were diagnosed with urinary tract infection (UTI), 19(8.3%) were diagnosed with skin infection, 7(3.0%) were diagnosed with intra-abdominal infection and 8(3.5%) were diagnosed with septicaemia. Patients' demographics, type of infection and comorbidities are shown in Table 4-2-1.

The median levels of D-Dimer and HsCRP were all significantly higher in patients with acute infection when compared with patients without acute infection (P=0.000 and 0.000)(Table4-2-2).

When comparing cellular immunity related variables (CD ₃, CD ₄, CD ₈, CD ₄/CD ₈,CD ₁₆CD ₅₆ and CD ₁₉), we found significant differences of CD ₁₆CD ₅₆ and CD ₁₉ (P=0.008,P=0.018) between the two groups. CD ₁₆CD ₅₆ was markedly increased in acute infection patients, while CD ₁₉ was reduced (see Table 4-2-2).

Compared with the control group, the expression of integrin β1, β2 and β3 was markedly increased in the acute infection group (P=0.000, 0.002 and 0.001, respectively)(Table 4-2-3). The relative risk ratio (RR) of increased integrin β1, β2 and β3 in acute infection patients was 1.424 (95%CI: 1.156-1.755, P=0.001), 1.535, (95%CI: 1.263-1.865,P=0.000) and 1.20 (95%CI: 0.947-1.521, P=0.148), respectively (Table 4-2-3, 4).

Combined integrin β1, β2 and β3 analysis (integrin β1, β2 and β3 increased at the same time means rise, otherwise normal) showed the relative risk ratio (RR) of increase in acute infection patients was 2.962 (95%CI: 1.621-5.410, P=0.001) (see Table 4-2-4).

Acute infection is a risk factor of thrombotic diseases [9-12]. In 2006, Smeeth et al. reported [2] that the risk for DVT was increased by 1.91 folds within 2 weeks to 6 months after acute respiratory tract infection. Similar finding is also noted in patients after urinary infection. Recently, in two large case-control studies [3,4], results also demonstrate that acute infection increases the risk for VTE by 2~3 folds after adjustment of other risk factors of VTE, and this risk is the highest within 2 weeks after acute infection.

Our results showed that the expression of integrins β1, β2 and β3 was markedly increased in patients with acute infection. The risk for increased integrin β1, β2 and β3 expression in patients with acute infection was respectively 1.424, 1.535 and 1.20 folds higher than that in patients without acute infection. Combined integrin β1, β2 and β3 analysis showed that the relative risk for patients with acute infection was 2.962 folds higher than that in patients without acute infection. These results may explain the increased risk of VTE in acute infection patients. For patients with acute infection and increased integrin β1, β2 and β3, early treatment and prevention should be given, in order to reduce the incidence of VTE in high-risk groups.

Integrins are cell adhesion receptors, and they play an important role in the interaction between cells and extracellular matrix (ECM), and cell-cell interactions [13].Integrins are heterodimers consisting of noncovalently linked α and β transmembrane glycoprotein subunits. They consist of at least 18 α and 8 β subunits, producing 24 different heterodimers [14]. The α and β subunits separate from each other once the integrin is activated, and then the α subunit binds the ligand. The β1 subunit is expressed mainly on cell surface of lymphocytes, and its ligands consist of laminins,collagens, thrombospondin, vascular cell adhesion molecule 1 and fibronectin [14,15].The β2 subunit is distributed on cell surface of neutrophils and monocytes, and ligands for this subunit include fibrinogen, complement component iC3b, intracellular adhesion molecule-1, factor X and so on [16,17]. The β3 subunit is observed on platelets, and this subunit binds fibrinogen, fibronectin, vitronectin von Willebrand factor (vWF) and thrombospondin [18,19]. At rest, the integrin receptor does not bind to corresponding ligand. When the α subunit is separated from the β subunit, the integrin is activated. The α subunit mainly mediates the reversible binding of receptor to corresponding ligand.The β subunit is responsible for the signal transduction and the regulation of integrin affinity [20-22].

In addition, our results revealed that the acute infection patients had a tendency toward disordered cellular immunity. Our previous studies [23,24] also showed that the VTE patients had compromised cellular immunity. These findings suggest that acute infection patients with compromised cellular immunity have an increased risk for VTE. A weakened immune system could be the basic condition of VTE occurrence.When the immune system can not timely and ef f ectively remove intravenous antigens of heterotypic cells, the platelets and white blood cells will be activated and bound to fibrinogens to form the biof i lter-like grid structure of thrombi, which are filled with red blood cells, forming red thrombi. The disease process was from the body's defense to venous thrombosis.

