Open access peer-reviewed chapter

Thrombosis-Related DNA Polymorphisms

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Nouha Bouayed Abdelmoula and Balkiss Abdelmoula

Reviewed: June 4th, 2021Published: May 4th, 2022

DOI: 10.5772/intechopen.98728

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Venous and arterial thrombosis are complex disorders involving several genetic inherited thrombotic and environmental risk factors as well as many mechanistic pathways including those of hemostatic, inflammatory and oxidative homeostasis. To provide an overview of genetic polymorphisms associated with thrombotic disorders, we studied related pathways and mechanisms of venous and arterial thrombosis along with their genetic polymorphisms in association with their clinical significance. We considered classical polymorphisms in the coagulation pathway factors, particularly the thrombophilia predisposition factors: Factor V, Prothrombin and MTHFR as well as PROC, PROS and antithrombin III. Other known and novel genetic polymorphisms having an impact on the pathogenesis of and the susceptibility to venous and/or arterial thrombotic disorders, in particular those involving inflammatory, immune and oxidant/antioxidant/redox signaling systems, were reviewed.


  • ACE
  • Antioxidant systems
  • Arterial thrombosis
  • Coagulation pathway factors
  • Factor V
  • Genetic polymorphisms
  • Hemostatic systems
  • Leiden
  • Predisposition factors
  • Prothrombin
  • Venous thrombosis

1. Introduction

Thrombotic disorders and their related diseases, particularly cardiovascular diseases, are among the most common causes of morbidity and mortality in the world, causing a heavy burden on public health.

At the era of precision medicine ecosystem, omics technologies are increasingly being used to provide new molecular taxonomy of diseases and more precise approaches to assess risks, to predict and diagnose, and to monitor prognosis, therapeutic management and progression of these diseases.

During the genomic and the post-genomic phases and after the success of the Human Genome project, many genetic markers of complex common multifactorial diseases, in particular thrombosis and cardiovascular diseases have been tested using genetic association studies.

Multiple single and combined genetic variations and polymorphisms especially in the genes of coagulation and hemostasis pathways, as well as in the genes of inflammation and other genes interacting with lifestyle and environmental factors such as immune and oxidative systems were considered.

However, some of those variants remain under debate and clinical genotype–phenotype correlations continue to be uncertain.

To provide an overview of thrombosis-related DNA polymorphisms, we reviewed thrombosis related mechanisms as well as genetic variants associated with arterial and venous thrombosis and embolism and their clinical manifestations.

Classical polymorphisms in the hemostasis and coagulation pathways factors are reported in this first chapter. Besides, other types of genetic polymorphisms and variants having an impact on the susceptibility to venous and arterial thrombotic disease will be documented in a second future part.


2. Definitions and stratification of thrombotic disorders

2.1 Thrombosis vs. embolism

Thrombosis is defined as the formation, development, or presence of a blood clot, known as a thrombus, within a blood vessel that may be either a vein or an artery. The prefix “thrombo” come from the Greek “thrombos” meaning a lump or clump.

When thrombosis detaches and travels through blood vessels to another part of the body, it becomes an embolism.

Thrombosis has catastrophic complications by obstructing blood flow leading to ischemia and even infarction of the tissues supplied by the occluded blood vessels.

Embolism is often considered more dangerous than thrombosis because of its predilection to obstruct the entire blood vessel.

2.2 Arterial thrombosis vs. venous thrombosis

Usually, thrombotic diseases are classified according to their occurrence in the venous system of low flow and pressure or in the arterial system of high-flow and pressure.

Venous and arterial thrombotic disorders have long been viewed as separate pathophysiological entities partly because of the recognizable anatomical differences, despite the idea of a common pathogenesis of all thrombosis which is fundamentally the disturbance of hemostasis [1].

In fact, arterial thrombosis has long been apprehended to be a phenomenon of platelet activation, whereas venous thrombosis has been mainly held to be secondary to the activation of the clotting system [1].

Differences observed in the composition of the thrombi, which are platelet rich thrombi in arterial thrombosis and fibrin rich thrombi in venous thrombosis, and the presence of vascular wall damage in particular atheroma in arterial thrombosis reinforced this dichotomy in the concepts of arterial vs. venous thrombosis [2].

Arterial thrombosis involves the formation of white platelet-rich thrombi that occurs after the rupture of atherosclerotic plaques and the exposure of procoagulant material such as lipid-rich macrophages, collagen, tissue factor and/or endothelial breach, in a high shear environment [3].

In contrast, venous thrombosis is usually associated with plasma hypercoagulability and activation of the clotting system with expression of procoagulant factors on an intact endothelium. This activation is the result of the inflammatory process associated or not to a reduced blood flow or a stasis subsequent to prolonged immobility [1].

Actually, the distinctions are not absolute, and there are many evidences that arterial and venous thrombosis have many common underlying mechanisms involving biological factors either responsible for activating coagulation or inflammatory pathways in both the arterial and the venous systems [4].

Moreover, it was shown that patients with venous thromboembolism are at a higher risk of arterial thrombotic complications than matched control individual supporting the interplay between venous and arterial thrombosis pathogenesis [5].

2.3 Arterial vs. venous thrombotic related diseases

Arterial and venous thrombosis and embolism are associated to a variety of diseases and clinical manifestations such as systemic arterial thrombosis or embolism, ischemic strokes and acute infarction as well as superficial vein thrombosis and acute peripheral venous phlebitis, deep vein thrombosis and acute pulmonary embolism [6]. Obstetrical and placental thrombosis is another clinical presentation of thrombotic disease.

2.4 Coagulopathies vs. thrombophilia thrombotic related states

The definition of the term coagulopathies is controversial. They are various conditions in which the aptitude of blood to clot is impaired. However, the term of coagulopathies is used by many health-professionals to design thrombotic states and disorders of coagulation [7].

The definition of the term thrombophilia is more consensual and refers to inherited defects leading to enhanced coagulation, especially of the venous system. On the other hand and for many authors, thrombophilia may be, inherited or acquired, and the hypercoagulability state may arise from an excess or hyperfunction of a procoagulant or a deficiency of an anticoagulant factor [8].

2.5 Microthrombosis vs. macrothrombosis

Microvascular thrombosis is defined by the occurrence of microthrombi within the microcirculation. This microthrombogenesis process is associated to various severe clinical diseases such as thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, and antiphospholipid syndrome as well as other thrombotic microangiopathies. It is also observed during systemic infections, cancer, myocardial infarction, stroke and neurodegenerative diseases [9].

Microvascular thrombosis often occurs subsequently to disordered clot formation and disordered inflammation pathway. Recently, during the coronavirus pandemic, it was shown that the novel severe acute respiratory syndrome coronavirus 2, is characterized by a dysregulated immune system and hypercoagulability recognized on the basis of profound d-dimer elevations and evidence of microthrombi and macrothrombi, both in venous and arterial systems. The complex crosstalking between the innate immune system and coagulation pathways culminates in the model of immunothrombosis, ultimately causing microthrombotic complications [9]. Microvessel thrombosis can then cause greatly differing symptoms that range from limited changes in plasma coagulation markers to severe multi-organ failure. Immunothrombosis is critically supported by neutrophil elastase and the activator molecules of blood coagulation tissue factor and factor XII. Identification of the biological driving forces of microvascular thrombosis should help to elucidate the mechanisms promoting pathological vessel occlusions in both microvessels and large vessels [10].


3. Thrombosis pathogenesis

Thrombosis is a pathologic phenomenon related to the disturbance of the dynamic balance of hemostasis. In fact, under normal circumstances, there is a fine balance between the procoagulant, anticoagulant and fibrinolytic pathways.

Hemostasis is the physiological mechanism aiming to protect the vascular system and to keep it intact after injury. The dynamic hemostatic balance comprising interactions between endothelial cells, thrombocytes, coagulation, and fibrinolysis prompts the regulation of hemostasis in order to assure the function of tissues and organs. This mechanism ensures the control of hemorrhage and thrombosis pathway activation and provide a matrix in wound healing and tissue repair. The amount of fibrin layers, at a site of injury inducing the progress of the tissue repair, is controlled by hemostasis balance [11].

When this equilibrium is disturbed under any condition, the physiologic process becomes pathologic leading to bleeding or to thrombotic troubles.

Numerous genetic, acquired and environmental factors can disturb the balance in favor of coagulation, leading to the pathologic formation of thrombi in veins, arteries, or cardiac chambers [1, 12].

The German pathologist Rudolf Virchow recognized that if this dynamic balance was altered by venous stasis, abnormal coagulability and vessel wall damages, microthrombi could propagate to form macroscopic thrombi. This understanding of thrombosis formation has been baptized as the triad theory in 1856 (Figure 1) [13]. Mechanisms of stasis, hypercoagulability, and endothelial dysfunction have been, subsequently elucidated as well as the different major factors involved in the hemostasis and coagulation cascade and fibrinolytic system, both at the biochemical and genetic levels [14].

Figure 1.

The triad contributing together to venous thrombosis described by Rudolph Virchow.

