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.

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