(Published: International Journal of Medical Sciences2015; 12(8): 639-643)

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22.Humphries MJ. Integrin structure. Biochem Soc Trans. 2000;28(4):311-39.

23.Lemin Wang, Haoming Song, Zhu Gong, Qianglin Duan, Aibin Liang. Acute pulmonary embolism and dysfunction of CD ₃CD ₈ T cell immunity. Am J Respir. Crit. Care Med.2011;Dec 184:1315.

24.Haoming Song, Lemin Wang, Zhu Gong, Aibin Liang, Yuan Xie, Wei Lv, Jinfa Jiang, Wenjun Xu, Yuqin Shen.T cellmeiated immune def i ciency or compromise in patients with CTEPH. Am J Respir Crit. Care Med.2011;183(3):417-8.

Cancer is one of the most common risk factors of VTE. The incidence of VTE in patients with cancer is about 4%~20%, and it has been a leading cause of death in cancer patients[1-3]. There is evidence showing that about 20% clinical first-episode patients with idiopathic VTE have been diagnosed malignant tumor in 6 months to 2 years. The prevalence of VTE in patients with malignancy is 4-7 times higher than that of patients without malignancy [4,5]. VTE has been an important contributor to morbidity and mortality among patients with cancer [6]. Why do malignancy patients have a high incidence of VTE? The molecular mechanisms are not clear. Acute venous thrombosis is red thrombus, which is composed of red blood cells, platelets, white blood cells and plasma proteins. In 2011, we reported that the main component of red thrombus in acute PE patients was fibrinogen, rather than fibrin, with only a small quantity of cellular cytoskeletal and plasma proteins [7]. In our further studies, genomics, proteomics and bioinformatics analyses of acute venous thrombi of PE patients confirmed that integrins β1, β2 and β3 were the core proteins of acute venous thrombi [8,9]. Integrin β1 is mainly localized on lymphocytes, integrin β2 is mainly localized on neutrophils and integrin β3 is mainly localized on platelets. Moreover, activated integrin β3 is involved in the accumulation of platelets and the receptors of integrins β2 and β3 are bound to fibrinogens to form the biof i lter-like grid structure of thrombi, which are filled with red blood cells to form red thrombi. We also found that the filamentous mesh-like structure was widespread in the veins of cancers, and a large amount of red blood cells and cancer cells were found in this biof i lter-like grid structure [10]. Integrin β1, β2, β3 subunits are core proteins and potential biomarkers of VTE [11]. Is there any relevance between core proteins of acute venous thrombi-integrin β1, β2 and β3 and cancer? In this study we will explore the expression of Integrin β1, β2, β3 subunits in patients with cancer and investigate their clinical importance.

A total of 144 inpatients with cancer diagnosed from April 2011 to April 2012 in the affiliated Tongji Hospital of Tongji University were recruited into this study, including 90 males and 54 females, aged 25-91 years, with a mean age of 67.36 years old. Cancers included lung cancer, intestinal cancer, hepatic cancer, gastric cancer, prostate cancer,breast cancer, esophageal cancer, pancreatic cancer, cervical cancer, kidney cancer,ovarian cancer, bladder cancer, nasopharyngeal cancer and laryngeal cancer. All cancers were confirmed by imaging or pathology. Meanwhile, 200 cases of age and gender matched inpatients without cancer were recruited as the control group, including 114 males and 86 females, aged 21-93 years (mean 68.17 years). Cancer was excluded in the control group by clinical symptoms, signs and imaging. Patients with acute infection,autoimmune disease or patients taking immunosuppressive drugs were excluded.Patients with clinical symptomatic venous thrombus were also excluded. This study was approved by the Ethics Committee of Affiliated Tongji Hospital of Tongji University,and informed consent was obtained before study.

Detailed clinical data were collected from each cancer patient and control patient on admission. Blood routine test, hsCRP and d-dimer were detected. HsCRP was detected by immune scatter turbidimetry, using Siemens BNII specific protein and auxiliary reagent. D-dimer was detected by Latex enhanced immune turbidimetric turbidity method, using SYSMEX CA1500 automatic blood coagulation analyzer. Fasting venous blood (2 ml) was collected from the cubital vein in the morning and anti-coagulated with EDTA. Two hours later, the anti-coagulated blood was processed as follows.

