Acute venous thrombus is characterized by easy degradation and autolysis. Delayed thrombolysis is effective on acute massive pulmonary embolism (PE); usually only low molecular heparin is used to dissolve thrombus in submassive PE. Interventional fragmentation is adopted to quickly lyse the massive thrombus and effectively recover the blood flow in the pulmonary artery when thrombolysis is contraindicated in patients with acute massive PE. However, the arterial thrombus lacks this kind of characteristics.

Acute venous thrombus is red thrombus, which is composed of red blood cells,platelets, white blood cells and plasma proteins under the microscope (Figure 3-1-1).

Although the principle in the treatment of venous and arterial thrombosis is identical,the thrombolytic time window differs largely. Thrombolytic therapy is effective within several hours after onset for arterial thrombosis but as long as several days, two weeks or even longer for venous thrombosis. The mechanism underlying the difference in the thrombolytic time window between venous and arterial thromboses still remains unclear.

Pulmonary artery catheter angiography was performed in a 31-year-old patient who got dyspnea without any definite cause and PE diagnosis was confirmed. The catheter was inserted to the pulmonary artery, and the embolus was obtained for mass spectrographic analysis of thrombus proteins. Results showed that a majority of proteins were less than 130 kDa with great differences in abundance. The proteins with the relative abundance of trace to 34.34% could be observed (Figure 3-2-1).

Mass spectrographic analyses showed that a majority of the proteins were fibrinogen, and the remaining included serum albumin and cytoskeletal proteins.

Early thrombolytic therapy may bring favorable effects [1-3]. Furthermore, some patients can benefit from thrombolytic therapy even 6~14 days after PE [4]. In our experience, when the thrombolytic therapy was applied in patients more than 2 weeks after PE, favorable outcome could still be achieved, although the mechanism underlying the wide thrombolytic time window in PE patients remains unclear. According to the phlebothrombosis theory, in veins the blood flow is slow, and the thrombus is rich in fibrin and red cells, only with a small amount of platelets. Ten days after the onset of acute PE, the embolus was found to be red embolus, flexible, elastic, and fragile. Our mass spectrographic analysis showed that the main component of thrombus in acute PE was fibrinogen, with some serum albumin and cytoskeletal proteins. Fibrinogen makes the embolus fragile, which explains the reason of wide thrombolytic time window and also explains why PE patients with stable hemodynamics may benefit from anticoagulant therapy alone, and interventional thromboclasis is still effective for acute PE patients [5]. The thrombus of VTE in pathology mainly includes red thrombus and mixed thrombus, with a small mount of white thrombus. The protein component of red thrombus in acute PE is mainly fibrinogen, which can convert to fibrin, but this needs some time. This change needing the time may explain why VTE patients may benefit from anticoagulant therapy alone and the reason of wide thrombolytic time window.

(Published: Am J Respir Crit Care Med 2011;184:145-6.)

1.Urokinase pulmonary embolism trial. Phase 1 results: a cooperative study. JAMA. 1970 Dec 21;214(12):2163-72.

2.Goldhaber SZ, Haire WD, Feldstein ML, Miller M, Toltzis R, Smith JL, et al. Alteplase versus heparin in acute pulmonary embolism: randomised trial assessing right-ventricular function and pulmonary perfusion. Lancet.1993;341:507-11.

3.Goldhaber SZ, Kessler CM, Heit JA, Elliott CG, Friedenberg WR, Heiselman DE, et al. Recombinant tissue-type plasminogen activator versus a novel dosing regimen of urokinase in acute pulmonary embolism: a randomized controlled multicenter trial. J Am Coll Cardiol. 1992;20:24-30.

4.Daniels LB, Parker JA, Patel SR, Grodstein F, Goldhaber SZ. Relation of duration of symptoms with response to thrombolytic therapy in pulmonary embolism. Am J Cardiol. 1997;80:184-8.

5.Wang L, Wei L, Liu Y, Li X, Guo X, Zhi J, et al. Optional therapeutic strategies based on clinically different types of acute pulmonary embolism. Chin Med J (Engl). 2003;116:849-52.

In 2011, we reported that the main protein component of red venous thrombus in APE was fibrinogen, rather than fibrin, with only a small quantity of cellular cytoskeletal and plasma proteins [1].