Typically, arterial thrombotic disease is interrelated to atherosclerosis and thrombosis, as well as their interaction designed by the term atherothrombosis. Acute arterial thrombosis occurs at the site of a ruptured, lipid-rich atherosclerotic plaque. This event contributes to the transition of a stable atherosclerotic disease to an acute state [3]. Depending on the localization of atherosclerotic plaques, arterial thrombotic disease may be an acute infarction such as myocardial or brain infarctions, ischemic stroke, or peripheral arterial occlusion leading to ischemia. Arterial thrombosis can also be due to other pathological conditions favoring arterial clotting and turbulences such as atrial fibrillation and antiphospholipid syndrome [1, 3].

In veins, Virchow’s triad is traditionally invoked to explain pathophysiologic mechanisms leading to thrombosis. In fact, abnormalities in blood composition with plasma hypercoagulability, alterations in the wall components of blood vessel and changes of the blood flow with stasis, are the three components involved in the development of venous thrombosis [13]. Clinical manifestations of venous thrombosis include acute peripheral venous thrombosis (phlebitis) and deep venous thrombosis or venous thromboembolism as well as pulmonary embolism, the most serious acute complication of deep venous thrombosis. Long-term complications represented by post-thrombotic syndromes are due to damages affecting the valves in the veins. Venous thromboembolism and superficial vein thrombosis account for about 90% of venous thrombosis. Other rarer forms include retinal vein thrombosis, splanchnic vein thrombosis, cerebral venous sinus thrombosis, renal vein thrombosis, and ovarian vein thrombosis [15].


4. Thrombosis related pathways

The complex thrombosis related pathways crosstalking implies hemostasis and coagulation, inflammation and immune system as well as the contributing role of Redox homeostasis and the interplay of oxidative/nitrosative stress to both inflammation and coagulation [16, 17].

4.1 Hemostasis and coagulation pathways

Understanding, components and factors as well as steps of hemostasis and coagulation pathways, is important in defining the molecular variants related to the thrombosis pathogenesis [18].

Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. Hemostasis, which is the physiologic response to vascular endothelial injury, encompasses a series of processes to maintain blood within the vascular system through the formation of a clot. It involves three basic steps: vascular spasm, platelet clot formation, and coagulation, in which activation of the coagulation cascade clotting factors promotes the formation of a fibrin clot. Fibrinolysis is the process in which a clot is degraded [19].

Hemostasis can be divided into primary hemostasis and secondary hemostasis corresponding to the coagulation process. The fibrinolytic pathway called the tertiary hemostasis interacts to regulate fibrin deposition and removal during healing [18, 20].

Primary hemostasis consists of the formation of the platelet clot and includes the blood vessel constriction or vasoconstriction, platelet adhesion, activation and aggregation at the site of the vessel injury. Secondary hemostasis is characterized by the transformation of fibrinogen into fibrin and changes the platelet clot into a stable fibrin clot [20, 21].

During hemostasis, three distinctive pathways can be involved: intrinsic, extrinsic, and common pathways. Activation of the intrinsic pathway is promoted through exposed endothelial collagen, while activation of the extrinsic pathway is stimulated through tissue factor released by various cells in particular the endothelial cells after external damage. These pathways initiate separately at the beginning but at a specific moment with the presence of factor X, the Stuart-Prower factor, they converge, leading to common pathway with the generation of the prothrombinase complex that cleaves the prothrombin into thrombin and then fibrin activation process and platelet clot stabilization with a fibrin webbing. Coagulation cascade involving conversion of inactive coagulation factors to their active forms following a series of enzymatic reactions including multiple cofactors and that ends with the conversion of fibrinogen to fibrin, leads to the formation of the definitive fixed blood clot with scrambled blood cells. The factors II, VII, IX, X, XI and XII circulate as zymogens that are activated into serine proteases to act as catalytic agents cleaving the next zymogen into more serine proteases, whereas factors V, VIII, XIII are not serine proteases [20, 21, 22].

To look at the multiple involved factors and actors in hemostasis pathways as well as their complex interactions; we reviewed recent literature reviews detailing the physiology of hemostasis and coagulation pathways. The biologic and molecular factors, cofactors and actors of the hemostasis pathways are summarized in Table 1 and Figure 2.

Clotting factorsAliases namesGeneChromosomeExonsGene ID
Factor IFibrinogenFGB4q31.382244
Factor IIProthrombinFII11p11.2142147
Factor IIIThromboplastin -Tissue FactorFIII1p21.362152
Factor IVIonized calcium
Factor VProaccelerinFV1q24.2252153
Factor VIUnassigned
Factor VIIProconvertin -Stable factorFvII13q34102155
Factor VIIIAnti-hemophilic factor AFVIIIXq28272157
Factor IXChristmas factorFIXXq27.182158
Factor XStuart-Prower factorFX13q3482159
Factor XIPlasma thromboplastinFXI4q35.2152160
Factor XIIHageman factorFXII5q35.3152161
Factor XIIIFibrin stabilizing factorFXIIIA16p25.1152162

Table 1.

Clotting factors and their encoding genes.

Figure 2.

The three distinctive pathways of hemostasis: intrinsic, extrinsic, and common pathways.

At the cellular level, hemostatic reactions involves plasma, platelet, and vascular components. After a blood vessel injury the extracellular matrix and the collagen become unprotected and in contact with the blood within an area of vasoconstriction, leading to the liberation of cytokines and inflammatory markers. Consequently, platelet adhesion, activation and aggregation at that site are sequentially mediated by interactions between various receptors including tyrosine kinase receptors, glycoprotein receptors, other G-protein receptors and proteins within the platelets [23]. Platelet degranulation induces the liberation of Adenosine diphosphate, thromboxane A2, serotonin, and multiple other activation factors. The conversion of fibrinogen to fibrin and the formation of a platelet-fibrin hemostatic clot ends by a coagulation cascade involving the formation of fibrin polymer mesh catalyzed by activated factor XIII that stimulates the lysine and the glutamic acid side chains causing the cross-linking of the fibrin molecules and the formation of a stable fibrin clot [24].

The fibrinolytic pathway called the tertiary hemostasis interacts to regulate fibrin deposition and removal during healing. The activities of thrombin and other serine proteases are modulated by the serine protease inhibitors (serpins), including antithrombin III and heparin cofactor II which are important in regulating the physiological anticoagulant action of glycosaminoglycans at the endothelium [25].

Under normal circumstances, there exists a fine balance between the procoagulant and anticoagulant pathway and hemostasis is under the inhibitory control of several inhibitors that limit clot formation, thereby avoiding thrombus propagation. This balance is disturbed whenever the procoagulant activity of the coagulation factors is increased, or the activity of naturally occurring inhibitors is decreased [14, 26]. As thrombin acts as a procoagulant, it also acts as a negative feedback by activating plasminogen (Serpine 1 Gene ID: 5054 7q22.1 with 9 exons) to plasmin and stimulating the production of antithrombin (Serpinc1 Gene ID: 462, 1q25.1 with 9 exons). Plasmin acts directly on the fibrin mesh and breaks it down. Antithrombin decreases the production of thrombin from prothrombin and decreases the amount of activated factor X. Protein C or blood coagulation factor XIV (PROC Gene ID: 5624, 2q14.3 with 8 exons) and protein S (PROS1 Gene ID: 5627 3q11.1 with 16 exons) act to prevent coagulation, mainly by inactivating factors V and VIII. The Kunitz-type protease inhibitor tissue factor pathway inhibitor (TFPI Gene ID: 7035, 2q32.1 with 13 exons) limits the diffusion of the coagulation cascade. TFPI binds to FXa or the TF-FVIIa-FXa complex to restrict coagulation function [,].

Under abnormal circumstances, the formation of thrombi happens in a not breached vessel, in particular in venous thrombosis where thrombi are formed subsequently to the activation of the clotting system. However, in arterial thrombosis, thrombi are considered typically as the result of a phenomenon of atherothrombosis after rupture of atherosclerotic plaques leading to platelet activation and interactions between platelet activation, tissue factor vesicle expression from plaque macrophages, and then activation of the coagulation cascade [27].

4.2 Inflammatory and immune pathways

Understanding the role of inflammation in thrombosis disorders is important in defining the molecular variants related to the thrombosis pathogenesis.

The complex pathways of inflammation and hemostasis appear to have a common evolutionary origin and interrelated pathophysiologic processes [28].

In fact, there is an inflammation-hemostasis cycle in which each activated process promotes the other, and the two systems function in a positive feedback circle. The mechanisms responsible in the relations between thrombosis and inflammation involve all components of the hemostatic system including associated cells and plasma coagulation/fibrinolysis cascades [1, 28, 29].

The first event in thrombus formation is probably the stimulation of an inflammatory response with the activation of endothelial cells, platelets, and leukocytes. Initiation of inflammation leads to the formation of microparticles that activate the coagulation system through the induction of tissue factor. In fact, throughout the inflammatory response, various inflammatory mediators, in particular proinflammatory cytokines play a central role in ever-changing the hemostatic activity towards procoagulant state by triggering endothelial cell dysfunction, increased platelet reactivity, activation of the plasma coagulation cascade, impaired function of physiologic anticoagulants and inhibited fibrinolytic activity [30].

