European Journal of Cardiovascular Medicine

Venous thromboembolism is a multifactorial disease and is associated with multiple genetic and acquired factors. Globally the VTE incidence is 0.75 -2.69 per 1000 population and in the elderly population, it is 2.7 per 1000.  Venous thrombosis includes deep vein thrombosis (DVT) and Pulmonary embolism (PE), where DVT is the most common and invariably associated with morbidities and mortalities, whereas, PE is a life-threatening condition with a survival rate of less than 60% [1]. The occurrence of recurrent VTE is about one-third in VTE patients.  The diagnosis of VTE is a challenging point in the case of severely ill patients and it is more difficult in PE cases. The pathophysiology of VTE is still in the exploratory stage.  Thrombosis formation is mostly initiated by coagulation due to activation, adhesion and aggregation of platelets and deposition of fibrins.

Platelet deposition forms an obstruction at the site of injury. In thrombosis formation and development of VTE, primary hypercoagulability due to deficiencies in anticoagulant proteins is attributed to 5- 10% whereas, acquired factors such as surgery, malignancy, and immobilization are more associated with VTE[2]. Early diagnosis and treatment of different types of thrombosis are of paramount value. Thereby, the predictive marker for low and high risk for VTE may improve the diagnosis efficiency and early therapeutic action. The role of pro-inflammatory biomarkers and acute phase reactant proteins could give an insight into the pathophysiology and mechanism of coagulation at different sites.

Coagulation cascade and disease manifestation:

Blood coagulation is a series of protease conversions, to form thrombin by converting soluble fibrinogen to insoluble fibrin monomer followed by cross linked fibrin polymer and fibrin clot. Protease activator receptors play a major role in the cascade through extrinsic, intrinsic and Common pathway with functional components of tissue factors (TF). TF is released into the micro particle circulation [3]. Under pathological condition, elevated level of TF trigger the thrombosis formation and leads to a sequence of events including DVT, PE and other thrombosis. Figure 1 shows a schematic flow chart of thrombin formation, in which, extrinsic pathways are regulated by tissue factors VII, VIIa, X, Xa, III and II and the intrinsic pathway is by Tissue factors XII, XIIa, XI, XIa, IX, VIIIa and VWF. In common pathway, factor XIII (F13) and XIIIa are involved in Fibrin clot formation [4].    

Currently, the diagnosis of DVT, Pulmonary Embolism & VTE is confirmed by Doppler, CT angiography, pulmonary angiography, or pathological examination of thrombus extract during surgery or autopsy. Despite extensive research, not a single biomarker reliably predicts VTE occurrence or recurrence because the pathophysiology of VTE is multifactorial with a complex interaction between genetic, environmental and acquired risk factors [6]. 

Potential Protein biomarkers differentially expressed in VTE manifestation: A total of 44 potential biomarkers were reviewed based on their expression level in VTE. A meta-analysis has reported 5 inflammatory proteins in 1385 cases of VTE, which revealed that monocytes, CRP and IL6 are significantly elevated in acute conditions. The authors concluded that hs-CRP and CRP were found to be significant markers for VTE recurrence [8]. A meta-analysis of blood markers detected in VTE cancer patients revealed 174 biomarker measurements and concluded with 9 biomarkers, which were significantly associated with VTE [9]. Based on other available literature, the differentially expressed proteins identified through different detection methods from plasma/serum are listed in the table 1. 