The report explained why the delayed thrombolytic therapy and thrombus fragmentation through a catheter are effective for acute VTE. However, the location and distribution of fibrinogen in thrombosis remain unclear. In addition, it has been reported that the use of antiplatelet drug aspirin alone for prevention and treatment of VTE cannot achieve good outcomes [2], suggesting that the role of platelets in the occurrence of VTE needs to be re-clarified.

Pathologically, there is mainly red venous thrombus in acute VTE, being composed of erythrocytes, platelets, leukocytes and proteins such as fibrinogen. Fibrinogen is a key protein in the coagulation system, and it consists of a symmetrical heterodimer. The binding of fibrinogen to leukocytes and platelets in the venous thrombus is involved in the pathogenesis of venous thrombus. We hypothesized that, due to the binding of fibrinogens (ligands) and activated receptors on the surfaces of leukocytes, platelets and lymphocytes, the thrombus protein network is constructed and red venous thrombus forms with erythrocytes and plasma components being filled in the protein network spaces.

Several 5-15mm red venous thrombi weighing 10-20 g were extracted from the pulmonary artery of 4 male patients (39, 45, 50, 61 years) with APE and the femoral vein of a 50-year-old male by femoral venous puncturing using a 7F catheter (Metronic USA).Tandem mass spectrometry was performed for 2 cases, and pathological analysis was performed for the other 3 cases.

Acute PE thrombi-MS/MS (LTQ, Thermo Finnigan USA) (sample preparation,sample pre-isolation Figure 3-3-1 (left), peptide segment enzymolysis and fragment sequence data Figure 3-3-1 (right))-database-retrieve proteins - corresponding genes-Gene Ontology analysis - differential genes - differential proteins - KEGGPathwaygeneNetwork - the core proteins of the thrombus protein network.

The data on peptides following tandem mass spectrometry were subjected to bioinformatics analysis, the proteins corresponding to peptides were precisely determined, and the corresponding genes were searched.

The ways in which interaction is performed were integrated from 1) the KEGG database in which protein interaction, gene regulation and protein modification are shown [4]; 2) the studies with high throughout detection; 3) studies reporting the interaction among genes. The pathways in KEGG database were employed to analyze the interaction among genes with a software from KEGGSOAP [5] (http://www.bioconductor.org/packages/2.4/bioc/html/KEGGSOAP.html), including the following three relationships:

ECrel: enzyme-enzyme relation, indicating two enzymes catalyzing successive reaction steps; PPrel: protein-protein interaction, such as binding and modification.

GErel: gene expression interaction, indicating relation of transcription factor and target gene product.

The interaction among genes is not confined to a specific pathway, which is different from the KEGG-Pathway database. On the pathways in which gene interaction acts, the downstream and upstream genes of screened genes are searched.The overlapping genes between screened genes and their downstream and upstream genes are further analyzed, and the pathways in which screened genes interact with other genes are identified. The genes are symbolized with circle which is then marked with different colors depending on the up-regulation/down-regulation, difference/non-difference. One or more pathways may be present and expressed with lines characterized by arrows with different shapes. Binding among proteins: two proteins bind to form a complex, which has no direction and a line without arrow is used.Binding induced activation leading to increase in expression: protein A may activate the gene transcription of protein B leads to the increase in gene expression, which has a direction and is expressed with an arrowheaded line. Activation: protein A may activate the functions of protein B via interaction, which has a direction and is expressed with an arrowheaded line. Inhibition: protein A may inhibit the functions of protein B via interaction, which has a direction and is expressed with a “T” shaped line.

Informatics showed that the core proteins were integrins in the protein network of embolus of APE (Figure 3-3-2).

Venous red thrombus is constituted by coralline skeleton and fibrinogenic filamentous sieve to possess the function of biological venous filter. The filter is filled with blood cells, mainly erythrocytes, so red thrombus is formed. Our findings demonstrate that the subunits β1, β2 and β3 in integrins are the core proteins of the network of venous red thrombi. In the red thrombi, β1 is localized on the platelets and lymphocytes, β2 is mainly found on the leucocytes and β3 is predominantly observed on the platelets. Results from bioinformatics analysis are consistent with those from immunohistochemistry. Integrins are important members in cell adhesion molecule family and mediate the adhesion between cells and between cells and extracellular matrix (ECM).