On the other side, coagulation cascade augments inflammation by means of thrombin-induced secretion of proinflammatory cytokines and growth factors. Platelets may also trigger inflammation, in particular by activating the dendritic cells. In abnormal circumstances, other inflammatory factors are implicated such as chemokines, adhesion molecules, platelet-derived mediators linking thrombosis and atherosclerosis and thrombosis, infection and immunity [28, 31].

Recently, there is a consensus that vascular thrombosis diseases are simultaneously, triggered by biological stimuli responsible for activating coagulation and inflammatory pathways in both the arterial and the venous systems [32]. In fact, while it is commonly recognized that the pathogenesis of arterial thrombotic disease is related to the chronic lipid-driven inflammatory disease of the arterial wall characterized by the involvement of the innate and adaptive immune systems or atherosclerosis, it is only recently that inflammation has been accepted as a common pathway of venous thrombosis formation [33, 34].

The most well described pathophysiologic process, in which there are an established relation between inflammation and hemostasis is the arterial atherothrombosis generated consequently to ruptured atherosclerotic plaque. Besides, chronic inflammation may cause endothelial damage, resulting in the loss of physiologic anticoagulant, antiaggregant and vasodilatory properties of endothelium. There are, many systemic inflammatory diseases characterized by thrombotic tendency in the absence of vessel wall damage, including chronic autoimmune diseases and vasculitis, such as Behçet disease, antineutrophilic cytoplasmic antibody-associated vasculitis, Takayasu arteritis, rheumatoid arthritis, systemic lupus erythematosus, antiphosholipid syndrome, familial Mediterranean fever, thromboangiitis obliterans and inflammatory bowel diseases [28, 35, 36].

Inflammation-induced venous thrombosis developed in the absence of vessel wall damage, in particular during malignancies, is also well demonstrated. Malignant proliferation induces prothrombotic substrates such as tissue factor and production of inflammatory cytokines, including tumor necrosis factor (TNF Gene ID: 7124, 6p21.33 with 4 exons) and interleukin-1 (IL1A Gene ID: 3552, 2q14.1 with 8 exons and IL1B Gene ID: 3553, 2q14.1 with 7 exons). This leads to the shift of the hemostatic state to procoagulant state that predisposes to the development of venous thrombosis [1, 37].

On the other hand, during the coordinated intravascular coagulation response of platelets in response to various blood pathogens and consequent tissue damage recently termed immunothrombosis, the risk of thrombosis that manifests as arterial or venous thrombosis (and may contribute to atherosclerosis). During this process of immunothrombosis, inflammation-dependent activation of the coagulation system is part of the host response to pathogens, aiming to limit their systemic spread in the bloodstream [38, 39]. This response is achieved through an interplay between innate immune cells and platelets, triggering the activation of the coagulation system and the releasing of the complement system. Platelets and immune cells form, in fact, a physical barrier of confinement preventing dissemination of pathogens and potentially leading to activation of the innate and adaptive branches of the immune system [40]. Interestingly, platelets mediate the crosstalk between the hemostatic and the immune system utilizing similar pathways. The dysregulated and excessive activation of immunothrombosis results in thromboinflammation, causing tissue ischemia by microvascular and macrovascular thrombosis. Pulmonary immunothrombosis in severe COVID-19 correlating with a systemic prothrombotic phenotype provides clinical evidence for the partnership between inflammation and thrombosis [12, 41].

4.3 Oxidative pathways and redox homeostasis

There is now a strong evidence for the participation of reactive oxygen and nitrogen species in the pathogenesis of thrombosis as well as solid proofs of an interplay of oxidative and nitrosative stress, inflammation and thrombosis [42].

First, it is well established that reactive oxygen species (ROS) participate in vascular cell signaling and proatherogenic gene expression by modulation of oxidation–reduction (Redox) reactions pathways [43, 44, 45].

The cellular redox state, or balance between cellular oxidation and reduction reactions, serves as a vital antioxidant defense system that is linked to all important cellular activities. Redox homeostasis is thought to be achieved by careful regulation of both ROS formation and removal from the body system [46].

Recently, redox processes in cell signaling imply, beside ROS, Reactive Nitrogen Species (RNS). In fact, ROS and RNS were identified as key players in initiating, mediating, and regulating the cellular and biochemical complexity of oxidative stress either as physiological or as pathogenic processes [47].

Oxidative stress is a term associated with both enhanced production of ROS and reduced efficacy of protection by antioxidant enzymes. Nevertheless, after the discovery of NO as a biological entity and the powerful role of superoxide radicals O2- and NO as oxidants via ONOO- formation, oxidative stress has become unavoidably related to nitrosative stress and RNS. The key species associated with oxidative and nitrosative stress as well as their interactions have been reviewed and it was suggested that ROS are the initial reactants produced from an ionization event, whereas RNS are the effectors/activators of redox-dependent cellular signal transduction pathways [48, 49].

Depending on the severity of the oxidative stress, adaptive processes occur by increasing antioxidant capacities and by growing the capacity of the oxidative damages reparation. In extreme cases, metabolic processes shift away from oxidative metabolism towards glycolytic metabolism. In the case of chronic metabolic oxidative stress along with the accumulation of oxidative damage to critical biomolecules, potentially pathological conditions can develop due to the cumulative oxidative damage interacting with proteins, lipids, and DNA [48, 50].

An excessive ROS generation or a defect in the antioxidant defense system impacts a wide variety of biological molecules, lipids in the plasma and mitochondrial membranes, causing lipid peroxidation that impairs membrane selective permeability, proteins (resulting in structural instability and damage to their enzymatic function) and nucleic acids, thus inducing pathways of apoptosis [51].

In particular, oxidative stress is responsible for the disruption of the coagulation cascade at various stages leading to anomalies in blood coagulability and platelet reactivity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family (NOX) enzymes appears to be the most important source for ROS involved in processes related to thrombosis [52]. On the other hand, oxidative stress contributes to the development of atherosclerosis leading to atherothrombosis [53].

Oxidative pathways of thrombosis can occur throughout endothelial apoptosis, impairment of red blood cells quality and function, endothelial dysfunction and damage of endothelial cell lining, activation of platelets and leukocytes, and consequently by affecting the clotting system. The endothelial cell lining is essential in triggering the prothrombotic events as an intact endothelial cell lining prevents platelets adherence and activation. Furthermore, endothelial injury-associated oxidative stress promotes tissue factor expression having a potent procoagulant activity. In fact, ROS increases the expression of tissue factor in endothelial cells, monocytes and vascular smooth muscle cells, with essentially the contribution of NOX enzymes [54]. Whereas TFPI, which is the only physiologic regulator of tissue factor activity, can be inhibited by oxidative stress and exerts a procoagulant effect. ROS can also directly inactivate major anticoagulant proteins such as protein C and its upstream agonist thrombomodulin (THBD Gene ID: 7056, 20p11.21 with one exon). The stimulation of protease-activated receptors (PARs) may also lead to endothelial tissue factor induction via mitochondrial ROS signaling [55].

ROS stimulates platelet reactivity and during this ROS generation/platelet reactivity, platelet ROS are mostly generated by reduced NADPH oxidase. NOX2 expressed in platelets is an important regulator of platelet activation associated thrombosis. Engagement of primary platelet receptors, GPIb-IX-V and GPVI, that initiate thrombus formation, leads to a rapid increase in intracellular ROS above basal levels, and is a key step in platelet activation following exposure to physiological ligands such as von Willebrand factor (VWF)/collagen [55, 56, 57].

Thus, in contrast to endothelial injury-associated thrombus, platelet-dependent thrombus formation may also be influenced by alteration of platelet redox state or other cells or vascular redox state. Settings and pathways that influence the formation of superoxide and nitric oxide, as well as their metabolism, may specifically influence platelet function and thrombus formation [58].

Many studies emphasis that ROS influences venous thrombus formation and resolution through the modulation of the coagulation, the fibrinolysis, the proteolysis and the complement system, as well as the regulation of effector cells such as platelets, endothelial cells, erythrocytes, neutrophils, mast cells, monocytes and fibroblasts. During antiphosholipid syndrome for example, venous thrombosis occurs in patients having alterations in their redox homeostasis [42].

Reactive free radicals are defined as any chemical species capable of independent existence that contains one or more unpaired electrons. Reactive oxygen species (ROS) and Reactive nitrogen species (RNS) are free radicals that are associated with the oxygen atom (O) and other molecules, with stronger reactivity. Other biologically important free radicals exist such as lipid hydroperoxide (ROOH), lipid peroxyl radical (ROO), and lipid alkoxyl radical (RO), which are associated with membrane lipids and thiol radical (RS), which has an unpaired electron on the sulfur atom [59].

ROS consists of radical and non-radical oxygen species formed by the partial reduction of oxygen, including superoxide anion (O2-), hydroxyl radicals (OH), singlet oxygen (1O2) and hydrogen peroxide (H2O2). Generally, ROS are generated endogenously as natural by-products of aerobic metabolism during mitochondrial oxidative metabolic rate and by the means of cytochrome p450, cyclooxygenase, lipoxygenase and NOX enzymes. ROS are, also generated in response to stimulation such as by cytokines and other inflammatory mediators [59, 60].

