Venous thromboembolism (VTE) includes pulmonary thromboembolism (PE) and deep venous thrombosis (DVT). Venous thromboembolisms that could be diagnosed in clinics are called dominant VTE. The clinical spectrum of VTE is relatively wide, because VTE can occur in different organs and tissues. However, dormant VTE that is hard to be diagnosed in clinics is often found in autopsy. VTE, including cerebral cortical vein, cerebral venous sinus thrombosis, acute PE, chronic thromboembolic pulmonary hypertension (CTEPH), hepatic venular occlusive disease, Butchart's syndrome,mesenteric venous thrombosis, pelvic venous thrombosis, DVT and PE after surgeries as well as clinically common lower limb DVTs, distributes in diverse clinical disciplines and is a disease faced by almost all the clinical disciplines. Among them, PE has become a global medical care problem due to its high morbidity, misdiagnosis rate and mortality[1, 2]. VTE can be divided into two categories, genetic VTE and acquired VTE. According to the results of epidemiological investigations, the incidence of genetic VTE is relatively low, while most of the VTEs are acquired VTEs. Both of them can be called symptomatic VTE (Figure 2-1-1,2,3) and are hard to be distinguished.

Many risk factors of VTE have been identified by organizations such as the American College of Chest Physicians (ACCP), which has published nine editions of guidelines for VTE diagnosis [3], treatment and prevention from 1995 to 2012. Proposed VTE risk factors include advanced age, infection, malignancy, autoimmune disease,surgery, trauma, pregnancy, long trip syndrome, family history and so on. ACCP has raised the risk stratification of surgical patients. Different measures should be taken in patients with different stratification to prevent VTE. Actually, only a small part of the patients with same risk stratification and same external environment have had VTE. In 2008, Shackfore [4] reported that 84% of the 37619 surgical patients were partly or totally treated and prevented according to the guideline. From 1995, when the first ACCP was published, to 2004, the numbers of symptomatic VTE increased rather than decreased,and there was segregation between preventing risk factors and VTE occurrence.

Thus, here the questions come. Why does the incidence of VTE increase as the age increases? Why does the incidence of VTE stay high in patients with malignancies? Why does only a small part of patients with the same infection develop VTE? Sudden death caused by acute PE resulting from surgeries, pregnancy, delivery or long trip syndrome is always hard to prevent. However, the vast majority of the population will not develop VTE under the same conditions. Both being thrombus, acute arterial thrombus is white thrombus but acute venous thrombus is red thrombus. What does the pathological difference mean? Thrombolytic therapy is effective for arterial thrombosis within several hours after onset, but venous thrombosis, with a wide thrombolytic time window, can be delayed to several days, 2 weeks, or even longer. What causes the difference in the thrombolytic time window between acute venous and acute arterial thrombus? Acute venous thrombus can autolyze, while arterial thrombus cannot. For VTE patients, oral anticoagulants are usually recommended for 3, 6 or 12 months and occasionally lifelong. Currently, there are no objective criteria for individual evaluation that complicates the selection of anti-coagulation therapy by physicians. Furthermore, even with standard anti-coagulation therapy and international normalized ratio (INR), some patients still develop CTEPH. Thus, the physicians are extremely puzzled about anticoagulant usage.

Query raised in clinics usually originates from clinical practices. The risk factors of VTE are only the clinical phenomenon of incident VTEs and the summary of evidencebased medicine, not the essence of this disease. So far, the medical resources put into the global prevention of VTE have not had the predicted effects. The reasonable explanation of this separation phenomenon between clinical prevention and treatment guidelines of VTE and clinical practices is that the etiology and pathogenesis of VTE are still unclear.

The acute venous thrombosis freshly taken out from the body is red to the naked eyes and is fragile. It is composed of red blood cells, platelets, white blood cells and plasma proteins under microscope.

Mass spectrographic analyses have shown that a majority of the proteins in the thrombus are fibrinogen, the the remaining being mainly serum albumin and cytoskeletal proteins [5]. The reversible combinations between the receptor and their ligand-- fibrinogens suggest that acute venous thrombus is easy to autolyze and delayed thrombolysis is effective, so it should be easy to lyse the thrombus through interventional fragmentation.