Integrin is a transmembrane heterodimer composed of subunits α and β at a ratio of 1∶1. To date, a total of 18 α subunits and 8 β subunits have been identified and they can form 24 functional heterodimers which may be classified into 8 groups (β1-β8) on the basis of β subunit. In the same group, the β subunit is identical, but the α subunit is distinct. At rest, the α subunit is covered by the β subunit and thus the integin is unable to bind to ligands. Following activation, the extension of the β subunit exposes the α subunit. The α subunit mainly mediates the specific and reversible binding between integrins and their ligands, and the β subunit dominates the signal transduction and regulation of affinity of integrins [6,7,8] (Figure 3-3-3,4,5).

The β1 subunit is mainly found on the lymphocytes and platelets, and its ligands include laminin, collagen, thrombospondin, fibronetin and VCAM-1 [6,7]. The β2 subunit is mainly distributed on the neutrophils and monocytes, and its ligands include fibrinogen, ICAM, factorX and ic3b [6,7,10,11]. The β3 subunit is mainly observed on the platelets, and its ligands include fibrinogen, fibronetin, vitronectin, vWF and thrombospondin [6,7,12].

The activated integrins in β1, β2 and β3 groups can bind to corresponding ligands via the α subunit, mediating the adhesion between cells and between cells and ECM.

From the receptor and ligand relationship, β1 intergrins mediate the homing of lymphocytes, intercellular adhesion and adhesion between cells and matrix protein,and also mediate the adhesion between platelets and blood vessel endothelium. β2 integrins mediate adhesion between cells and matrix protein. β3 intergrins mediate the aggregation of platelets and the adhesion of platelets to the basement membrane involving in thrombosis. The formation of venous red thrombus can be explained as a process of adhesion between receptors and corresponding ligands on cells. Two receptors on platelets bind to one ligand fibrinogen in a β3 dependent manner leading to the aggregation of platelets [13-16], and the skeleton of thrombus is formed. The results suggest that the platelets aggregated to become the skeleton structure of a thrombus.The β3 subunit on platelets and β2 subunit on leucocytes can bind to the fibrinogen,forming a filamentous sieve in which blood cells are filled. The skeleton structure and the filamentous sieve lead to the formation of biological venous filter. The main protein of thrombus is fibrinogen, the receptor of which is β3 intergrin. The platelets aggregated coralline skeleton structure and the filamentous sieve are both related to β3 intergrins.So, integrin β3 plays important roles in venous thrombosis. We have found the reason why the antiplatelet drug aspirin alone for prevention and treatment of VTE cannot achieve good outcomes, which is because aspirin binds to different receptors of platelets,not β1 or β3 receptors.

Bioinformatics analysis was employed to analyze the data from tandem mass spectrometry of proteins in thrombi, and the core proteins in thrombi were determined.

1.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.

2.Watson HG, Chee YL. Aspirin and other antiplatelet drugs in the prevention of venous thromboembolism. Blood Rev. 2008;22(2):107-16.

3.Jin WH, Dai J, Li SJ, Xia QC, Zou HF, Zeng R. Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification. J Proteome Res. 2005; 4(2):613-9.

4.Kanehisa M, Goto S, Furumichi M, Tanabe M, Hirakawa M. KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 2010;38(Database issue):D355-60.

5.Antonov AV, Schmidt EE, Dietmann S, Krestyaninova M, Hermjakob H. R spider: a network-based analysis of gene lists by combining signaling and metabolic pathways from Reactome and KEGG databases. Nucleic Acids Res.2010;38(Web Server issue):W78-83.

6.Takada Y, Ye X, Simon S. The integrins. Genome Biol. 2007;8(5):215.

7.Var der Flier A, Sonnenberg A. Functions and integrins. Cell and tissue research.2001;305:285-298.

8.Xiong JP, Stehle T, Diefenbach B, et al.Crystal structure of the extracellular segment of integrin alpha Vbeta3.Science. 2001; 294(5541):339-45.

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

10.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.

11.Gerber DJ, Pereira P, Huang SY, Pelletier C, Tonegawa S. Expression of alpha v and beta 3 integrin chains on murine lymphocytes. Proc Natl Acad Sci U S A. 1996; 93(25):14698-703.