Imbalance between oxidative stress and antioxidant status may be the result of an up-regulation of ROS-producing enzymes, such as NADPH oxidase and myeloperoxidase, along with down-regulation of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx). Antioxidant defenses modulate the steady state balance of ROS with the implication of other several antioxidant enzymes such as catalase, glutathione peroxidases, heme oxygenase, thioredoxin system as well as small molecule antioxidants such as glutathione, vitamins A, C and E. These enzymes are produced to scavenge ROS, thereby limiting their detrimental effects [60, 61].

For example, age-dependent increased venous thrombosis is related to age-related endothelial dysfunction involving upregulation of the NADPH oxidase and cyclooxygenases (COXs)-dependent oxidative stress pathways. On the other hand, overexpression of the antioxidant enzyme GPX-1 protects from age-dependent increased venous thrombosis. Moreover, during aging, abnormal aged red blood cells may also adhere to the endothelium or extracellular matrix, activate platelets and other cells, and enhance local thrombin generation during thrombosis [52].

Second, in nitrosative stress, RNS involve various species such as nitric oxide (NO), nitrogen dioxide (NO2) and peroxynitrite (ONOO-). NO is generated in biological tissues by specific nitric oxide synthases (NOSs) and acts as an important oxidative biological signaling molecule in defense mechanisms and immune regulation. In the extracellular milieu, NO reacts with oxygen and water to form within an enzymatic cascade more reactive products. Immune cells, including macrophages and neutrophils, simultaneously release NO and superoxide into their phagocytic vacuoles. Other inflammatory cells can also produce reactive chemicals that can result in 3-NT formation, including the peroxidases in activated neutrophils and eosinophils. 3-NT is a characteristic marker of nitrosative stress and, commonly, inflammation [47].

NO was originally discovered as a vasodilator product of the endothelium and later as a factor having important antiplatelet actions, inhibitor effects of leukocyte adhesion and migration, and other inflammatory cells adhesion. By activating guanylyl cyclase, inhibiting phosphoinositide 3-kinase, impairing capacitative calcium influx, and inhibiting Cox-1, endothelial NO limits platelet activation, adhesion, and aggregation. Platelets are also an important source of NO, and this platelet-derived NO pool limits recruitment of platelets to the platelet-rich thrombus [62, 63].

A deficiency of bioactive NO is associated with arterial thrombosis in individuals with endothelial dysfunction and patients with a deficiency of the extracellular antioxidant enzyme GPx-3. Impaired NO availability also seems to be caused by inactivation of NOS and the levels of an endogenous inhibitor of NOS such as asymmetric dimethylarginine (ADMA). On the other hand, it seems that the decline in vascular NO production may be a characteristic feature of mammalian aging [47, 64].

Low NO availability is incriminated in thrombus formation. In fact, in the presence of low NO availability, endothelium-dependent vasodilation is impaired, which leads to abnormal red blood cells adhesion, and may contribute to increased platelet activation [65].

NO is considered as having a dual role as a protective or harmful molecule depending on tissue concentration levels and interaction with oxidative stress. In the endothelium, NO has antiatherogenic actions related to the inhibition of platelet function and inflammatory cell adhesion, promotion of fibrinolysis, and attenuation of smooth muscle cell proliferation. Oxidative stress and enhanced ROS production seem to be involved in the down-regulation of the protective NO pathway [48].

Finally, ROS/RNS generated during inflammation and inflammatory response and crosstalk between cellular redox state and the ROS/RNS network during inflammation constitute an emerging field. In states of inflammation, NO production by the vasculature increases considerably and, in conjunction with other ROS, contributes to oxidative stress [47].

ROS/RNS have also emerged as important modulators of intracellular transduction signaling. These radicals interact with redox-sensitive signaling molecules including protein tyrosine phosphatases, protein kinases and ion channels, regulating cellular processes like growth factor signaling, hypoxic signal transduction, autophagy, immune responses, and stem cell proliferation and differentiation. Moreover, the level of miRNAs can be modulated at the transcription and/or processing level by stress-induced factors like p53 or NF-kB as well as the presence of intracellular hydrogen peroxide levels [43, 44, 51, 66].


5. Thrombosis-related DNA polymorphisms

Among the many important insights derived from completion of the Human Genome Project was the recognition of the abundance of single nucleotide polymorphisms (SNPs) as a major source of genetic variation. Studies over the past last years have resulted in increasing recognition of the critical role of structural genetic variation in particular of copy-number variation in modulating gene expression and disease phenotype. Recently, genome-wide surveys and association studies are being widely applied to identify genetic factors that affect complex diseases or traits. However, while for SNP-association studies there are well-developed available resources, resources for structural genetic variation identified via genome-wide association studies are still in their early phases [67, 68, 69].

Arterial and venous thrombosis, with their clinical manifestations classified as complex multi-factorial diseases are related to various genetic variations that are both deleterious mutations and disease-susceptibility sequence polymorphisms, since the last century. In fact, beyond deficits in coagulation inhibitors, which have been known for a long time, the two most known thrombogenic mutations have been discovered in 1994 and 1996 [69].

Genetic studies in thrombosis started with the conception of the term thrombophilia by Jordan and Nandorff in 1956 [70]. Nine years later, antithrombin deficiency was identified as a genetic risk factor of thrombosis [71]. In the 1990s, activated PC resistance and the causal genetic variation of factor V Leiden, the first-born thrombogenic mutation, were revealed in a family setting. Factor V Leiden risk factor was also the first prothrombotic defect in a procoagulant protein. In 1996, case–control studies revealed the common prothrombin 20210 G > A mutation [72].

Using association studies and commonly PCR-RFLP tools, many genetic variations in almost all coagulation proteins were tested. However, until the beginning of the new century, only few gene loci were significantly associated with thrombotic diseases. The majority of the genetic variations was identified in the coding genes of hemostasis and coagulation factors and was shown as important risk factors for particularly venous thrombosis [73]. In contrast, in arterial thrombosis disease commonly related to atherosclerosis, many polymorphisms were identified, with significant relationships demonstrated between genotypes and plasma phenotypes. However, the exact contribution of genotypes to clinical phenotypes remains regularly uncertain. These variants associated in majority to a small risk, if any risk at all, have a limited usefulness as relevant biomarkers of the thrombotic diseases that must be prescreened to guide prevention, prognosis and treatment [74].

Nowadays, despite the success of genome-wide association studies in identifying new genetic factors determining many thrombosis-related diseases such as coronary artery disease, results for arterial and venous thrombosis have yielded little success. One of the reasons for the limited number of loci identified is most likely the lack of power due to the small sample sizes of studied cases [75].

Since the underlying pathogenic mechanisms are only partially known, regarding the complex interplay of many pathways in thrombosis-related pathogenesis as shown in the chapter 3 and the emerging role of intricate modulators of intracellular transduction signaling, there is mounting evidence indicating the challenging struggle of the mapping disease-susceptibility genes in thrombotic disorders. Genetic variations within thrombosis related pathways involving hemostasis and coagulation, inflammation, and immune system as well as Redox homeostasis and oxidative/nitrosative stress might be potential risk factors. Subsequently, many new loci need identification as risk factors as well as characterization in the future, through studies of candidate genes and genome-wide association studies.


6. Classic thrombophilia-related DNA polymorphisms

Thrombophilia traditionally refers to rare inherited defects leading to enhanced coagulation, especially of the venous system. Thrombophilia may be inherited or acquired. Acquired causes of thrombophilia include trauma, surgery, pregnancy, use of oral contraceptives, antiphospholipid syndrome, paroxysmal nocturnal hemoglobinuria and heparin induced thrombocytopenia. Inherited causes of thrombophilia are related to the hypercoagulability state that may arise from an excess or hyperfunction of a procoagulant or a deficiency of an anticoagulant factor [72].

In 1937, Nygaard and Brown first used the term thrombophilia, when they described sudden occlusion of large arteries, sometimes with coexistent venous thrombosis. Investigation of thrombophilia causes started by familial setting within families characterized by a predisposition to thromboembolic diseases and a strong tendency to venous thrombosis. This approach leaded to the early description of the deficiency of antithrombin causing venous thromboembolism at a young age related to thrombophilia entity in several members of a Norwegian family. Deficiencies of protein C and protein S were discovered after few years like the novel hereditary thrombophilia risk factors in other anticoagulant proteins. The inherited risk factors implying prothrombotic factors for thrombophilia were identified in following years like the underlying causes linked to venous thrombosis. They included FV Leiden variant linked to resistance to activated protein C (APC resistance) and FII 20210 G/A transition linked to elevated levels of Prothrombin (Table 2) [82, 83].

The factor V G1691A and prothrombin G20210A polymorphisms in arterial disease have been subjects of numerous reports. Many of these—some large—concerned with their association with arterial disease in young, middle-aged, and elderly populations had negative results. In contrast, some studies report positive associations, particularly when the interaction of these polymorphisms with environmental factors were formally evaluated [82, 83].

