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.)

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