Monoclonal antibodies against integrin β1 (CD29), β2 (CD18) and β3 (CD61) (BD company) were used to detect the integrins β1, β2 and β3, respectively. Three tag monoclonal antibodies (BECKMAN-COULTER) were used for detection of CD ₃, CD ₄ and PC5, FITC, and PE label were used for CD ₈, CD ₃, CD ₄ and CD ₈, respectively. CD ₁₆CD ₅₆ and CD ₁₉ also used PE label. In brief, 100 µL of EDTA treated blood was added to each tube and control tube was also included. Then, 20 µL of mouse IgG1-PC5, IgG1-FITC or IgG1-PE was added (20 µL of IgG2-PE was mixed with CD29), followed by addition of corresponding fluorescence antibodies (20 µL). Following vortexing, incubation was done in dark for 30 min at room temperature. Then, 500 µL of hemolysin (BECKMANCOULTER) was added, followed by incubation at 37°C for 30 min. Following washing,500 µL of sheath fluid was added to each tube, followed by flow cytometry (EPICS XL-4; BECKMAN- COULTER). The PMT voltage, fluorescence compensation and sensitivity of standard fluorescent microspheres (EPICS XL-4; BECKMAN-COULTER) were used to adjust the flow cytometer and a total of 10000 cells were counted for each tube. The corresponding cell population in the scatterplot of isotype controls was used to set the gate, and the proportion of positive cells was determined in each quadrant (%).SYSTEM-II was used to process the data obtained after flow cytometry.

SPSS18.0 statistical software was used for statistical analysis. Normality test was performed for all measurement data using the Kolmogorov-Smirnov test, with P>0.05 as normal distribution. Data of normal distribution were expressed as means ± SD and were compared with student's t-test between groups. Corrected t-test was applied when heterogeneity of variance. Non-normal data were expressed as median P ₅₀ and interquartile range (P ₂₅-P ₇₅), and group comparison was analyzed using nonparametric test (Mann-Whitney U test). Measurement data were compared using chi-square test.The association degree between two categorical variables was analyzed by calculating the relative risk (Relative Risk, RR). P<0.05 was considered statistically significant for all tests.

A total of 144 patients with cancer and 200 patients without cancer matched in age and sex were enrolled into this study. The 144 cancer patients included, 43 (29.86%)lung cancer, 25 (17.73%) intestinal cancer, 17 (12.06) hepatic cancer, 13 (9.22%) gastric cancer, 11 (7.8%) prostate cancer, 10 (7.09%) breast cancer, 6 (4.26%) esophageal cancer,6 (4.26%) pancreatic cancer, 3 (2.13%) cervical cancer, 2 (1.42%) kidney cancer, 2 (1.42%)ovarian cancer, 2 (1.42%) bladder cancer, 2 (1.42%) nasopharyngeal cancer and 2(1.42%) laryngeal cancer. Patients' demographics, type of cancer and comorbidities are shown in Table 4-3-1. The median levels of D-Dimer and HsCRP were all significantly higher in patients with cancer when compared with patients without cancer (P=0.000 and 0.000). In comparisons of the cellular immunity related variables (CD ₃, CD ₄, CD ₈,CD ₄/CD ₈, CD ₁₆CD ₅₆ and CD ₁₉), significant differences were found between the two groups: CD ₃, CD ₄, CD ₄/CD ₈ and CD ₁₉ were markedly decreased in patients with cancer(P=0.004, P=0.000, P=0.000 and P=0.000 respectively), while CD ₈ and CD ₁₆CD ₅₆ were increased (P=0.000 and P=0.035) (Table 4-3-2). When compared with the control group,the expression of integrins β1 and β3 were markedly increased in patients with cancer(P=0.000 and P=0.008), while integrin β2 was only mildly increased in patients with cancer (P=0.274) (Table 4-3-3). The relative risk ratios (RR) of increased integrins β1, β2 and β3 in patients with cancer were 1.655 (95% CI: 1.321-2.074, P=0.000), 1.314, (95% CI:1.052-1.642, P=0.021) and 1.852 (95% CI: 1.097-3.126, P=0.028), respectively. Combined integrin β1, β2 and β3 analysis showed (integrin β1, β2 and β3 increased at the same time means rise, otherwise normal) the relative risk ratio (RR) of increase in patients with cancer was 4.895 (95% CI: 1.645-14.563, P=0.002) (Table 4-3-4).

