**3. Genetic risk factors for venous thrombosis**

Like all proteins produced in the body, clotting factors and other proteins of the Coagulation and Fibrinolysis processes are encoded by genes in the DNA of human cells. Any genetic abnormalities may lead to lower or no production of these proteins, or the production of molecules with abnormal structure and/or functions, although the quantity of which may be normal. Many of these abnormalities were found to cause venous thrombosis. For example, genetic defects in the genes of the natural anticoagulants may lead to lower

forever and the clot may enlarge until it blocks the whole lumen of the blood vessel. Therefore, the Coagulation process should be limited to the area of blood vessel injury and should be prevented from extending abroad. This is achieved by three main proteins that circulate normally in the blood, namely protein C (PC), protein S (PS) and antithrombin (AT). Together they are called "natural anticoagulants" since they function as antagonists to clotting. They exert their function after a blood clot is formed to prevent excessive clotting. They also interfere with the Coagulation process if it starts working accidently inside intact blood vessels. To explain more, AT, as its name indicates, inactivates thrombin, and therefore stops the process of Coagulation. PC, which is first activated into activated protein C (APC), tends to breakdown co-factors V and VIII and therefore slows down the Coagulation process. For APC to function normally, PS is involved as a cofactor. Phospholipids and calcium ions also assist in this process. Another inhibitor specific for the Extrinsic pathway, namely Tissue Factor Pathway Inhibitor (TFPI), limits the action of TF in activating factor VII (Kalafatis et al., 1994; Novotny, 1994; Davie, 1995; Esmon et al., 1997; Rosing & Tans, 1997; Cella et al., 1997; Hoffbrand et al., 2001; Escobar et al., 2002; Laffan &

Regarding the control of the Fibrinolysis process, there are many proteins involved. For example, plasminogen activator inhibitors (PAI) prevent tPA from activating plasminogen and therefore stop the initiation of fibrinolysis. This is important to avoid early removal of blood clot before the completion of blood vessel healing. Another anti-fibrinolysis protein is α2-antiplasmin (AP) which is a major inhibitor of plasmin. Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) is a protein that is activated by thrombin to prevent the binding of plasmin to fibrin and therefore stops plasmin from breaking down the clot. The control actions of these fibrinolysis antagonists are illustrated in figure 2 (Hoffbrand et al., 2001;

For normal healthy clotting/anticlotting results, the Coagulation and Fibrinolysis processes, with their control systems, should work in a highly balanced manner. Any abnormalities may disturb this balance leading to serious consequences. Abnormalities can be quantitative (deficiency or increase in quantity) or qualitative (abnormal structure or function [loss, lowering or gain]) that may affect any of the proteins involved. For example, abnormalities in clotting factors may lead to bleeding problems (termed haemophilia), while abnormalities in the natural anticoagulants may lead to increased clotting tendency (termed

In the following sections, different genetic and acquired abnormalities affecting the Coagulation and Fibrinolysis processes are discussed. Only those leading to thrombosis are included in accordance with the scope of this chapter. These abnormalities are usually referred to as "risk factors" since they put forth clinical manifestations in patients suffering

Like all proteins produced in the body, clotting factors and other proteins of the Coagulation and Fibrinolysis processes are encoded by genes in the DNA of human cells. Any genetic abnormalities may lead to lower or no production of these proteins, or the production of molecules with abnormal structure and/or functions, although the quantity of which may be normal. Many of these abnormalities were found to cause venous thrombosis. For example, genetic defects in the genes of the natural anticoagulants may lead to lower

hypercoagulability) leading to thrombosis, with certain exceptions in both (figure 3).

Manning, 2002a).

from these abnormalities.

Escobar et al., 2002; Laffan & Manning, 2002a).