In 1988, Kang et al. [84] described a heat-labile form of MTHFR associated with mild hyperhomocysteinemia. The C677T polymorphism of the MTHFR gene has led to the identification of many more cases of thrombophilia.

Hereditary thrombophilia manifests more or less severely and early depending on the genotype, which may be heterozygous (a single affected allele), homozygous or composite heterozygous (two affected alleles) or associated with several different genetic risk factors.

Association studies between inherited thrombophilia and venous thromboembolic disease showed dominance of factor V Leiden, and factor II G20210A variant in comparison with coagulation protein deficiencies (Table 3).

6.1 Factor V mutations

The factor V gene (Gene ID: 2153) is located on the long arm of chromosome 1 at q24.2. It consists of 25 exons that span a region of approximately 80. The FV gene encodes a propeptide of 2224 amino acids containing a 28-residue signal peptide, excised after translocation into the endoplasmic reticulum. This propeptide, called proaccelerin, is a monomeric protein of 330 kDa. It is characterized by a domain structure with 3 A domains (330aa), 2 C domains (150aa) and a B domain [N-A1-A2-B-A3-C1-C2--COOH]. Proaccelerin consists of two calcium-stabilized non-covalent chains: a heavy chain (110,000) and a light chain (74,000–71000). It is highly N- and O-glycosylated (about 13–25% of the mass, i.e. 37 N-glycosylation sites: 25 at the B-domain, 9 heavy chains and 3 light chains). Factor V exists in two forms V1 and V2, related to the heterogeneity of the molecular mass of the light chain and caused by the partial glycosylation at the Asn2181 residue (3 and 2 carbohydrate chains respectively for FV1 and FV2). These 2 forms differ in their functions: FV2a would have more affinity for membrane phospholipids and thrombin generation. Factor V is synthesized by hepatocytes and megakaryocytes. Approximately 80% of pro-accelerin is circulating in plasma (with a concentration of 5-10 mg/l = 21 nM) and only 20% is stored in the α-granules of platelets (i.e. between 4600 and 14000 molecules/platelet) in association with BPM (binding protein multimerin). This platelet fraction is released during platelet activation [91, 92].

Proaccelerin has a dual function: procoagulant and anticoagulant. Procoagulant action: Factor Va is part of the prothrombinase complex (Xa;Va;PL;Ca) that converts prothrombin (FII) to thrombin (FIIa), which is responsible for thrombus formation. It is initially activated by thrombin, which eliminates the B domain, and then its Xa cofactor binds to the C2 domain. At the molecular level, factor V performs its procoagulant function after proteolysis by thrombin and FXa at three arginine residues (Arg709, Arg1018, and Arg1545). This results in the elimination of the B domain. Thus FVa is formed by a heavy chain (105 KDa) [A1-A2] and a light chain (74–71 KDa) [A3-C1-C2] stabilized by calcium [92].

Once activated, FV complexed with FXa on membrane phospholipids and in the presence of Calcium forms the prothrombinase complex that converts prothrombin to thrombin. Anticoagulant action: Factor V binds to protein S and is a synergistic cofactor for inhibition of VIIIa by activated protein C. The anticoagulant action requires the inactivation of FVa. This is mediated by protein C which successively proteolyses it at Arg506, Arg306 and Arg 679. The first cleavage at Arg506 reduces the activity of FVa (25–40%) as well as its affinity for FXa, this partial inactivation is completed after cleavage at Arg 306, however Arg 679 is less important in this process. Thus, factor V will be fragmented into FVai (composed of the A1 domain associated with the light chain) and two fragments derived from the A2 domain (A2N and A2C respectively on the N and C terminal side). Alternatively, the inactivation of FVa is mediated by thrombin, which cleaves Arg643 in the presence of endothelial cells, resulting in a reduction of affinity between the two heavy and light chains [91, 92, 93].

On the other hand, activated protein C can degrade intact FV and thus confers an anticoagulant property (FVac). Thus, the latter would be a cofactor of APC and protein S in the degradation of FVIIIa. This anticoagulant property requires cleavage at different sites (Arg306, Arg506, Arg679 and Lys994). However, only Arg 506 is required for the expression of the FV-APC cofactor activity [93]. This functional duality of FV in the coagulation process is dependent on the local concentration of procoagulant and anticoagulant enzymes such as thrombin, FXa and APC, which are responsible for the conversion of FV into a procoagulant or anticoagulant cofactor [94].

Various mutations affecting the FV gene have been described in association with a thrombotic phenotype. Among these missense mutations, the most prevalent is the Factor V Leiden mutation, initially described by Bertina et al. (Table 4) [76].

Thrombophilia risk factorPrevalence in the general population (%)Mode of transmissionReference
FV Leiden2–15DominantBertina et al. [76]
FII G20210A2–3DominantPoort et al. [77]
MTHFR C677T1–11RecessiveFrosst et al. [78]
ATIII deficiency0.02–2DominantEgeberg [79]
PC deficiency0.2–0.5DominantGriffin et al. [80]
PS deficiency0.1–2.1DominantComp and Esmon [81]

Table 2.

Prevalence and mode of transmission of inherited thrombophilia.

Thrombophilia n/NAbsence of thrombophilia n/NOR (CI = 95%)
FV Leiden heterozygote
Dilley et al. [85]8/932/10718.75 (2.25–156.15)
Gerhardt et al. [86]47/6572/2877.80 (4.26–14.28)
Martinelli et al. [87]22/2897/3238.54 (3.36–21.73)
Murphy et al. [88]3/1629/5564.19 (1.13–15.54)
Tormene et al. [89]6/941/815.45 (0.64–46.29)
TOTAL (95%)86/212231/13548.94(2.32–50.79)
Dilley et al. [85]4/436/11218.86 (0.99–359.76)
Gerhardt et al. [86]20/2398/32115.17 (4.41–52.24)
Martinelli et al. [87]7/14112/3372.01 (0.69–5.87)
TOTAL (95%)31/41246/77012.01(2.03–139.29)
Dilley et al. [85]5/1322/631.16 (0.34–3.99)
Murphy et al. [88]1/579/2230.42 (0.05–3.42)
Ogunyemi et al. [90]2/1228/480.14 (0.03–0.72)
ATIII Deficiency
Gerhardt et al. [86]6/883/2124.66 (0.92–23.65)
Martinelli et al. [87]1/2118/3491.96 (0.12–31.58)
PC Deficiency
Gerhardt et al. [86]15/2491/3124.05 (1.71–9.58)
(Ogunyemi et al. [90]2/228/585.35 (0.25–116.31)
PS Deficiency
Gerhardt et al. [86]13/2492/3092.79 (1.2–6.45)
Martinelli et al. [87]2/3117/3483.95 (0.35–44.00)
TOTAL15/27209/6573.37 (0.77–25.22)

Table 3.

Association studies between inherited thrombophilia and venous thromboembolic disease.

ARG306GLY1090A-GFV HONG KONGChan et al. [95]
ARG306THR1091G-CFV CAMBRIDGEWilliamson et al. [96]
ILE359THR1250 T-CFV LIVERPOOLSteen et al. [97]
Arg506Gln1691G-AFV LEIDENBertina et al. [76]

Table 4.

Various mutations affecting the FV gene.

6.1.1 Factor V Leiden

Many studies have focused on the pathogenicity of VF in the occurrence of thrombophilia. Since its discovery in 1994 by Bertina et al. [76], FV Leiden represents the major anomaly in thromboembolic patients. It is a 1691G-A transition in exon 10 of the FV gene resulting in an Arg506-to-Gln substitution (R506Q) (Figure 3). This mutation is responsible for resistance to activated protein C, since it affects the potential site of cleavage by activated protein C, both for FVa degradation and FV-APC cofactor activity for FVIIIa inactivation [98, 99].

Figure 3.

Sanger sequence of the G1691A Factor V Leiden mutation in exon 10 of the FV gene.

The thrombotic risk depends on the form of expression of the FV Leiden allele: it is 5–7 times in heterozygotes, 30 times in homozygotes and intermediate in pseudo homozygotes. The latter is a particular form, where the FVL allele is associated with another deficient or null allele [98].

Different mutations have been described in association with FV Leiden. The first mutation is a A4070G transition at exon 13 of the FV gene, resulting in a His1299Arg substitution, described in 1997 [100]. This mutation is an allelic form characterized by a moderate deficiency in Factor V not counterbalanced by FV Leiden; hence the occurrence of thrombosis.

In 1998, it was identified a null mutation that consists of a C2308T transition, at exon 13 of the FV gene affecting codon Arg 712(CGA), and producing a stop codon (TGA) resulting in a truncated protein at the level of its light chain (A3, C1, and C2 domains) unable to perform its anticoagulant function. As a result, only FV Leiden molecules are present and thus responsible for thrombosis [101].

The geographic distribution of FVL is extremely heterogeneous: it is absent in Asians, Africans, Americans, and Australians; however, it is prevalent in the Caucasian population. The existence of a single haplotype of FV Leiden worldwide suggests a single mutational event that occurred about 30,000 years ago, after the migration from Africa and the segregation of Asians from Europe. The age of factor V Leiden is estimated at 21,340 years [102].