Integrins are a kind of widespread cell surface receptors, which mediate interactions between cells and cells, and between cells and extracellular matrix (ECM). As signal receptors, integrins play an important role in the cell growth, migration, proliferation and differentiation of many aspects, and are one of the key members of the family of cell adhesion molecules [12]. Integrins are heterodimers consisting of noncovalently linked α and β transmembrane glycoprotein subunits. They consist of at least 18 α and 8 β subunits, producing 24 different heterodimers [13]. The β1 subunit is expressed mainly on cell surface of lymphocytes, and its ligands consist of laminins, collagens,thrombospondin, vascular cell adhesion molecule 1 and fibronectin [14]. The β2 subunit is distributed on cell surface of neutrophils and monocytes, and ligands for this subunit include fibrinogen, complement component iC3b, intracellular adhesion molecule-1,factor X and so on [15]. The β3 subunit is observed on platelets, and this subunit binds fibrinogen, fibronectin, vitronectin von Willebrand factor (vWF) and thrombospondin[16].

Cancer is a risk factor of VTE, and VTE is an important cause of death in cancer [17-19]. This study explored the expression of integrin β1, β2, β3 subunits in patients with cancer, the results showed that integrin β1, β2, β3 subunits were all increased in patients with cancer, among which integrin subunits β1 and β3 were increased significantly. The relative risk ratios (RR) of increased integrins β1, β2 and β3 in patients with cancer were 1.655, 1.314 and 1.852 respectively. Combined integrin β1, β2 and β3 analysis showed that the relative risk ratio (RR) of increase in patients with cancer was 4.895. As core proteins of venous thrombosis, the increased expression of integrins β1, β2 and β3 in patients with cancer may explain the relatively high risk of VTE in cancer patients.

The plasma levels of hsCRP and d-dimer were all significantly higher in patients with cancer in this study. As nonspecific inflammation markers, hsCRP was associated with venous thrombosis [20]. Elevated levels of serum hsCRP are a risk factor of VTE in cancer patients, which shows the role of nonspecific inflammation in the prone of VTE in patients with cancer [21]. Our study has shown that the incidence of VTE in patients with malignant tumor is the result of nonspecific inflammatory repair of small veins after being destroyed by tumor cells invasion, as demonstrated by morphological examination and immunohistochemistry [10]. This is different from infective inflammation. D-dimer is a degradation product of cross-linked fibrin that is formed immediately after thrombin-generated fibrin clots are degraded by plasmin and reflects a global activation of blood coagulation and fibrinolysis. Being the best-recognized biomarker for the initial assessment of suspected VTE, d-dimer has a high sensitivity of 83%-96%, but a poor specificity (around 40%) [22-24]. As core proteins of venous thrombosis, integrins β1, β2 and β3 have been proven to be a new useful biomarker of VTE both with a high sensitivity and an approving specificity in our previous study [11].For patients with cancer who have increased integrins β1, β2 and β3, early treatment and prevention should be given in order to reduce the incidence of VTE in high-risk groups.

In this study, the cellular immune function was reduced or disordered in patients with cancer. Our previous studies had shown that VTE patients had association with compromised cellular immunity [25,26]. A weakened immune system could be the basic condition of VTE occurrence. These findings suggest that malignant tumor patients with compromised cellular immunity possess the intrinsic basic conditions for VTE and thus have an increased risk for VTE.

(published: Int J Clin Exp Med 2015;8(2):2772-2777)

1.Chew HK, Wun T, Harvey D, Zhou H, White RH. Incidence of venous thromboembolism and its effect on survival among patients with common cancers. Arch Intern Med 2006; 166: 458-64.

2.Khorana AA, Liebman HA, White RH, Wun T, Lyman GH. The risk of venous thromboembolism in patients with cancer. American Society of Clinical Oncology. Cancer Thromb; 2008. pp. 240-8.

3.Chew HK, Wun T, Harvey DJ, Zhou H, White RH. Incidence of venous thromboembolism and the impact on survival in breast cancer patients. J Clin Oncol 2007; 25: 70-6.

4.Wun T, White RH. Epidemiology of cancer-related venous throm-boembolism. Best Pract Res Clin Haematol 2009;22: 9-23.

5.Blom JW, Doggen CJ, Osanto S, Rosendaal FR. Malignancies, pro-thrombotic mutations, and the risk of venous thrombosis. JAMA 2005; 293: 715-22.