Acute venous thrombus is red and is composed of red blood cells, platelets, white blood cells and fibrinogen. But how does fibrinogen bind to blood cells in the formation of thrombus? MS/MS and bioinformatics analyses of thrombus in patients with acute PE have shown that subunits β1, β2 and β3 in integrins are the core proteins of acute venous thrombus.

Integrins, important members in cell adhesion molecule family, mediate the adhesion between cells and between cells and extracellular matrix (ECM) and are involved in the bidirectional signaling transduction between cells and ECM. They combine to different ligands in various cellular processes, either physiological or pathological, such as angiogenesis, invasion, metastasis, inflammation, wound healing and coagulation [6].

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 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 subunits are identical but the α subunits are distinct. At rest, the α subunit is covered by the β subunit and thus the integrin 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 the integrins [7-9].

The β1 subunit is mainly found on the lymphocytes and platelets, and its ligand includes laminin, collagen, thrombospondin, fibronetin and VCAM-1. The β2 subunit is mainly distributed on the neutrophils and monocytes, and its ligand includes fibrinogen, ICAM,factor X and ic3b. The β3 subunit is mainly observed on the platelets, and its ligand includes fibrinogen, fibronetin, vitronectin, vWF and thrombospondin [10-12].

In a study, the authors collected the thrombi from patients with acute PE, and detected the expression and distribution of integrins β1, β2 and β3 in thrombi and ligands of integrin subunits β1, β2 and β3 by immunohistochemistry [13]. They found that the dark-brown integrin β1 was expressed on the lymphocytes, but no expression of laminin, fibronectin, collagen-Ⅰ or collagen-Ⅱ was observed on the lymphocytes.Meanwhile the dark-brown integrin β2 was expressed on the neutrophils and bound to fibrinogen. ICAM, factor X and iC3b were expressed on neutrophils, whereas the darkbrown integrin β3 was expressed on platelets, which aggregated as a coral-like structure to become thrombotic skeleton, and these platelets bound fibrinogen to construct mesh structure(Figure 2-3-1). No expression of fibronectin, vitronectin or vWF was observed on the platelets; the dark-brown factor Xa was distributed on the mesh-like structure,which was composed of fibrin/fibrinogen.

More than 30 years ago, people invented different types of artificial vena caval filter used in clinics, the mechanism of which is preventing the genesis of PE by blocking the deep venous thrombi flowing back to pulmonary arteries through the filter. Core proteins of thrombi, integrins β2 and β3, bind their ligand fibrinogen to construct mesh structure, which becomes a nest-like filter in thrombi.

As a precise and perfect life entity, the human body always functions towards the beneficial aspects and the balance, stability and extension of internal and external environments. The construction of intravenous biological filter is the result of self-regulation of human body. But what are the effects of biological filter on human body? And what are the meanings of the body regulation and the construction of a biological filter?

The author of a recent report found a biological filter in veins of the resected sigmoid colon adenocarcinoma tissues [14], in which malignant cells were found in the biological filter and interfered with hematogenous metastasis of cancer cells.

We previously reported virus-like microorganisms in the T lymphocytes of peripheral blood of a PAH and VTE patient with low CD ₃ and CD ₈ [15] and rod-like bacteria in the phagocytes of peripheral blood in patients with recurrent PE [16]. The heterophilic antigens (pathogenic microorganisms or cancerated cells) cannot be timely or effectively cleared, indicating that the human body needed to build a new defense line when losing functions of immune cells.

The defense system inside the human body is the immune system. Simply speaking,it is the function of immune system that removes all the foreign agents from human body, including external pathogenic microorganisms, implants, foreign bodies and toxins from wounds, and internal senile cells and malignant cells. Through a long-term evolution, the immune system with both inside and outside functions has developed perfect tissue structures and exceptional functions. The immune system can be divided into innate immune system and adaptive immune system. The innate immunity,also called congenital immunity, is the oldest existing functionality in the biological evolution, which eliminates the encountered foreign bodies immediately through its components, macrophages, granulocytes, natural killer cells and complement system.However, the adaptive immunity, also called specific immunity, is acquired after birth with a characteristic of having memories, which can specifically attack the same invasive foreign agents.