FV Leiden is a remarkable genetic anomaly in more ways than one. It is common in the general population (2–15%) and affects 20–25% of patients with at least one episode of venous thromboembolism. Carriers of the anomaly are heterozygous in the vast majority of cases, which increases their risk of thrombosis by a factor of 5. However, homozygotes are not rare (0.05 to 0.25%) in the general population. They have a significant risk of venous thromboembolism (RR in the range of 20–30), but generally have no thrombotic events in childhood. They may remain asymptomatic, even in the homozygous state as the Factor V Leiden mutation has incomplete penetrance [103].

6.1.2 Factor V Hong Kong

In the Chinese population, it was identified a 1090A-G substitution in exon 7 of the FV gene resulting in an Arg306Gly (R306G) substitution and the mutation is named Factor V Hong Kong. It is one of the sites of cleavage by activated protein C that is affected, leading to a loss of the procoagulant activity of FVa. However, no predisposition to thrombosis was detected [95].

6.1.3 Factor V Cambridge

In 1998, at Addenbrooke’s Hospital (Cambridge, England), Williamson et al. [96] identified a new mutation of the FV gene in a thrombophilic patient. It is a G to C transversion that results in an Arg306Thr (R306T) substitution. This mutation affects the APC cleavage site and is responsible for a loss of procoagulant activity of FVa. Unlike FV Hong Kong, FV Cambridge is associated with resistance to activated protein C.

6.1.4 Factor V Liverpool

In 2004, Steen et al. [97] identified a new mutation affecting the FV gene, called FV Liverpool. It is a 1250 T-C transition resulting from an Ile359Thr substitution (I359T). This mutation reduces the susceptibility of FVa to proteolysis by APC, since it alters the N- glycosylation at the asn357 residue of the A2 domain. Thus, the anticoagulant activity of FV is decreased and resistance to protein C is observed.

6.2 Factor II or prothrombin gene mutations

The factor II gene (Gene ID: 2147) is about 21 Kb long. It is located on chromosome 11, near the centromere (band 11p11.2). It has 20,241 bp, 14 exons of 25 to 315 bp and 13 introns of 84 and 9447 bp.

Factor II protein or prothrombin is one of the coagulation factors whose hepatocyte synthesis depends on vitamin K. It is a globular protein of about 72 kDa. It consists of a polypeptide chain of 579 amino acids, formed by functional domains found in several coagulation factors. The propeptide (residues −43 to −1), encoded by exons 1 and 2, is cleaved before secretion of the protein by hepatocytes. The Gla domain (residues 1 to 37), encoded by exon 2, is characteristic of vitamin K-dependent proteins: it contains 10 glutamic acid (Glu) residues that are converted to c-carboxy-glutamic acid (Gla) post-translationally by a vitamin K-dependent carboxylase present in the endoplasmic reticulum of the hepatocyte. Gla residues are involved in binding to anionic membrane phospholipids (mainly phosphatidylserine) in the presence of calcium. Two “Kringle” domains (Kringle 1: residues 65–143; Kringle 2: residues 170–248) are encoded by exons 5–6 and exon 7 respectively. Kringle 2 is involved in the binding of prothrombin to factor Va. The C-terminal part of prothrombin carries the serine protease domain, encoded by exons 8–14 [104].

Thrombin is the active form of factor II. It is composed of two polypeptide chains joined by a disulfide bridge: an A chain (residues 284–320) encoded by exons 8 and 9 and a B chain (residues 321–579) encoded by exons 9 to 14. Prothrombin synthesized in hepatocytes is found in the bloodstream. Prothrombin is activated by the prothrombinase complex into thrombin, a potential factor in the coagulation cascade. The prothrombinase complex consists of an enzyme (factor Xa, or FXa), a protein cofactor (factor Va, or FVa) and anionic phospholipids. FXa performs two cleavages at the Arg271/Thr272 and Arg320/Ile321 peptide bonds, giving rise to thrombin and activation peptides (F1, F2, F3) [104, 105].

Thrombin, a serine protease, is both multifunctional and highly selective. It has procoagulant properties, converting fibrinogen to fibrin, activating factor XIII, and amplifying its own formation (activation of platelets and factors V, VIII, XI). It is also anticoagulant, since by binding to thrombomodulin, it becomes capable of activating protein C, the negative regulator of coagulation. It activates not only platelets but almost all cell types (blood, vascular and non-vascular cells), thus intervening in many processes other than hemostasis (inflammation, angiogenesis, tissue remodeling, etc.…) [106].

Various mutations have been described associated with the occurrence of thrombophilia (Table 5). These mutations affect both the regulatory and the splicing system and lead to thrombosis and hyperprothrombinemia respectively. Nevertheless, the G 20210A polymorphism first described by Poort et al. [77] is the most incriminated in thrombophilia.

3’UTRC20209TWarshawsky et al. [107]Thrombosis
3’UTRC20221TWylenzek et al. [108]Thrombosis
3’UTRG20210APoort et al. [77]Thrombosis
A19911GCeelie et al. [109]Hyperprothrombinemia

Table 5.

Various mutations affecting the FII gene.

6.2.1 G20210A In the 3’UTR region of the FII gene

The G20210A mutation affects the 3’UTR region of the FII gene. It leads to transcriptional efficiency through facilitated polyadenylation, resulting in increased prothrombin synthesis (

The geographic distribution is due to a founder effect that dates back to 23,720 years [102]. The G20210A prothrombin mutation is quite common in the general population (2–3%). It is found in about 10% of patients with venous thrombosis, with carriers having a 3–4 fold increased risk. Homozygotes are rare and are likely to have a higher risk [103].

6.2.2 Other molecular polymorphisms of the FII gene

In 2001, Ceelie et al. [109] focused on the genetic causes of hyperprothrombinemia by analyzing variations in the prothrombin sequence in homozygous 20210-GG subjects. A homozygous 19911-G mutation is associated with an elevated prothrombin level. However, it does not affect thrombotic risk. On the other hand, this risk is potentially increased in association with the G20210A mutation (OR = 1.6 for 19911A versus 4.7 for 19911G). In the same year, Wylenzek et al. [108] detected a new mutational point in a Lebanese family: a C20221T substitution in the 3’UTR region of the F2 gene. In addition, in 2002, Warshawsky et al. [107] identified a mutation in four patients of African American origin presenting venous thrombosis. It is a C20209T substitution of the 3’UTR region of the FII gene [110].

6.3 Polymorphisms of the methylenetetrahydrofolate reductase MTHFR

The MTHFR gene (Gene ID: 4524) is located on chromosome 1 (1p36.22) and consists of 12 exons ( Several transcriptional start sites, alternative splicing and polyadenylation sites have been observed for MTHFR. Transcription start sites are located in two regions, and two promoters have been characterized. The latter contain multiple binding sites for transcription factors [111].

5,10-methylenetetrahydrofolate reductase (MTHFR) is a 150 kDa dimer comprising two isoforms of varying sizes: 77 kDa and 70 kDa. It consists of 656 AA and has two domains: catalytic on the N-terminal side and regulatory on the C-terminal side. The MTHFR protein is a homodimer with a βα structure. Each monomer is formed by 8 alpha helices and 8 beta sheets. It is the cofactor of the flavine adenine dinucleotide (FAD) [111].

MTHFR is a cytoplasmic enzyme found in the spleen, lymph nodes and bone marrow. Dimeric flavoprotein 5,10-methylenetetrahydrofolate reductase (MTHFR) is an NADPH-dependent enzyme that catalyzes the reduction of 5,10-MTHFR, the major carbon donor in nucleotide biosynthesis, to 5-MTHFR, which is the predominant form of folate and the donor of the methyl radical in the reaction of homo-cysteine remethylation to methionine [111].

Indeed, homocysteine is a sulfur-containing amino acid formed during the conversion of methionine to cysteine (demethylation). The catabolism of homocysteine follows two pathways: on one hand, transsulfuration (conversion to cystathionine and then to cysteine) involving cystathionine betasynthase (CBS) and, on the other hand, remethylation (regeneration of methionine) involving methionine synthase (MS) and methylene tetrahydrofolate reductase (MTHFR). These enzymes have as enzymatic cofactors certain vitamins of the B group (B6, folic acid, B12) [112].

The 5,10-methylene tetrahydrofolate reductase (MTHFR) catalyzes the irreversible reduction of 5,10-methylene tetrahydrofolate (CH2THF) to 5-methyltetrahydrofolate (CH3THF). MTHFR activity thus affects the availability of CH2THF, which influences RNA and DNA synthesis. CH3THF is required for the remethylation of homocysteine to methionine (MET), which in turn is involved in protein synthesis and methylation of DNA and other compounds (CH3-X) [113].

Hyperhomocysteinemia may be both a genetic and acquired abnormality. Homocystinuria and hyperhomocysteinemia can be caused by rare inborn errors of metabolism that result in marked elevations of plasma and urine homocysteine concentrations [114].

Genetic polymorphisms result from common mutations that are usually ignored because they are often benign. However, some polymorphisms are not without health consequences. Two SNPs are described for MTHFR: C677T polymorphism and A1298C polymorphism of the MTHFR. Recently, rare variants in MTHFR have been detected by whole exome sequencing in association with the occurrence and the recurrence of pulmonary embolism [115].