6.Lyman GH, Khorana AA, Falanga A, Clarke-Pearson D, Flowers C, Jahanzeb M, Kakkar A, Kuderer NM, Levine MN,Liebman H, Mendelson D, Raskob G, Somerfield MR, Thodiyil P, Trent D, Francis CW; American Society of Clinical Oncology. American Society of Clinical Oncology Guideline:recommendations for venous throm- boembolism prophylax is and treatment in patients with cancer.J Clin Oncol 2007; 25: 5490-5505.

7.Wang L, Gong Z, Jiang J, Xu W, Duan Q, Liu J, Qin C. Confusion of wide thrombolytic time window for acute pulmonary embolism: mass spectrographic analysis for thrombus proteins. Am J Respir Crit Care Med 2011; 184:145-146.

8.Xie Y, Duan Q, Wang L, Gong Z, Wang Q, Song H, Wang H. Genomic characteristics of adhesion molecules in patients with symptomatic pulmonary embolism. Mol Med Rep 2012; 6: 585-590.

9.Wang LM, Duan QL, Yang F, Yi XH, Zeng Y, Tian HY, Lv W, Jin Y. Activation of circulated immune cells and inflammatory immune adherence are involved in the whole process of acute venous thrombosis Int J Clin Exp Med 2014; 7: 566-572.

10.Le-Min Wang, Qiang-Lin Duan, Xiang-Hua Yi, Yu Zeng, Zhu Gong, Fan Yang. Venous thromboembolism is a product in proliferation of cancer cells. Int J Clin Exp Med 2014; 7: 1319-1323.

11.Yanli Song , Fan Yang, Lemin Wang, Qianglin Duan, Yun Jin, Zhu Gong. Increased expressions of integrin subunit β1, β2 and β3 in patients with venous thromboembolism: new markers for venous thromboembolism. Int J Clin Exp Med 2014; 7: 2578-2584.

12.Barczyk M, Carracedo S and Gullberg D. Integrins. Cell Tissue Res 2010; 339: 269-280.

13.Cavers M, Afzali B, Macey M, McCarthy DA, Irshad S and Brown KA. Differential expression of beta1 and beta2 integrins and L-selectin on CD ₄ and CD ₈ T lymphocytes in human blood: comparative analysis between isolated cells, whole blood samples and cryopreserved preparations. Clin Exp Immunol 2002; 127: 60-65.

14.Lityńska A, Przybylo M, Ksiazek D, Laidler P. Differences of alpha3beta1 integrin glycans from different human bladder cell lines. Acta Biochim Pol 2000; 47: 427-34.

15.Solovjov DA, Pluskota E, Plow EF. Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2. J Biol Chem 2005; 280: 1336-45.

16.Gerber DJ, Pereira P, Huang SY, Pelletier C, Tonegawa S. Expression of alpha v and beta 3 16-integrin chains on murine lymphocytes. Proc Natl Acad Sci USA 1996; 93: 14698-703.

17.Sorensen HT, Mellemkjaer L, Olsen JH, Baron JA. Prognosis of cancers associated with venous thromboembolism.N Engl J Med 2000; 343: 1846-50.

18.Prandoni P, Falanga A, Piccioli A. Cancer and venous thromboembolism. Lancet Oncology 2005; 6: 401-10.

19.Khorana AA, Francis CW, Culakova E, Kuderer NM, Lyman GH. Thromboembolism is a leading cause of death in cancer patients receiving outpatient chemotherapy. J Thromb Haemost 2007; 5: 632-4.

20.Vormittag R, Vukovich T, Schönauer V, Lehr S, Minar E, Bialonczyk C, Hirschl M, Pabinger I. Basal high-sensitivity-C-reactive protein levels in patients with spontaneous venous thromboembolism. Thrombosis and Haemostasis.2005; 93: 488-93.

21.Kröger K, Weiland D, Ose C, Neumann N, Weiss S, Hirsch C, Urbanski K, Seeber S, Scheulen ME. Risk factors for venous thromboembolic events in cancer patients. Annals of Oncology 2006; 17: 297-303.

22.Bounameaux H, Cirafici P, de Moerloose P, Schneider PA, Slosman D, Reber G, Unger PF. Measurement of D-dimer in plasma as diagnostic aid in suspected pulmonary embolism. Lancet 1991; 337: 196-200.

23.Bozic M, Blinc A, Stegnar M. D-dimer, other markers of haemostasis activation and soluble adhesion molecules in patients with different clinical probabilities of deep vein thrombosis. Thromb Res 2002; 108: 107-114.