12.Lityńska A, Przybyło M, Ksiazek D, Laidler P. Differences of alpha3beta1 integrin glycans from different human bladder cell lines. Acta Biochim Pol. 2000; 47(2):427-34.

13.Hughes PE, Pfaff M. Integrin affinity modulation. Trends Cell Biol. 1998 Sep;8(9):359-64.

14.Kasirer-Friede A, Kahn ML, Shattil SJ. Platelet integrins and immunoreceptors. Immunol Rev, 2007;218: 247-264.

15.Mendolicchio GL, Ruggeri ZM. New perspectives on von Willebrand factor functions in hemostasis and thrombosis. Semin Hematol, 2005; 42(1): 5-14.

16.Quinn MJ, Byzova TV, Qin J, et al. Integrin alphaIIbbeta3 and its antagonism. Arterioscler Thromb Vasc Biol,2003; 23(6): 945-952.

We have reported that the main component of acute venous thrombi is fibrinogen[1]. In our previous study, the thrombi were collected from patients with acute PE,and tandem mass spectrometry and bioinformatics were employed to determine that integrin subunit β1, β2 and β3 are core proteins of acute venous thrombi.Immunohistochemistry was performed to investigate the expression and cell distribution of integrins β1, β2 and β3 in acute venous thrombi and the binding with different ligands in these cells. We aimed to explore the role of immune cells in the process of acute venous thrombosis.

Thrombi (n=5-8; 5-15 mm in length and 10-20 g in weight) were collected from the pulmonary artery of a patient with acute PE and samples were prepared for pathological examination.

After preparation of thrombus samples, HE staining, immunohistochemistry and Masson staining were performed. The following reagents were used in this study:integrin β1∶1∶50 (abcam B3B11), integrin β2∶1∶150 (abcam MEM-48), integrin β3∶1∶150 (abcam PM6/13); ligand anti-Laminin antibody 1∶50 (abcam14055),ligand anti-Fibronectin antibody 1∶50 (abcam2413), ligand anti-Collagen Ⅰ antibody 1∶50 (abcam34710), ligand anti-Collagen Ⅱ antibody 1∶50 (abcam34712), ligand anti-fibrinogen antibody1∶100 (abcam ab34269), ligand anti-Factor X antibody1∶50(abcam ab11871), ligand anti-Factor Xa heavy chain antibody 1∶50 (abcam ab140112),ligand anti-C3/C3b antibody1∶50 (abcam ab11871), ligand anti-ICAM1 antibody 1∶50(abcam124759), ligand Von Willebrand Factor antibody 1∶50 (abcam11713), ligand anti-Vitronectin antibody1∶50 (abcam28023).

(1) Acute venous thrombi were red thrombi in which there are cord-like structures,and the spaces were filled with a large amount of aggravated red blood cells and nucleated blood cells (Figure 3-4-1).

(2) Immunohistochemistry showed that integrin β1 was expressed on the lymphocytes (Figure 3-4-2A), but no expression of Laminin, Fibronectin, Collagen Ⅰ or Collagen-Ⅱ (receptors of integrin β1) was observed on the lymphocytes (Figure 3-4-2B, C,D, E).

(3) Immunohistochemistry showed that integrin β2 was expressed on the neutrophils (Figure 3-4-3A), which bound to fibrinogen (Figure3-4-3B). The ICAM, factor X and iC3b were expressed on neutrphils (Figure 3-4-3C, D, E).

(4) Immunohistochemistry showed that integrin β3 was expressed on platelets,which aggregated to be thrombotic skeleton (Figure 3-4-4A) and coral-like structure(Figure 3-4-4B); these platelets bound fibrinogen to construct mesh structure (Figure 3-4-4C). No expression of Fibronectin, Vitronectin or vWF was observed on the platelets(Figure 3-4-4D, E, F).

(5) The thrombi had mesh-like structure (Figure 3-4-5A, Masson staining), in which a large amount of red blood cell dominant blood cells filled (Figure 3-4-5C, Masson staining). In colon cancer tissues, there are widely distributed dark-brown mesh-like structures in the venules (Figure 3-4-5B anti-fibrinogen antibody1:100), in which a variety of cancer cells filled (Figure 3-4-5D).