6.3.1 C677T polymorphism of MTHFR

In 1995, Frosst et al. [78] identified a C to T substitution of nucleotide 677 that converts an alanine to a valine in 222. This mutation affects the catalytic domain of the MTHFR protein responsible for the generation of a thermolabile enzyme whose enzymatic activity is reduced by half at 37°C and absent at 46°C. This thermolability depends on the transmitted form: in homozygotes, the residual activity is only 18–22% while it is 56% in heterozygotes. On the other hand, the presence of the mutation in the homozygous state alters the metabolism of folates and induces a moderate increase in plasma homocysteine concentrations. The study of the biochemical characteristics of this thermolabile factor revealed a tendency to segregate into monomers as well as the dissociation of its cofactor FAD in solution [116].

The C677T mutation of MTHFR is a common polymorphism in the general population (allelic frequency is 0.38). Its frequency in the homozygous state varies between 1 and 21% with a significant heterogeneous distribution among different ethnic groups [117]. The homozygous TT genotype is particularly prevalent in northern China (18%), eastern Italy (18%) and California (21%). In addition, there is a geographical gradient in Europe (increase from north to south) and China (decrease from north to south). Furthermore, the genotypic frequency is low in African ancestors, intermediate in Europeans and prevalent in Americans [111].

6.3.2 A1298C polymorphism of the MTHFR

In 1998, Van der Put et al. [118] identified another SNP less frequent than C677T (allelic frequency is 0.33): it is a 1298A-C substitution that converts Glu429 to Ala (E429A) and thus destroys the restriction site for MboII. This mutation affects the regulatory domain of the protein and is associated with a more pronounced reduction in MTHFR enzymatic activity in homozygotes than in heterozygotes. However, the E429A protein is biochemically similar to the normal protein [116].

6.4 Protein C deficiency

Protein C (PROC), protein S (PROS1), and antithrombin (Serpinc1) have been demonstrated to play important roles in the anticoagulation process and thrombophilia [69, 72, 119].

PROC gene is located on chromosome 2 (2q14.3) and consists of 8 exons. Gene ontology annotations related to this gene include calcium ion binding and peptidase activity. An important paralog of this gene is PROZ (

This gene encodes a vitamin K-dependent plasma glycoprotein, produced and secreted by hepatic cells as a zymogen. Along with its cofactor protein S (PS), activated protein C plays the role of an inactivator of the coagulation factors and an important factor of the regulation of the blood clotting pathway. It has a proteolytic effect on of the activated forms of coagulation factors V and VIII (Va and VIIIa). Protein C is a multi-domain glycoprotein composed of a non-catalytic light chain linked to the catalytic heavy chain by a single disulfide bond. The light chain harbors the vitamin K-dependent N-terminal g-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor (EGF)-like domains. The C-terminal catalytic heavy chain with a trypsin-like substrate specificity is preceeded by an activation peptide, which is removed during the activation of protein C by the thrombin- thrombomodulin complex. The encoded protein C is cleaved to its activated form or APC by the thrombin-thrombomodulin complex. APC with the serine protease domain leads to the degradation of the coagulation factors Va and VIIIa in the presence of PS acting as a non-enzymatic cofactor, calcium ions and phospholipids. Protein C exerts also a protective effect on the endothelial cell barrier function [119, 120, 121].

Various conditions have been shown to cause acquired protein C deficiency. These conditions include vitamin K deficiency, warfarin therapy, severe liver disease, disseminated intravascular coagulation, severe bacterial infections in the young, and some chemotherapy drugs [122].

In contrast, inherited protein C deficiency is caused by genetic variations in the PROC gene. Prevalence of hereditary PROC deficiency is estimated at 0.2–0.5%. The milder form is caused by an alteration in one PROC gene and is inherited in an autosomal dominant manner. The severe form is caused by an alteration in both PROC genes (homozygous or compound heterozygotes) and is inherited in an autosomal recessive manner. In the other hand, heterozygous mutations in many adults may be asymptomatic for life but other heterozygous protein C deficiencies are characterized by recurrent venous thrombosis. Individuals with decreased amounts of protein C are classically referred to as having type I deficiency and those with normal amounts of a functionally defective protein as having type II deficiency [123].

PC deficiency is found in 3% of patients with primary venous thromboembolic disease. However, regarding the complex forms of inherited PC deficiency, studies of thrombophilic patients have shown that the prevalence of PC deficiency associated with thrombosis is between 1/16,000 and 1/36,000 [124]. A much higher prevalence of asymptomatic PC deficiency has been shown in a healthy blood donor population (1/200 to 1/700) [125].

Mutations in PROC gene have been long associated with thrombophilia with an increased tendency toward thromboembolic disease risk. In 1981, it was first described by Griffin et al. [80] that hereditary PROC deficiency was responsible of an hypercoagulability state. Hereditary PROC deficiency considered as autosomal dominant by familial studies arises from several distinct mutations in the PROC gene. PROC mutations leading to homozygous deficiency are detected during neonatal purpura fulminans [126].

Protein C database analysis suggests that there are about 380 mutations of PROC gene and that the mutations are scattered on both light and heavy chains and involve all functional domains of the protein (Gla, EGF1, EGF2 and catalytic domains), and recurrent venous thrombosis [127]. ClinVar database records that mention thrombosis and PROC showed 200 genetic variations. PROC variants occurring as the result of these genetic changes can lead to severe intracellular impairments and ineffective PROC release or non-functional PROC release.

PROC gene neighboring sequence contains several transcriptional regulatory regions. Distinct polymorphic loci were identified on promoter region of the human PROC gene. It was shown that polymorphic regions of the PROC gene: -1654C > T, -1641A > G and -1476A > T were associated with deep venous thromboembolism in some countries. Pulmonary embolism incidence in Chinese population seems to be associated with TT phenotype of -1654C > T polymorphism of PROC gene [128].

Recently, it was shown that PC as well as its PS cofactor are not only partners in the anticoagulant system, but also proteins closely involved in the mechanisms of inflammation, apoptosis, and in vascular permeability [129].

6.5 Protein S deficiency

PROS1 gene is located on chromosome 3 at 3q11.1 with 16 exons. There are two genes with 98% homology: an active gene with 15 exons spanning more than 80 kb and a non-coding pseudogene b, which is very close to the PSa gene ( This gene encodes Protein S (PS), the major cofactor of PC. It is a single-stranded, vitamin K-dependent glycoprotein of 69 kDa. PS is produced by the liver, but has also been localized in the endothelial cell, the megakaryocyte and the Leydig cell. It is synthesized as a 676 AA precursor comprising a leader sequence eliminated before secretion, a hydrophobic signal peptide, and a propeptide with the carboxylase recognition site analogous to that of other vitamin K-dependent factors. The mature form of PS (635 AA) consists of a GLA domain with 11 GLA residues, a binding peptide, a thrombin-sensitive loop (TSL), four EGF domains, and a carboxyterminal region with areas of homology to the hormone-binding globulin (SHBG) [130, 131, 132, 133].

PS acts to prevent coagulation, mainly by inactivating factors V and VIII. PS increases the affinity of PCa for negatively charged phospholipids, forming a membrane-bound PCa-PS complex that makes factors Va and VIIIa more accessible to cleavage by PCa. PS circulates in the plasma partly under the influence of PCa. Free form (40% of circulating PS) is active in the coagulation system. Whereas, 60% is in the complexed form with C4b-binding protein (C4bBP), a protein of the complement system that binds PS at the SHBG domain. C4bBP-bound PS has no cofactor effect on PCa [131].

Other mechanisms of action independent of PC have been suggested for PS but their physiological importance is not firmly established; in particular, PS may have direct anticoagulant activity through its ability to bind and inhibit factors Xa, Va, and VIIIa and to compete with procoagulant factors for binding to phospholipids. It may also stimulate inhibition of the tissue factor pathway inhibitor or TFPI.

PS deficiency is found in 2–3% of thrombophilic patients. The prevalence in the general population may be in the range of 0.05% to 0.1% [130].

6.6 Antithrombin III deficiency

Antithrombin belongs to the serine protease inhibitor superfamily: the serpins. It exerts its physiological function by inhibiting procoagulation factors, such as thrombin, factor Xa, factor IIa, and other factors of the blood coagulation system. It contributes to the regulation of clot formation both by inhibiting thrombin activity directly and by interfering with earlier stages of the clotting cascade [134].

Antithrombin (AT) is a single-stranded plasma glycoprotein with a molecular weight of 58 kDa and 432 amino acids (AA) and four oligosaccharide side chains. AT is synthesized by the liver. It mainly inactivates thrombin and activated factor Xa, but also, in the presence of heparin, factors VIIa, XIa and XIIa [135].

Inactivation of the protease involves the formation of an irreversible bond between the active site of the enzyme and the reactive site of the inhibitor, formed by Arg 393 and Ser 394 (P1-P1’). The AT acts as a pseudosubstrate for the enzyme. Indeed, cleavage of the P1-P1’ linkage induces a major conformational change in the AT, which can then form a stable complex with the target protease by incorporation of the AAs located upstream of Arg 393 into a b-sheet structure consisting of five strands in the uncleaved form and six strands in the cleaved form, with the sixth strand being the P1-P14 segment [136].