The process of immune balance is a complicated process. When the functions of immune cells blancing collapse, the human body will lose the defensive functions of immune system, external pathogenic microorganisms or tumor cells will intrude into the circulating system, and then the alternative defensive barrier is activated inevitably. It is a basic principle of releasing compensatory reserved functions. The formation of intravenous biological filter indicates the existence of external pathogenic microorganisms or tumor cells in the veins and the supplement of the lost immune functions.

Human genomics is the study of human genetics with characteristics of wholeness,comprehensiveness and directivity. Although there is difference in the gene-guided protein synthesis among individual proteins, which requires to be validated by proteomic and cytological studies, comparisons of gene expression patterns among different groups and functional analysis of differentially expressed genes may provide a general view and a direction for the understanding of mechanisms underlying the pathogenesis of diseases. This is a unique feature of genomics and cannot be replaced by other methods.

Gene Ontology analysis of the gene expression in PE group targets the significant downregulations of T cell immune complexes and T cell immune functions when compared with the controls [17].

Phagocytes, NK cells, complement system and cytokine related gene expressions in both PE group and control group were compared.

mRNA expressions of pattern recognition receptors (TLR2, TLR4,CD14, MYD88, MRC1L1, MRC2, MSR1, SCARA5, SCARB2, SCARF1 and SCARF2) and opsonic receptors (CR1, FCGR2A, FCGR2B, FCGR3A and FCGR3B) were up-regulated in phagocytes of the PE group compared with controls, among which TLR4, CD14,MYD88, SCARB2, SCARF2, CR1 and FCGR2A were significantly up-regulated (P<0.001),indicating the increased functions of neutrophils and monocytes [18].

Compared with control group, NK cells related gene expressions declined overall in PE group, among which mRNA expressions of seven tenth of the lectin-like receptors and Natural cytotoxic receptors were significantly down-regulated(P<0.05), suggesting reduced functions of NK cells killing target cells directly [18].

There are 14 genes of complement early components. In PBMCs from PE patients, expression of the genes encoding C1qα, C1qβ, C4b and Factor P was significantly greater (P<0.01) than that in controls. Gene expression of MBL and MASP1 was lower (P<0.05) in PBMCs from PE patients compared with controls, and seven genes of the complement late components were detected. In PE patients, mRNA expression of C5 was significantly up-regulated (P<0.05), whereas C6, C7 and C9 were significantly down-regulated (P<0.05) compared with controls. In PE patients, expression levels of all the seven genes mRNAs were up-regulated, and mRNA expressions of CR1,integrin αM, integrin αX and C5aR were significantly up-regulated (P b 0.01) compared with controls. Gene expressions of complement regulators C4b binding protein, α(C4BPα), C4b binding protein, β (C4BPβ), Factor H, Factor I, CD59, CD55 and CD46 in PBMCs from PE patients and controls were detected. CD59 and CD55 mRNAs were both significantly up-regulated (P<0.05), while Factor I mRNA was significantly downregulated (P<0.05) in PBMCs from PE patients than controls, and the other 3 genes showed down-regulated trend. mRNA expression of various components, receptors and regulators of the complement pathways were unbalanced in PE patients, indicating the interruption of complement system cascade reactions and the loss of complement mediated membrane attacking functions [19].

In PBMCs from PE patients, the expression levels of genes encoding IFNα5,IFNα6, IFNα8, IFNα14, IFNκ, IFNω1 and IFNε1 were significantly lower than those detected in PBMCs from controls (P<0.05). IFNγ mRNA expression was significantly downregulated in PBMCs from PE patients compared with controls (P<0.01) [20].

A total of 37 interleukin genes were detected. In comparison with the control, the expression levels of 12 genes were downregulated specifically IL1A,IL9, IL17B, IL19, IL23A and IL25 (P<0.05), IL2, IL3, IL13, IL22, IL24 and IL31 (P<0.01),and those of two of the genes, IL10 and IL28A, were upregulated (P<0.05), in the PE patients. The imbalance of Th1/Th2 manifests as reduced cell-mediated immunity [20,21].