(6) Dark-brown Factor Xa was distributed on the mesh-like structure, which was composed of fibrin/fibrinogen (Figure 3-4-6A, B).

The integrin family was initially recognized as adhesion molecules mediating the adhesion between cells and extracellular matrix, which leads to the integration of cells.Integrins are widely distributed in human body. A kind of integrin can be distributed in a variety of types of cells, and one cell may have the expression of several integrins. The expression of integrins varies from activation status and differentiation status of cells [2].Integrin is a transmembrane heterodimer composed of α and β subunits at a ratio of 1:1 via the non-covalent bond. A total of 8 β subunits (β1-β8) have been identified in human.Under the quiescent condition, the β subunit covers the α subunit, and thus the integrin fails to bind ligand. After activation of integrin, the β subunit extends and then the α subunit is exposed. The α subunit mainly mediates the reversible binding of integrin to its ligand. The β subunit is responsible for signal transduction and regulation of integrin's affinity [3]. Integrin β1 is mainly expressed on lymphocytes [4], and its ligands include laminin, fibronetin, collagen, thrombospondin and VCAM-1 [5]. The binding of Integrin β1 and its ligands is involved in immune cell adherence, which can provide costimulation for activation of T cells. Integrin β2 is mainly expressed on the neutrophils and monocytes [6], and its ligands include fibrinogen, ICAM, factor X and C3b [7]. The binding of Integrin β2 and ligands is involved in immune cell adherence, inflammation and phagocytosis. Integrin β3 is expressed on the platelets [8] and its ligand includes fibrinogen, fibronetin, vitronectin, VWF and thrombospondin [9]. The binding of Integrin β3 and its ligands is involved in activation and aggregation of platelets.

Many cells are involved in inflammatory immune responses, including lymphocytes, neutrophiles and platelets. Light microscopy showed that the thrombi in acute pulmonary thromboembolism were red thrombi. Immunohistochemistry revealed that integrin β1 was distributed on lymphoctes. Laminin, fibronetin, collagen Ⅰ andⅡ, ligands of integrin β1, were not expressed on these cells. Integrin β2 was mainly distributed on neutrophils. The binding of activated integrin β2 with fibrinogen results in the formation of filamentous mesh. The ligands of integrin β2 (ICAM, factor X and C3b) were expressed on neutrophils, suggesting that the binding of integrin β2 with the ligands is involved in the thrombosis. Integrin β3 is distributed on platelets gathered in different shapes, which bind with fibrinogen to construct the filamentous mesh. No expression of fibronetin, vitronectin or vWF was observed on the platelets. The main protein component of acute venous thrombi is fibrinogen [1]. The result indicates the binding of platelet integrin β3 and neutrophil integrin β2 with ligand fibrinogen in thrombi is the early form of venous thrombosis.

In the thrombi, neutrophils and platelets are activated and bind to corresponding ligands, leading to inflammatory immune adhesion, which finally constructs filamentous mesh, a framework of venous thrombus. When the filamentous mesh is fully filled with red blood cell dominant blood cells, a red thrombus is formed. In the circulation, except for red blood cells, platelets and neutrophils have the largest amount. The binding of integrins on the membrane of platelets and neutrophils and their ligands is directly involved in the formation of acute venous thrombus. The binding of neutrophils and factor X can trigger the coagulation process and the activated factor X is converted to Xa and distributed on the fibrinogen, promoting soluble fibrinogenic thrombi to be transformed to fibrinic thrombi. Acute venous thrombosis is a main activation process of circulating neutrophils and platelets, and it is a whole process of integrin subunits β2 and β3 binding with their ligands, and a process of inflammatory immune adherence triggering coagulation reaction.

Thirty years ago, investigators developed and applied transient or permanent inferior vena cava filter in clinical practice to block the flow back of venous thrombi into the pulmonary artery, which may prevent the occurrence of PE [10]. In the study, the mesh-like structure in thrombi is similar to a biological filter, but what is the function of this mesh-like structure?