Based on the functionality and differential expression in VTE, a total of 44 proteins were searched from the literature of 40 published articles. The identified proteins are classified into different sections such as lipoprotein, glycoprotein, Coagulation factors, inflammatory proteins and other cytosolic proteins. Lipoproteins regulate the transportation of dietary lipids, from the liver to peripheral tissue. It plays a major role in cardiovascular diseases especially lipoprotein a (Lpa). An elevated level of Lpa is strongly associated with atherosclerotic cardiovascular disease (ASCVD) [32]. Pro-inflammatory phospholipids are involved in disease manifestation due to the presence of lysine binding sites and is similar to plasminogen, which inhibits fibrinolysis. This inhibition strongly plays a significant role in pulmonary embolism [33]. Other lipoproteins are also differentially expressed in pulmonary embolism such as haptoglobin, serum amyloid A1 (SAA1), retinol-binding protein, and inter alpha trypsin inhibitor heavy chain H4 [10,11]. Biological processes such as protein folding, secretion, adhesion, stability in circulation and signal transduction are associated with glycoprotein. The majority of proteins glycosylated during the synthesis of carbohydrates and a change in glycosylation pattern is a signature of disease. Platelet glycoproteins are actively participate in thrombosis formation [34]. The present study reviewed many published articles, which revealed that glycoproteins such as osteopontin, fibronectin precursor, alpha 1 microglobulin, clusterin, leucine-rich alpha 2 glycoprotein are elevated in pulmonary embolism and lipopolysaccharide-binding proteins are upregulated in VTE [10,11,12-15]. Coagulation factors such as FII, FV, FVII, FVIII, FIX, FX, FXI and Von Willebrand are commonly involved in blood clot formation. Any disproportional change in the expression level leads to thrombosis formation majorly in the deep vein and pulmonary artery and vein [35]. Under VTE manifestation, differentially expressed coagulation factors are FVIII, FIX, and FXI. Factor VIII is an acute phase response protein and the expression level increases in inflammation conditions. In cases of provoked VTE, the level of Factor VIII increases five fold and is identified as a risk factor for recurrent VTE [36]. Soria et al. reported that protein C and FVIII were jointly influenced in thrombosis cases [37]. Factor IX is a vitamin K-dependent glycoprotein that plays a role in hemostasis. It is activated by tissue factors and leads to form a fibrin clot. B Furie et al suggested that Factor IX and Factor VIII have similar effects of high levels of clotting factors on thrombotic risk [38]. Factor X is one of the coagulation factors and occupies a central position in the coagulation system and is a key driver in thrombin formation. In extrinsic and intrinsic pathways factor X is converted to FXa [39]. Factor XI is a 160 KDa disulfide-linked dimeric protein, activated by FXIa and contributes to hemostasis. It is a target site for the therapeutic action of VTE. 53 clinical trials based on FXI inhibitors have been registered. Among them, abelacimab is in phase II trials in comparison to apixaban or dalteparin towards prevention, and treatment of tumor-associated VTE and the completed trials have shown that FXI / FXIa inhibitors are safe and effective in therapeutic application prospects. Arterial thrombosis and inflammation are interlinked; DVT and arterial thrombosis share the same risk factors with having elevated C - reactive protein (CRP). CRP promotes a prothrombotic effect and induces tissue factors, which leads to an extrinsic coagulation cascade. Several studies have reported the elevation of inflammatory markers like CRP, interleukins, ILBeta, IL6, IL8, IL10, IL17, Il15 Receptor, interferon (IFN gamma) and ST2 and monocyte chemotactic protein 1 in acute VTE  patients [17,18,25-28]. Other inflammatory proteins such as D-dimer, selectin family (E-selectin, L-selectin and P-selectin) and micro particles, are reported with a significant increase in expression in VTE manifestation and disease progression [24,28,30,31]. The decreased levels of Calprotectin were reported in VTE patients [11] As D-dimer is a degradation product of cross-linked fibrin, formed when thrombin converts fibrinogen into fibrin, so D-Dimer is considered a significant diagnostic marker for VTE. Several studies reported that D-dimer is a negative predictor and non-specific to DVT [40,41]. Thus it remains as controversial. After completion of anticoagulation therapy, the levels were significantly reduced to half from 500 ng/ml among 247 patients. Few articles reported that VTE recurrence occurred due to discontinuation of oral anticoagulation therapy [43].The association between CRP and VTE has been reported in different prospective studies as observed in the oncologic and obese population. In the acute stage of VTE, CRP activates immune cells (neutrophils, monocytes and macrophages) through the classical complement pathway. As CRP is a pentameric non-glycosylated protein consisting of five identical subunits synthesized in hepatocytes [43,44] and a family member of of Pentraxin along with serum amyloid P component protein (SAP) and Female protein (FP). SAP binds with amyloid fibrils, DNA, chromatin, fibronectin, C4-binding protein and glycosaminoglycan. In response to inflammation, the production of CRP is stimulated by different types of cytokines (IL 6, IL 1, and TNF). Thus, CRP is known to be an acute phase reactant. CRP rises in wide varieties of infections and inflammations.  It initiates precipitation, agglutination and capsular swelling. It has been shown that altered CRP reacts with the FC receptor of monocytes, and lymphocytes and activate the platelet. Therefore, CRP is considered a diagnostic biomarker in monitoring chronic inflammatory conditions promising biomarker for risk stratification in case of short-term mortality, bleeding cases and recurrence due to anticoagulant withdrawal [45]. Selectins are carbohydrate-binding adhesion molecules that actively participate in immune regulation. E selectin is recognized as a cytokine-inducible glycoprotein, which is mostly involved in the new adhesion of molecules.  A strong association of E-Selectin with recurrent VTE has been observed in which a single nucleotide polymorphism “Ser128Arg” was detected in  E-Selectin. An in vitro analysis shows that this polymorphism alters ligand affinity, regulates the interaction of leukocyte-endothelial cell interactions and enhances tissue factor-mediated coagulation, atherosclerosis, myocardial infarction and endotoxin-trigger [46].