24.Di Nisio M, Squizzato A, Rutjes AW, Büller HR, Zwinderman AH, Bossuyt PM. Diagnostic accuracy of D-dimer test for exclusion of venous thromboembolism: a systematic review. J Thromb Haemost 2007; 5: 296-304.

25.Haoming S, Lemin W, Zhu G, Aibin L, Yuan X, Wei L, Jinfa J, Wenjun X, Yuqin S. T cell-mediated immune deficiency or compromise in CTEPH patients. Am J Respir Crit Care Med 2011; 183: 417-8.

26.Wang L, Song H, Gong Z, Duan Q, Liang A. Acute Pulmonary Embolism and Dysfunction of CD ₃ CD ₈ T Cell Immunity. Am J Respir Crit Care Med 2011; 184: 1315.

In this study, we report that there was expression of specific proteins in patients with symptomatic VTE, and the expression of these proteins was compared between VTE group patients and those with different risk factor groups (acute infection, malignancy,autoimmune diseases, trauma /surgery). In addition, the role of these proteins in the risk for VTE was assessed.

The subjects included patients with a definite diagnosis between March, 2011, and Feb,2012, in Tongji Hospital of Tongji University. Patients were grouped by specific diseases based on the national standards by professionals, and the results were analyzed by statistics experts without knowledge of the clinical status of the patients. Treatment decisions were not influenced by the findings of this research.

A total of 1006 subjects (male=53%; female=47%; mean age=67.40±16.26) were enrolled from Departments of Cardiology, Internal Emergency, Oncology, Rheumatology, and Surgical Emergency of Shanghai Tongji Hospital, and divided into six groups.

The VTE group (within 3 months of onset, n=72) consisted of patients with DVT and/or PE, and the diagnosis was confirmed by imaging, and the patients received anticoagulant therapy with low molecular heparin or warfarin orally.

The acute infection group (n=330) included patients with infections of respiratory tract (pneumonia or bronchitis, n=168), urinary tract (n= 54), skin, soft-tissue (n= 25),intra-abdominal (gastrointestinal or hepatobiliary infections) (n=64), or sepsis (no site identified) (n= 19). The patients received anti-infection and comprehensive therapy.

The malignancy group (clinical stage Ⅲ-Ⅳ, n=144) included lung cancer (n=56),gastric cancer (n=23), esophagus cancer (n=12), rectum cancer (n=14), pancreatic cancer(n=4), hepatic cancer (n=9), breast cancer (n=18), and brain cancer (n=8). A total of 47 cancer patients received chemotherapy, and the others received comprehensive therapy including chemotherapy, radiotherapy, immunization therapy and treatment by Chinese herbs.

The autoimmune diseases group (mild to moderate, n=103) included rheumatoid arthritis (n=39), systemic lupus erythematosus (n=32), Sjogren's syndrome (n=17), and connective tissue disease (n=15). They all received conventional immunoregulation and/or Chinese medicine treatments. The trauma /surgery group (n=111) included 79 cases of several trauma (multiple injuries, n=36; head injury, n=28; traumatic fractures, n=15) and 32 surgical patients (cervical or lumbar vertebra surgery, n=16; gastric cancer surgery,n=5; intestinal cancer surgery, n=11) under general or local anesthesia for more than 30 min. The control group (non-risk factor group) included patients with atherosclerotic heart disease or/and hypertension (n=246), but without clinically symptomic VTE, and patients with acute infection, cancer, autoimmune diseases, trauma or recent surgical treatment were excluded.

1) Levels of integrins β1, β2 and β3 in all individuals were detected and analyzed by EPICS XL-4 flow cytometry (Beckman-Coulter) using System Ⅱ software. Fluorescent antibodies were provided by BD Company.

2) Patients were placed in a sitting position, and peripheral blood (2 ml) was collected from the median cubital vein and anti-coagulated with EDTA. After mixing,flow cytometry was done within 2 h.

3) 100 µl EDTA was added to each tube for anti-coagulation. Isotype control was also included. Integrin β1 and integrin β2 were mixed with 20 µl mouse IgG1-PE, and integrin β3 was mixed with 20 µl mouse IgG2-PE. Then, 20 µl fluorescent antibody was added to the above solution. After misce bene, incubation was done at room temperature in dark for 30 min. After addition of 500 µl hemolysin, incubation was done at 37℃ for 30 min. Following washing, 500 µl sheath fluid was added and then detected by flow cytometry.