We have reported that virus-like microorganisms were observed in cytoplasm and intercellular substance of lymphocytes from peripheral venous blood of VTE patients with pulmonary hypertension and T cell immune dysfunction/disorder [11].We also observed rod-shaped bacteria like microorganisms in apoptotic phagocytes from peripheral venous blood of patients with repeated PE/DVT and T cell immune dysfunction/disorder [12]. We also found DVT in the veins of multiple organs (such as pulmonary artery, kidney, liver and pancreas) of a patient who died of SARS[13]. These findings indicate that the onset of VTE has the involvement of infection of microorganisms. Moreover, the mRNA expression of T cells and NK cells was significantly down-regulated in patients with symptomatic VTE, as demonstrated by genomics data [14]. The amounts of CD ₃, CD ₈ and CD ₁₆CD ₅₆ T cells reduced significantly,the increased CD ₄ level in patients with symptomatic VTE was consistent with findings in genomics [15]. The increased level of integrin subunit β1 in this study indicates the activation of lymphocytes, suggesting that the regulatory function of lymphocytes is enhanced. Malignancy is a disease related to immune dysfunction. Figures 3-4-5 B and D show that the mesh-like structure in the veins of cancer tissue is similar to that in venous thrombi. Furthermore, a variety of cancer cells were observed in this mesh like structure of veins in cancer tissue.

Acute venous thrombosis is an activation process of circulating lymphocytes,neutrophils and platelets, and it is a whole process of integrin subunit β1, β2 and β3 binding with their ligands, and a process of immune adherence, generating biological sieve and triggering coagulation reaction. Thus, we hypothesize that, when the infected cells or cancer cells can not be effectively and timely cleared in the presence of immune dysfunction/disorder, activated neutrophils and platelets bind to their ligands to construct biological filamentous mesh-like structure, which acts as a barrier to block the flow of infected cells or cancer cells. When the filamentous mesh-like structure was fully filled with red blood cell dominant blood cells, red venous thrombi occurred. The defensive biological filamentous mesh-like structure causes venous thrombosis.

(Published: Int J Clin Exp Med 2014;7(3):566-572.)

1.Wang L, Gong Z, Jiang J, Xu W, Duan Q, Liu 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(1): 145-6.

2.Dorner M, Zucol F, Alessi D, Haerle SK, Bossart W, Weber M, Byland R, Bernasconi M, Berger C, Tugizov S, Speck RF, Nadal D. beta1 integrin expression increases susceptibility of memory B cells to Epstein-Barr virus infection. J Virol. 2010 Jul;84(13):6667-77.

3.van der Flier A, Sonnenberg A. Function and interactions of integrins. Cell Tissue Res. 2001; 305(3): 285-98.

4.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.

5.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.

6.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.

7.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.

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

9.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 ay;66(5):1946-52.

10.Athanasoulis CA, Kaufman JA, Halpern EF, Waltman AC, Geller SC, Fan CM.Inferior vena caval f i lters: review of a 26-year single-center clinical experience. Radiology. 2000 Jul;216(1):54-66.

11.Wang L, Gong Z, Liang A, et al. Compromised t-cell immunity and virus-like structure in a patient with pulmonary hypertension. Am J Respir Crit Care Med 2010; 182:434-5.

12.Wang L, Zhang X, Duan Q, Lv W, Gong Z, Xie Y, et al. Rod-like Bacteria and Recurrent Venous Thromboembolism.Am J Respir Crit Care Med. 2012; 186(7): 696.

13.Xiang-Hua Y, Le-Min W, Ai-Bin L, et al. Severe acute respiratory syndrome and venous thromboembolism in multiple organs. Am J Respir Crit Care Med 2010; 182:436-7.

14.Wang H, Duan Q, Wang L, Gong Z, Liang A, Wang Q, Song H, Yang F and Song Y. Analysis on the pathogenesis of symptomatic pulmonary embolism with human genomics. Int J Med Sci 2012; 9: 380-386.

15.Duan Q, Gong Z, Song H, Wang L, Yang F, Lv W, Song Y. Symptomatic venous thromboembolism is a disease related to infection and immune dysfunction. Int J Med Sci. 2012;9(6):453-61.

PE is an acute disease with high mortality and a sudden death rate of about 25% on autopsy [1]. To prevent the onset of PE, different kinds of filters such as nested vena cava filters were developed 30 years ago and had been applied in clinical practice to block DVT thrombi below the inferior vena cava reflowing to pulmonary artery.