In thrombus formation, the interaction of P selectin and PSGL-1 plays a major role.  P selectin influences fibrin deposition in the thrombus. Rectenwald et al. reported that P-selectin concentration is high in patients with acute DVT and has the clinical applicability to assess the risk of DVT [46]. It is a transmembrane glycoprotein (M. W~140kDa) present in storage granules of endothelial cells (weibal-palade bodies) and stimulated by thrombin and other mediators, intersected with L and E selectin. CD 62 is known to be associated with platelet association [47,48]. L-selectin (Leu-8-TQ1) mostly circulates in lymphocytes, Neutrophils and monocytes. CD36 increases the number of circulating micro particles [49]. Tissue factor activity is significantly correlated with micro particles. In the mice model authors reported that micro particles amplify, the coagulation by binding factor VIIa and are considered an independent risk factor during acute thrombogenesis [50].

Protein-protein interaction construction:

A literature review on differentially expressed proteins as involved in VTE manifestation revealed that maximum number of proteins are lipoproteins, glycoproteins, inflammatory proteins and a few are cytosolic proteins. A STRING search tool was used to construct a protein-protein network between differentially expressed proteins in VTE manifestation,  as mentioned in table 1. As VTE is a multifactorial disease, the network speculates the connection and constructed 31 nodes and 116 edges with PPI enrichment p-value <1.0e-16. (Figure 2) Based on their pleiotropic effects, antagonistic master cytokines, CRP, IL6, CXCL8(IL8), IL17 A, SPP, selectins, VWF and SERPINE 1 are centrally located in the network with direct connection with tyrosine-protein kinase JAK 2, PDGEFRB, Tissue factors (TFPI, F12, F8, F9), S100AB, SAA1 and HLA-G.  These clusters are indirectly connected to other clusters leucine-rich glycoprotein (LRG1), fibrinogen protein (FGG), extracellular chaperon (CLU), 35 KDa inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), haptoglobin (HP), Calcium 2⁺ dependent actin filament serving protein (SCIN) and retinol transporter (STRA6). Majorly all the proteins are over-expressed except a few such as S100A8, which are downregulated under pulmonary embolism and deep vein thrombosis condition but this is closely connected to IL6, serum amyloid protein (SAA1) and plasminogen activator inhibitor (SERPINE). Five proteins such as Bleomycin hydralase (BLMH), Extracellular matrix protein -Tenascin N (TNN), Follistain-related protein 3 (FSTL 3), deoxyribonuclease gamma (DNASE 1 L3) and Calcium/calmodulin-dependent protein kinase type 1 (CAMK1) are independently activated and not connected the identified network. These proteins can be a potential target point for disease management [51].