Inhibition of the enzyme by AT is catalyzed by heparin and proteoglycans of the vascular endothelium. This interaction accelerates thrombin inhibition by a factor of approximately 2000. In the presence of heparin, the AT reactive site loop is more exposed at the protein surface and more readily fits the catalytic site of certain activated factors such as factor Xa [137]. In the case of thrombin, which like AT has binding sites for heparin, a ternary complex is formed that brings the enzyme closer to its inhibitor. The heparin-binding domain of AT comprises the region of AA 41–49 on the one hand and AA 107–156 on the other. Both regions are rich in basic AAs that can interact with the sulfate groups of heparin. They are similar in the tertiary structure of the protein [138].

SERPINC1 is the gene encoding antithrombin. It is located on chromosome 1 at 1q25.1 with 9 exons. There are ten Alu sequences in the introns, representing 22% of the intronic sequences, four times more than in the entire human genome. These repetitive elements may contribute to the occurrence of many mutations and deletions in the gene (

Antithrombin deficiency, a rare autosomal dominant disorder (MIM#107300), is caused by rare genetic variations of SERPINC1 gene. There are two types of antithrombin deficiency. In type I antithrombin deficiency, functional and antigenic levels are proportionally decreased. In type II antithrombin deficiency, antigenic levels are normal while the functional activity is abnormal. In around 0.02–0.25% of a healthy population with antithrombin deficiency, there is a 5- to 50-fold increased risk of developing venous. AT deficiency is found in 1–2% of patients with primary venous thromboembolic disease. The prevalence of symptomatic AT deficiency in the general population is between 1:2000 and 1:5000 [139].

The first variation linked to antithrombin deficiency was characterized in 1983 and, to date, more than 200 variants have been reported to be associated with the risk of thrombosis. The homozygous variant (Phe229Leu) of SERPINC1 leading to spontaneous antithrombin polymerization in vivo has been shown to be associated with severe childhood thrombosis [140]. The heterozygous variant is mainly associated with a high risk of venous thrombosis [141].

However, most of SERPINC1 genetic variants (currently 399 different mutations reported) are rare, usually found in a single family baptized as private or orphan and occasionally discovered in more than one population. For example, in a Dutch population, Bezemer et al. [142] reported the 5301G > A polymorphism of SERPINC1 gene, to be associated with the risk of venous thrombosis. The frequencies of this rs2227589 polymorphism were around 0.10 and 0.329 in the East Asian population. In Spanish Caucasian population, it was shown the presence of a functional effect of the 5301G > A on antithrombin levels [143]. All of these conclusions were debated in different other studies [139].

Although thrombophilia can be identified in about half of all patients presenting with venous thrombosis, genetic testing or screening for hereditary thrombophilia is indicated only in selected cases [144, 145].


7. Conclusions

This chapter has focused on thrombosis-related genetic polymorphisms, particularly the variations of hemostatic genes involved in classical inherited thrombophilia diseases. They are the earliest and the most studied polymorphisms in the field.

Despite the increasing knowledge about thrombosis-related genetic polymorphisms, genetic testing for inherited thrombophilia remains considered, most often, not helpful to guide clinical decisions and not recognized on a routine basis.

The current knowledge of the contribution of thrombosis-related genetic polymorphisms showed an accumulation of understanding over the years for more than half a century that has led to robust results regarding their roles in thrombotic disorders and their potential clinical consideration, particularly as genetic markers of the diseases.

Despite their recognition as risk factors with well-established frequencies and sufficiently convincing associations, the implementation of the genetic testing as diagnosis/prognosis tools failed to attain an international consensus for clinical application. In fact, even though, genetic and genomic testing and screening are expected to have a greatly increased role in healthcare with a gradually likely to be ordered in routine for many diseases, genetic test reports in thrombosis miss the ability to deliver with the results, their clinical implications clearly and unambiguously. Guidelines and recommendations on thrombosis related genetic polymorphisms laboratory analysis remained limited to a narrow range of specific clinical situations and patients and are not uniform worldwide. The conditions under which genetic testing for thrombophilia have been in fact defined, were engaged, validated and published by some working groups and medical associations [144, 145, 146, 147, 148, 149, 150, 151, 152, 153].

Furthermore, literature review showed that the AT, PC and, PS deficiencies, as well as FV Leiden, prothrombin mutation and MTHFR polymorphisms mentioned above, are considered as having an increased risk for venous thrombosis but have little or no effect on arterial thrombosis. In fact, the available evidence indicates that Leiden FV variant is not a major risk factor of any sort in arterial thrombosis and micro thrombosis, including myocardial infarction and strokes. The same conclusions were demonstrated for the other thrombophilia polymorphisms [154, 155, 156]. However, hyperhomocysteinaemia is still considered as a mixed risk factor for both arterial and venous thrombosis [1, 154].

Additionally, analysis of the literature revealed the description of several other polymorphisms that predisposes to the development of thrombosis, mainly those involving the hemostatic pathway factors like fibrinogen, factors VIII and VII, factor XIII, activated protein C receptor, thrombomodulin, plasminogen activator inhibitor, tissue plasminogen activator, Thrombin-activatable fibrinolysis inhibitor and platelet receptors (GPIIb-IIIa, GPIb-IX-V, GPIa-IIa, GPVI and others) etc… Two exhaustive reviews reporting these polymorphisms and especially their clinical significance were identified [146, 157].

In contrast, there is little clarity in relation to arterial thrombotic disease and the initial promise that genetic risk factors might contribute appreciably to an explanation of the development of arterial thromboses has largely been unfulfilled. As well, the expectations raised by early reports of positive associations have been tempered by inconsistent results with almost the majority of the studied hemostatic genes. In reality, the most consistent associations that have been found involve fibrinogen and the factor XIII [146]. Recently, most association studies of arterial thrombosis-related genetic polymorphisms are focused more much towards genes and factors involved in the other pathogenic pathways leading to thrombosis, i.e., inflammatory, immune and oxidative pathways.

Interrogation of the NCBI ClinVar database with an inquiry linking polymorphism and thrombosis revealed more than 3500 variations. The pathogenicity and the cause-and-effect relationship of these genetic variations was not strongly validated in the majority of cases. Most of related studies involved limited number of patients with untested statistical associations on a large scale. Additionally, some of the problems in identifying causal genetic markers are related to difficulties associated with the precise definition of the clinical phenotype under study. The sampling of patients in the studies was characterized by obvious heterogeneity both in terms of pathologies and in terms of the physiopathogenic origins of considered thrombosis. Indeed, there are many problematical uses of the terminology and other difficulties regarding the disorders stratification and the thrombosis typology. These difficulties of clinical and biological heterogeneity limit fundamentally the statistical homogeneity of the studied subgroups and the comparative effectiveness during the associative relationship approaches.

To overcome these shortcomings and to reach effective associations, several other technical and biological obstacles must be considered and defeated.

Translational medicine and research findings in the field of polymorphisms during thrombotic disorders are promising by the use of omics approaches and genome-wide association analysis, which will permit the identification of new risk loci. They will provide mechanistic insights into the genetic pathogenesis of thrombotic entities and put on view greater overlap among venous, arterial and microvascular thrombotic disorders than previously thought. The next step will be at the interactomic level to disclose binary and complex interactions between genes, factors and actors of thrombosis pathways. Indeed, it has been evident through this review that there are permanent interactions between the different pathogenic factors at the origin of thrombosis and almost permanent functional dualities for each factor under the influence of the dynamic homeostatic states of the organism and of the cells facing various situations to establish adaptive equilibriums. The importance of environmental influences and the complexity of the processes involved in vascular thrombotic disease suggest another myriad of other interacting metabolic factors. This understanding further increases the importance of lifelong risk interactions and may suggest an explanation for some of the inconsistencies in case–control studies. It is also important to consider that studies of population genetics of polygenic disorders, such as thrombotic disease, would ideally require prior knowledge of the relationship between the protein level/receptor density and disease, the degree of heritability of variance in the plasma levels of the protein/receptor density, and the genetic determinants of heritability.

It should be emphasized that despite the difficulties delaying the approval of genetic polymorphisms as reliable markers of thrombotic disorders, some of them have already been integrated into the preventive approaches of precision medicine, while others could be adopted in the near future in the predictive approaches of precision medicine.

Finally, it is important to mention that the most advantageous achievements of the studies on the associated thrombosis genetic variations are perceived through the pharmaceutical and biotechnological industries discovers in the field of thrombosis. These progresses on drug discoveries during the past, the present and the future are closely related to the deep understanding of the pathogenic mechanisms of thrombosis as well as their complex interplay.


Conflict of interest

Nouha Bouayed Abdelmoula and Balkiss Abdelmoula declare they have no conflict of interest.


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Written By

Nouha Bouayed Abdelmoula and Balkiss Abdelmoula

Reviewed: June 4th, 2021Published: May 4th, 2022