In this study, several red thrombi were extracted via a 7F catheter from the pulmonary artery of a 50-year-old male with acute PE. Pathological sections were prepared and Masson staining was done. Light microscopy found spontaneous nest-like biological venous filter within the venous thrombus.

In the thrombus of acute PE, there distributes thrombus skeleton, which connects with filamentous grid to build a nest-like biological venous filter (Figure 3-5-1A), which is mainly filled with red blood cells (Figure 3-5-1B).

The sophisticated body always adjusts to the favorable direction of development and tends to balance stability and continuity between the internal and external environment.Biological venous filter is a result of the body's own regulation. What is the role?Manmade venous filter was used to block thrombus reflowing to pulmonary artery, what about the spontaneous nested biological venous filter within the thrombus blocking?

Smeeth et al. [2] have reported that acute infections were associated with an increased risk of VTE. We have reported that the functions of CD ₃, CD ₈, CD ₁₆CD ₅₆ and CD ₁₉ are compromised or disordered in more than 95% acute symptomatic VTE [3].

We reported that virus-like micro-organisms were detected under electron microscope in the cytoplasm of lymphocytes from the peripheral venous blood of VTE patients with pulmonary hypertension [4]. We also reported that electron microscopy showed rod-like bacteria in apoptotic phagocytic cells from the peripheral venous blood of a patient with recurrent PE/DVT [5]. We found biological venous filter formed in

veins surrounding cancer tissues (Figure 3-5-1C), and the filter was detained with cancer cells (Figure 3-5-1D).

These results indicated that heterophilic antigens (pathogenic microorganisms or cancer cells) can not be timely or effectively cleared, so the biological venous filter becomes the protective screen. This protective screen is the local physical defense line of the body. When the filter is obstructed by blood cells and the blood flow is interrupted,venous red thrombus forms.

In the venous thrombus, there are irregular, coralliform thrombus skeleton and fibrous net, forming the nest-like biofilter (Figure 3-5-2,3-5-3). Thrombus skeleton is a product of platelet aggregation and the fibrous net is made of fibrinogen.

The biological function of biofilter in the venous thrombus is still unclear. We have reported that there are cancer cells in this biofilter, suggesting that this biofilter may hinder the circulation of cancer cells and block the hematogenous metastasis of cancer cells. In addition, we also reported the virus-like microorganisms and rodshaped bacteria in the lymphocytes and neutrophils, suggesting the presence of intracellular infection. The formation of intravenous biofilter means the existence of cells with intracellular infection / cancer cells and the loss of ability of immune cells to effectively clear foreign materials. The intravenous biofilter is a new barrier that spontaneously forms in the vein, indicating the re-construction of human defense system.

1.Lucena J, Rico A, Vázquez R, et al. Pulmonary embolism and sudden-unexpected death: prospective study on 2477 forensic autopsies performed at the Institute of Legal Medicine in Seville. J Forensic Leg Med.2009;16(4):196-201.

2.Smeeth L, Cook C, Thomas S, et al. Risk of deep vein thrombosis and pulmonary embolism after acute infection in a community setting. Lancet 2006; 367:1075-9.

3.Duan Q, Gong Z, Song H, et al. Symptomatic Venous Thromboembolism Is a Disease Related to Infection and Immune Dysfunction Int J Med Sci. 2012; 9(6):453-461.

4.Wang L, Gong Z, Liang A, et al. Compromised t-cell immunity and virus-like structure in a patient with pulmonary hypertension. Am J Respir Crit Care Med, 2010, 182:434-435.

5.Lemin W, Xiaoyu Z, Qianglin D, Wei L, Zhu G, Yuan X, Aibin L, Yenan W. Rod-like Bacteria and Recurrent Venous Thromboembolism. Am J Respir Crit Care Med. 2012;186(7):696.

An 83 year old male received surgical intervention due to adenocarcinoma of the sigmoid colon. An 84-year old male underwent surgical intervention due to gastric cancer. HE staining and immunohistochemistry for fibrinogen (rabbit anti-human fibrinogen antibody [ab34269] abcam, 1∶100) were performed to observe the cancer cells and tissues (Figure 3-6-1, 3-6-2).