Twelve genes encoding CXC chemokines were detected. In PE patients, mRNA expression levels of Cxcl1, Cxcl2, Cxcl6, Cxcl13 and Cxcl14 were significantly upregulated (P<0.05), and Cxcl10 mRNA expression levels were significantly downregulated compared with controls (P<0.01). Twenty-three genes encoding CC chemokines were examined and the mRNA expression levels of CC chemokines were significantly lower in PE patients than controls (P<0.01) [20].

Thirty-eight genes encoding members of the TNF superfamily and TNF receptor superfamily were examined. In PE patients, the mRNA expression levels of TNF superfamily members 1, 9 and 13, and TNF receptor superfamily members 1A, 1B,9, 10B, 10C, 10D and 19L, were significantly upregulated (P<0.05), whereas TNF receptor superfamily members 11B, 19 and 25, were significantly downregulated compared with controls (P<0.05) [20].

Six genes encoding colony stimulating factors were detected and the mRNA expression levels of granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF),erythropoietin (EPO), thrombopoietin (THPO) and mast cell growth factor (KITLG)were significantly lower in PBMCs from PE patients than controls (P<0.05) [20].

Eight genes associated with transforming growth factor(TGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF)were detected. The mRNA expression levels of TGFβ1, TGFβ1-induced transcript 1,EGF and VEGF were significantly upregulated (P<0.01), whereas TGFβ3 mRNA was significantly downregulated (P<0.05) in PBMCs from PE patients compared with controls [20].

From the characteristics of a variety of cytokine mRNA expression levels in PE patients, we conclude that the immune function and the ability of clearing viruses,intracellular bacteria and parasites are reduced in PE patients [20].

In patients with PE, the expression of the majority of integrin mRNAs located on leukocytes and platelets was significantly upregulated. The expression of mRNAs related to L-selectin and P-selectin glycoprotein ligand was significantly upregulated,while the expression of mRNA related to E-selectin was significantly downregulated.The expression of mRNAs related to classic cadherins and protocadherins was downregulated, and the expression of mRNAs related to vascular endothelial cadherin was significantly downregulated; the expression of mRNAs related to the immunoglobulin superfamily had no obvious difference between the 2 groups.In conclusion, we demonstrated that, in symptomatic PE patients, the adhesion of leukocytes and platelets was enhanced; the activation of endothelial cells was obviously weakened; the adherens junctions among endothelial cells were weakened, with the endothelium becoming more permeable [22].

Among the 13 leukocyte-related integrin mRNAs, integrins β1 and β2 mRNAs expressions were upregulated in the PE group, compared with the controls (P<0.05). Of the 7 platelet-related integrin mRNAs, integrins β2 and β3 mRNAs expressions were upregulated in the PE group, compared with the controls (P<0.05). Among the 11 other integrin mRNAs, 6 were upregulated (of which 3 significantly) in the PE group (P<0.05)and 5 were downregulated (of which 3 significantly) (P<0.01). It can be concluded that most leukocyte- and platelet- related integrin mRNAs were upregulated in the PE group, as well as fibronectin- and fibrinogen- related integrin mRNAs [22].

T and B lymphocyte related gene expressions in both PE group and control group were compared.

Of the 6 genes of T cell immunological synapse, receptor complex, plasmalemma and receptors mRNAs, ZAP70, CD247 and GZMB mRNAs were downregulated in the PE group, compared with the controls (P<0.05), while GZMA,CD3G and CD3D mRNAs downregulated in the PE group, compared with the controls(P<0.01) [18].