The standardized BECKMAN-COULTER fluorescent microspheres were used to adjust the PMT voltage, fluorescence compensation, sensitivity, and the detection protocol was determined. A total of 10000 cells were collected in each tube. The scattered plot of isotype control was used for gating at corresponding cells. Integrin β1 and integrin β2 gating were lymphocytes, and integrin β3 gating was platelets. According to the fluorescence intensity in each quadrant, the proportion of positive cells was calculated (%). Data were analyzed with SYSTEM-II software.

Continuous variables were expressed as means ± SD or medians (interquartile range),and categorical variables were expressed as frequencies (percentages). Control ranges of integrin positive cells were determined from control individuals. The control ranges of integrins β1 and β3 positive cells were calculated as the mean ± 2SD, and were 3.14 to 13.90% and 4.39 to 14.7%, respectively (Table 4-4-1). Using the 5th-95th percentiles,we determined control range of integrins β2 positivity (non-normally distributed) of 73.40 to 95.30 (see Table 4-4-1). Integrin levels above the upper limit of the control range were considered positive, while levels below the upper limit of the control range were considered negative (see Table 4-4-1).

A total of 1006 inpatients were divided into: symptomatic VTE group, acute infection group, malignancy group, autoimmune diseases group, trauma /surgery group and control group. Expression of integrins was detected in these groups.

Compared with control group, the integrin β1 expression in VTE group and subjects with different risk factors (acute infection, malignancy and autoimmune diseases)increased markedly (P<0.001, <0.01). However, compared with control group, the integrin β1 expression in trauma /surgery group was not significantly different (P>0.05,Figure 4-4-1).

Compared with the control group, the integrin β2 expression in VTE group increased significantly (P<0.05). Compared with the control group, in different risk factor groups (acute infection, malignancy, autoimmune diseases and trauma/ surgery),the integrin β2 expression was not significantly different (P>0.05, see Figure 4-4-1).

Compared with the control group, the integrin β3 expression in VTE group was significantly elevated (P<0.05). However, the integrin β3 expression levels in different risk factor groups (acute infection, malignancy, autoimmune diseases, trauma/ surgery)were not significantly different (P>0.05, see Figure 4-4-1).

The principal findings in this study are firstly that there is an increased expression of integrins β1, β2 and β3 in patients with VTE. Secondly, in most patient groups traditionally considered at risk of VTE (infection, inf l ammation and malignancy) there was increased expression of β1 but not of β2 or β3. However this increased expression was not found in patients following trauma or surgery, calling into question that such patients are at increased VTE risk in the absence of other factors such as concomitant infection.

Integrins are cell adhesion receptors, which play an important role in the interaction between cells and extracellular matrix (ECM), and in cell-cell interactions [1].Integrins are heterodimers consisting of noncovalently linked α and β transmembrane glycoprotein subunits. They consist of at least 18 α and 8 β subunits, producing 24 different heterodimers [2]. The α and β subunits separate from each other once the integrin is activated, and then the α subunit binds the ligand. The β1 subunit is expressed mainly on cell surface of lymphocytes, and its ligands consist of laminins,collagens, thrombospondin, vascular cell adhesion molecule 1 and fibronectin [2, 3]. The β2 subunit is distributed on cell surface of neutrophils and monocytes, and ligands for this subunit include fibrinogen, complement component iC3b, intracellular adhesion molecule-1, factor X and so on[4,5]. The β3 subunit is observed on platelets, and this subunit binds fibrinogen, fibronectin, vitronectin von Willebrand factor (vWF) and thrombospondin [6,7].

Integrin β1 is mainly expressed on lymphocytes, and increased integrin β1 expression is related to the inflammation, thrombosis, homing of lymphocytes and metastasis of cancer cells. Integrin β2 is mainly distributed on neutrophils and monocytes, and increased integrin β2 expression is associated with inflammation.Integrin β3 is mainly expressed on platelets, and elevated integrin β3 expression suggests the platelet activation which is associated with platelet aggregation and thrombosis.

The study results showed that the expression of integrins β1, β2 and β3 increased signif i cantly in VTE group, and integrin β1 elevated markedly in risk factor groups (acute infection, malignancy and autoimmune diseases), but that of integrin β2 or β3 had no significant difference in all risk factor groups. This suggests that there was difference in the protein expression between VTE group and risk factor groups. The elevated expression of integrins β2 and β3 suggests the activation of neutrophils, monocytes and platelets, which is a basic process in the inflammation and thrombosis. Thus, we speculate that the risk factor groups have activated lymphocytes immune cells, and the VTE group may have activated lymphocytes, neutrophils and platelets. The trauma/surgery group may have no activation of immune cells and platelets and so may not be the “true” risk factor for VTE.