In the acute venous thrombus, the filamentous mesh-like structure formed by dark brown fibrinogens was identical to the filamentous mesh-like structure in the veins of cancers. This suggests that the pathogenesis of VTE in patients with cancer is related to the destruction of small veins and the intravenous formation of filamentous mesh-like structure by fibrinogen.

Our study showed the exudation of a large amount of red blood cells and a large amount of fibrinogens deposit in cancer tissues. These findings suggest that the cancer tissues damage the small veins and/or increase vascular permeability, which are characterized by hemorrhagic inflammation and fibrous inflammation. The small veins contain filamentous mesh-like structure formed by fibrinogens, in which cancer cells were found. This structure significantly interfered with the migration of cancer cells.

Fibrinogens convert to fibrins and deposit around cancer cells to form a barrier to block metastasis of cancer cells, which inhibit the migration of cancer cells.Electric microscopy and immunohistochemistry demonstrated the presence of fibrin in the primary cancer and metastatic cancer. These fibrins capsulated the primary cancer cells to inhibit the escape of cancer cells. In addition, these fibrinogens also formed stable skeleton in the extracellular matrix of cancer cells [1]. In the cancer,the intravenous fibrinogen formed mesh-like structure which becomes a barrier inhibiting the migration of cancer cells. The mesh-like structure not only inhibits the hematogenous metastasis of cancer cells but also blocks the back-flow of blood cells.The red blood cell dominant blood cells filling the mesh-like structure may cause VTE, which indicates the shift from defense to the opposite side. Our previous study showed that the main protein component of acute venous thrombi was fibrinogen[2]. Fibrinogens and fibrins constitute mesh-like structure, which becomes a nestlike filter in the veins. The blood cells stay in the filter forming red thrombi. The intravenous mesh-like structure in the cancer was consistent with the mesh-like structure in the venous red thrombi, as demonstrated by morphological examination and immunohistochemsitry.

The proliferation of cancer cells is usually faster than the growth of small blood vessels. Thus, the cancer is susceptible to ischemic necrosis, which is characterized by increase in vascular permeability and disruption of small blood vessels. The malignant tumor may invade the small blood vessels (mainly the small veins), which may also destroy the small vessels. Autopsy of patients with malignancies showed 50% of patients developed concomitant VTE [3]. We speculated that the prevalence of VTE in malignancy patients was higher than 50%. The morphological characteristics of proliferative cancer cells increase the risk for VTE in cancer patients, but the VTE may not be identified at early phases.

About 10-25% of VTE patients are diagnosed as malignancy within 2 years after diagnosis of VTE. Thus, patients with VTE of unknown cause might be a candidate of occult cancer with VTE as a first symptom [4]. This is of important significance for the diagnosis of cancer. On one hand, the cancerous VTE and non-cancerous VTE patients have obvious differences in the treatment, risk for VTE recurrence and survival time;on the other hand, malignant tumor may be diagnosed at an early phase due to the occurrence of VTE as an alarm, which promotes early diagnosis. On the basis of the above findings, the National Institute for Health and Clinical Excellence in England developed a CG144 guideline in 2012, which recommends the screening of malignant tumors in patients older than 40 years and with idiopathic VTE [4]. Roekshana regarded it as a milestone in the prevention and treatment of VTE [3].

Malignancy patients with concomitant VTE have identical nature in the occurrence of VTE to the occult cancer patients with VTE as an initial symptom. In these patients,VTE serves as a product in the proliferation of cancer cells and a result of focal fibrous inflammation after the disruption of small veins in cancers.

(Published: Int J Clin Exp Med 2014; 7: 1319-1323)

1.Hu L, Yang H, Su L, et al. Clinical research of the hypercoagulable state in 180 patients with malignant tumor.Med Res Edu 2010; 27(5): 30-32.

2.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.

3.Shaboodien R, Stansby G, Hunt BJ, Agarwal R. Unprovoked venous thromboembolism: assess for cancer. Lancet Oncol. 2012;13(10):973-4.

4.Chong LY, Fenu E, Stansby G, Hodgkinson S; Guideline Development Group. Management of venous thromboembolic diseases and the role of thrombophilia testing: summary of NICE guidance. BMJ.2012;344:e3979.