Gene ontology and pathway enrichment analysis:

The network analysis extended the views of pathway involvement through a KEGG pathway enrichment analysis which revealed that the majority of genes participated in the protein translation, which is similar to the pathways and part of the complement and coagulation cascade, malaria pathogenesis, signaling pathway in diabetic complications, other signalling pathway (IL17 & P13K-AKT) cell adhesion molecules, cellular senescence,  tyrosine kinase inhibitor, rheumatoid arthritis and ECM receptor interaction. Figure 3 and 4 explains the involvement of biological processes as derived from the gene ontology based on the protein network.  In Figure 4, it has shown that the interacted proteins are responsible for acute inflammation.

Figure 1: Schematic diagram of coagulation cascade revealing the activators and inhibitors in fibrin clot formation

Figure 2: A protein-protein interaction network consisting of 31 nodes and 116 edges was constructed by using the STRING online tool with PPI enrichment p-value <1.0e-16. Further analysis of k-means clustering reports three clusters; Red colour for cluster 1 corresponds to Acute inflammatory response, the Green colour for cluster 2 corresponds to the acute phase and the blue colour for cluster 3 as calcium-dependent (Ca²⁺) actin filament-serving protein (SCIN). The dotted lines are for edges. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins)[52]

Figure 3: KEGG pathway enrichment analysis in PPI (Protein protein Interaction) by using by using STRING Protein- protein interaction tool. The purple colour shows lower p-value while orange colour shows higher p-value (p-value < 1.0e-16)

Abbreviations: KEGG: Kyoto Encyclopedia of Gene and Genome 

Figure 4: Gene Ontology enrichment analysis in PPI by using STRING Protein- protein interaction tool. The purple colour shows lower p-value while orange colour shows higher p-value (Protein protein interaction enrichment p-value < 1.0e-16)

Tables & figure legends

Table 1: A list of differentially expressed proteins in venous thromboembolic disorder

At present, radiological diagnosis of VTE is one of the reliable ways to diagnose VTE, but it is difficult in severely ill patients because of the uncomfortable invasion and complex procedures. Though VTE is a multifactorial disease, pro-inflammatory biomarkers are more desirable for the early diagnosis of thrombosis and critically ill patients. As a therapeutic aspect, D-dimer and Factor VIII have been proven for the prediction of severity in VTE and treatment action on the duration of anticoagulant use. Lipoproteins, glycoproteins, and inflammatory proteins have the potential for diagnosis and therapeutic action. The interaction of proteins confirmed that CRP, IL6, IL8, IL17, selectin family, JAK-STAT group and tissue factor proteins including VWF proteins are significantly interconnected in disease manifestation. The mechanism of P-selectin, FVIII and IL-10 in coagulation gives a new insight into the pathophysiology and inhibition of anti-inflammatory cytokines in VTE, which may help in dealing with inflammation due to DVT /PE and will give some hope in immunotherapy implementation. Genetic factors are also essential for the pathogenesis of DVT. Further investigations, including full genome sequencing and genome-wide association studies (GWAS), lipidomics may help to elucidate the mechanisms underlying the risk of thrombosis and therapeutic strategies for precision medicine to reduce morbidity and mortality.

Author Contribution: Study design, conceptualization, literature search, Interpretation, Interaction analysis, KEGG pathway analysis and Original draft, done by BP.  Literature search and data collection done by BP & NR. Critical review and final approval done by CKRM, BP, HT and SA.

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