mRNA expressions of 82 genes involved in B cell activation were detected.(i)B cell receptor: In PE patients, expressions of LYN, CD22, SYK, BTK, PTPRC and NFAM1 were signif i cantly higher, whereas expressions of FYN, FCRL4 and LAX1 were significantly lower than the control group. (ii)T cell dependent B cell activation:In PE patients, mRNA expressions of EMR2, TNFSF9, CD86, ICOSLG, CD37 and CD97 were signif i cantly up-regulated, whereas SPN mRNA was signif i cantly downregulated compared with the control group. (iii)T cell independent B cell activation:LILRA1 and TLR9 mRNAs were signif i cantly up-regulated in PE patients compared with the control group. (iv)Regulators: In PE patients, expressions of the genes including CR1, LILRB4 and VAV1 were signif i cantly higher, whereas expression of SLAMF7 was signif i cantly lower than those in the control group. (v) Cytokines: In PE patients, expressions of genes including LTA and IL10 were signif i cantly higher,whereas expressions of L1A, IFNA5, IFNA6, IFNA8, IFNA14, IL2, IL13 and IFNG were signif i cantly lower than those in the control group. It is indicated that Deferential gene expressions in dif f erent stages of B cell activation suggest the decrease or disorders of B cell function [23].

The whole genomics results showed significantly decreased functions of T lymphocytes, disorganized functions of B lymphocytes and complements, and inflammations with enhanced immune adherence.

We have found that [24], among the familial VTE patients, pore forming protein gene mutations located in NK cells, T cells and complements, were detected in 3 patients with the results of a combined cell deficiency of killing targeted cells. NK cells, CD ₈ T cells and complements form the membrane attack complex. The steps of killing foreign or pathological antigen cells include membrane perforation and release of the granzyme.The mutation of pore forming protein gene can lead to membrane perforation function loss. The genomics of symptomatic PE patients showed the significant downregulation of T cell granzyme mRNA expression, which indicates the decreased function of killing cells. Both the mutation and downregulated granzyme mRNA expression suggest the decreased killing activities of immune cells.

Smeeth [25] reported that the risk for DVT was increased by 1.91 folds within 2 weeks to 6 months after acute infection. In two large case-control studies [26, 27], results also demonstrate that acute infection increases the risk for VTE by 2~3 folds after adjustment of other risk factors of VTE. We found DVTs in multiple organs including pulmonary arteries, kidney, liver and pancreas during autopsy of SARS patient [28], indicating that the genesis of VTE was related to virus infection.

In acute PE [29], the decreased CD ₃ and CD ₈ levels, and the increased CD ₄/CD ₈ ratio, were similar to those in CTEPH [30]. We have reported that the functions of CD ₃,CD ₈, CD ₁₆CD ₅₆ and CD ₁₉ were compromised or disordered in more than 95% acute symptomatic VTE [31].

T cells are key parts of immune cells, as they regulate the cellular and humoral immunity, and phagocyte functions in innate immunity through Th1, Th2 and Th17.The immune functions decrease when T cell functions decline. The process of immune balance is a complicated process. Different immune functioning status, including normal, decreased and disturbed immune functioning status, decides different body status. The collapse of the balancing functions of immune cells indicates that immune cells in the innate and adaptive systems have a state of no function or dysfunction.We observed significantly decreased T cell functions through cytology, and decreased whole immune cell functions through genomics, indicating the collapse of the balancing functions of immune cells in VTE patients.

Malignancy is one of the risk factors of VTE, and VTE is one of the leading causes of death in patients with malignancy [32-34]. The prevalence of VTE in patients with malignancy is 4-7 times higher than that of patients without malignancy [35, 36].

Malignancy results when cancer cells cannot be effectively and timely cleared by the immune system. The author of a recent study [14] reported that necrosis,granulation tissues, disruption of small veins, and dark brown fibrinogens in veins formed mesh-like structure in sigmoid colon adenocarcinoma. Also, a necrotic region in poorly differentiated gastric carcinoma presented with exudation of a large number of red blood cells. Venous thrombosis and hemorrhage serve as inevitable products in the proliferation of cancer cells and disruption of the surrounding veins and arteries.

In a previous study [38], we recruited a total of 1006 subjects and divided them into VTE group, risk factor groups and control (non- risk factor) group without a difference in age. Flow cytometry was performed to detect the expression of the core proteins in venous thrombi. The normal range of integrins β1, β2 and β3 were generated from healthy people.