In the present study, the elevated expression of integrins β1, β2 and β3 in VTE group patients was highly consistent with the findings from immunohistochemistry of red thrombus in acute PE patients. In the risk factor groups, those with acute infection,malignancy or autoimmune diseases have increased expression of integrin β1,but the expression of integrins β2 and β3 had no signif i cant dif f erence. This suggests that there is expression dif f erence of core proteins of red thrombus between VTE group and risk factor groups (acute infection, malignancy or autoimmune diseases, trauma/ surgery).In the screening of VTE patients, the increased expression of integrin β1 suggests the elevated risk for venous thrombosis, so prevention against VTE should be done in these patients. The increased expression of integrins β1, β2 and β3 has a value in the clinical diagnosis of VTE. The detection of the expression of integrins β1, β2 and β3 is helpful to diagnose VTE and screen risk people, thus, integrins β1, β2 and β3 may serve as new specif i c protein markers of VTE and risk people.

The current work is based on the previous studies on genomics [8], proteomics [9],bioinformatics and venous thrombosis core protein screened by immunohistochemstry[10,11]. The present study aimed to verify the core protein in VTE group and risk factor groups. Due to the limitations of sample size in our study, further larger-size or multicentral studies on the normal range of the venous thrombosis core protein are still needed.

(published: Am J Transl Res 2015;7(3):624-631)

1.Barczyk M, Carracedo S, Gullberg D. Integrins. Cell Tissue Res 2010;339:269-280.

2.Cavers M, Afzali B, Macey M, McCarthy DA, Irshad S, Brown KA. Differential expression of beta1 and beta2 integrins and L-selectin on CD ₄ and CD ₈ T lymphocytes in human blood: comparative analysis between isolated cells, whole blood samples and cryopreserved preparations. Clin Exp Immunol. 2002 Jan;127(1):60-5.

3.Fiorilli P, Partridge D, Staniszewska I, Wang JY, Grabacka M, So K, Marcinkiewicz C, Reiss K, Khalili K, Croul SE.Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation. Lab Invest. 2008 Nov;88(11):1143-56.

4.Rezzonico R, Chicheportiche R, Imbert V, Dayer JM. Engagement of CD11b and CD11c beta2 integrin by antibodies or soluble CD23 induces IL-1beta production on primary human monocytes through mitogen-activated protein kinase-dependent pathways. Blood. 2000;95(12):3868-77.

5.Schwarz M, Nordt T, Bode C, Peter K. The GP IIb/IIIa inhibitor abciximab (c7E3) inhibits the binding of various ligands to the leukocyte integrin Mac-1 (CD11b/CD18, alphaMbeta2). Thromb Res. 2002 Aug 15;107(3-4):121-8.

6.Fang J, Nurden P, North P, Nurden AT, Du LM, Valentin N, Wilcox DA. C560Rβ3 caused platelet integrin αII b β3 to bind fibrinogen continuously, but resulted in a severe bleeding syndrome and increased murine mortality. J Thromb Haemost. 2013 Jun;11(6):1163-71. doi: 10.1111/jth.12209.

7.Coburn J, Magoun L, Bodary SC, Leong JM. Integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells. Infect Immun. 1998 May;66(5):1946-52.

8.Xie Y, Duan Q, Wang L, Gong Z, Wang Q, Song H, Wang H. Genomic characteristics of adhesion molecules in patients with symptomatic pulmonary embolism. Mol Med Rep 2012;6:585-590.

9.Wang L, Gong Z, Jiang J, Xu W, Duan Q, Liu J,Qin C. Confusion of wide thrombolytic time window for acute pulmonary embolism: mass spectrographic analysis for thrombus proteins. Am J Respir Crit Care Med.2011;184:145-146.

10.Wang LM, Duan QL, Yang F, Yi XH, Zeng Y, Tian HY, Lv W, Jin Y. Activation of circulated immune cells and inflammatory immune adherence are involved in the whole process of acute venous thrombosis Int J Clin Exp Me 2014;7(3):566-572.

11.LeMin Wang, Qiang-Lin Duan1, Xiang-Hua Yi, Yu Zeng, Zhu Gong1, Fan Yang.Venous thromboembolism is a product in proliferation of cancer cells Int J Clin Exp Med 2014;7(5):1319-1323.