Compared with that in the 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 the control group, the integrin β1 expression in trauma /surgery group was not significantly dif f erent (P>0.05). The elevated expression of integrin β1 in VTE group, acute infection group, malignancy group and autoimmune diseases group was highly consistent with the increased core protein expression in thrombi, indicating that VTE patients shared the same protein expression with patients with acute infection, malignancy and autoimmune diseases. However, similar elevations of protein expression were not found in patients with trauma /surgery.

Compared with the control group, the integrin β2 expression in VTE group increased signif i cantly (P <0.05). However, compared with control group, the integrin β2 expression in subjects with different risk factors (acute infection, malignancy and autoimmune diseases,trauma /surgery group) was not significantly different (P>0.05), suggesting no same elevation of protein expression was found in patients with acute infection group,malignancy group and autoimmune diseases group, trauma /surgery group.

Compared with control group, the integrin β3 expression in VTE group was elevated (P<0.05). However, the integrin β3 expression in different risk factor groups(acute infection, malignancy, autoimmune diseases, trauma/ surgery) were not signif i cantly dif f erent (P>0.05).

We found that integrins β2 and β3 are the proteins to distinguish patient with VTE from patients with risk factors, and are also the key proteins for determining the occurrence of VTE. The increased integrin β1 is the characterized expression in patients with risk factors. However this increased expression of integrins β1, β2 and β3 was not found in patients following trauma or surgery, calling into question that such patients may have no VTE risk.

The author of a report [39] adopted ROC curve analysis to assess diagnostic performance of these core proteins in 120 VTE patients. 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, 0.821 and 0.731, respectively. Optimum cutoffs of integrin β1, integrin β2 and integrin β3 calculated according to Youden's index were 10.29pg/ml,91.10pg/ml and 10.35pg/ml, respectively. With these optimum cutoffs, the sensitivity,specificity, positive predictive value and negative predictive value of integrin β1 were 80.3%, 83.7%, 71.1% and 89.3%, respectively; integrin β2 78.6%, 73.7%, 59.4% and 87.6%;integrin β3 68.4%,71.2%, 54.3% and 81.8%. The AUC of combined three integrins was 0.916, the sensitivity, specificity, positive predictive value and negative predictive value were 84.6%, 90.8%, 81.7% and 92.0%, respectively. Clinical researches have confirmed significantly increased expression of integrins β1, β2 and β3 in VTE patients, which had relatively high specificity and sensitivity.

Taken together, with underlying conditions of collapsed immune cell balancing function, cells infected by pathogenic microorganisms and malignant cells may trigger intravenous immune adhesive inflammation, which is the defensive reaction in human body to establish the intravenous biological filter preventing infected cells and malignant cells from flowing back. However, the red thrombus forms and the defense transfers into thrombotic disease, when the filter is filled with red blood cells.

People with collapsed immune cell balancing functions are the vulnerable groups for venous thromboembolism. In a patient of venous thromboembolism,it is very possible that the genesis of venous thrombosis was triggered by infected cells or malignant cells. Only under the condition of immune cell balancing function collapse, the risk factors, such as advanced age, infection, malignancy, autoimmune disease, pregnancy, long trip syndrome, as well as family history may cause venous thrombosis. However, even with definable risk factors, there is no risk of getting venous thromboembolism in patients without collapsed immune cell balancing function.

Core proteins in acute venous thrombus are integrins β1, β2 and β3. The increased integrin β1 is the characterized expression in people with risk factors of VTE. However this increased expression of integrins β1, β2 and β3 was not found in patients following trauma or surgery calling into question that such patients may have no VTE risk.Therefore, trauma or surgery may be not the “true” risk factor for VTE.

It should be extended to the upstream of the disease from the middle and lower reaches to prevent the VTE, to decrease the incidence and to increase the cure rate.Hence, it is not enough only to prevent and reduce the known risk factors. Rather, the adjustment and improvement of immune cell balancing function, lowering the stress level inside human body, and restoring the balance of neuroendocrine functions, are new contents of prevention, treatment and rehabilitation of VTE.

(Published: Int J Clin Exp Med 2015;8(11):19804-19814)

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