**3. Genetic risk factors for venous thrombosis** 

Fig. 3. Balance between the Coagulation and Fibrinolysis processes, in health and disease (prepared and drawn by the author).

production of these proteins and therefore lower control over the Coagulation process. This usually leads to an increase in the rate of coagulation, a phenomenon called "hypercoagulability", which is usually manifested clinically in patients as VTE. On the other hand, the natural anticoagulants may be produced normally, but they can not exert their function normally on their targets, and therefore hypercoagulability and VTE are expected too. Also, certain genetic defect may affect the clotting factors themselves leading to overproduction of such factors causing hypercoagulability. Moreover, abnormalities in the Fibrinolysis process may lower the efficiency of removal of clot, which leads to accumulation of clots and formation of thrombosis. In the following lines, several genetic abnormalities (risk factors) leading to venous thrombosis are discussed. These usually cause VTE at relatively earlier ages (less than 40 years-old) and may be referred to as "familial or hereditary thrombophilia". Although the condition known as "Activated Protein C Resistance" is the most common genetic defect associated with VTE, this defect will be left till the end because it was discovered relatively more recently and it was found to be the most common and important genetic risk factor for VTE.

#### **3.1 Antithrombin (AT) deficiency**

Historically, Egeberg (1965) was the first to associate cases of venous thrombosis with a hereditary defect in the Coagulation system; namely AT deficiency. AT is an inhibitor for thrombin, and its inhibition action is largely enhanced by heparin as a co-factor. AT deficiency causes lower control over thrombin, and therefore the Coagulation process becomes overactive (hypercoagulability) leading to VTE. Also, decreased control over

defective. Type I is more common and has been found to be present in 1 to 14 % of cases having recurrent thrombosis. Type II is present in 10-15% of PC deficiency cases (Mohanty

PS acts as a co-factor in the process of inactivation of clotting co-factors V and VIII by APC, enhancing the process by 10-fold (ten Kate & van der Meer, 2008). PS has a very high affinity towards complement 4b binding protein (C4bBP). PS bound to C4bBP becomes inactive, and only free PS is active. Normally, the concentration of PS is more than C4bBP, and therefore only 60% of PS is present in an inactive form bound to C4bBP, while 40% remain as free active PS (Simmonds et al, 1998; Ehsan & Plumbley, 2002; Laffan & Manning, 2002b; Dahlbäck, 2008). First cases with hereditary PS deficiency were reported in 1984 (Comp & Esmon, 1984; Comp et al, 1984). Hereditary PS deficiency is an autosomal dominant disorder that has been associated with a 3- to 11-fold increased risk of venous thrombosis (Svensson and Dahlbäck, 1994; Hoffbrand et al, 2001; Ehsan & Plumbley, 2002; ten Kate & van der Meer, 2008; Bereczky et al., 2010). Similar to PC deficiency, homozygous cases with PS deficiency have tendency towards developing neonatal purpura fulminans and Warfarin-associated skin necrosis (Hoffbrand et al, 2001; Ehsan & Plumbley, 2002). In addition, PS deficiency has been linked to foetal loss (ten Kate & van der Meer, 2008). More than 200 genetic abnormalities in the PS gene were identified to cause PS deficiency, half of which were missense mutations and one-fifth were deletions or insertions (Bertina, 1997; ten Kate & van der Meer, 2008; Bereczky et al., 2010). The prevalence of PS deficiency is 0.03-2% in the general population and 1-13% in patients with VTE (Lane et al., 1996; Bertina, 1997; Seligsohn & Lubetsky, 2001; Dykes et al., 2001; Ehsan & Plumbley, 2002; Beauchamp et al., 2004; ten Kate & van der Meer, 2008; Bereczky et al., 2010). There are three types of hereditary PS deficiency. In Type I, total and free PS levels are lower than normal. Type II PS deficiency is the dysfunctional type of PS deficiency, in which the level of PS remains normal. A third type (Type III) is characterized by a mild deficiency in PS, and this is reflected in lower free PS level (Ehsan & Plumbley, 2002; ten Kate & van der Meer, 2008; Bereczky et al., 2010). Type I and III are the quantitative types of PS deficiency while Type II is the qualitative type. Certain sources refer to Type II as Type IIb and Type III as Type IIa (Ehsan & Plumbley, 2002). The majority of hereditary PS deficiency are Type I while 5-15%

TFPI is a protease that inhibits TF-VIIa complex in the presence of factor Xa, thereby regulating the Extrinsic pathway of coagulation. Only 10% of TFPI is present as a free active form in the blood while the majority is in combination with lipoproteins. Deficiency in TFPI may lead to a hypercoagulable state and hence VTE (Novotny et al., 1989; Novotny, 1994; Samama et al, 1996; Cella et al, 1997; Ehsan & Plumbley, 2002). TFPI decreased activity was noticed to contribute in developing thrombosis in women using oral contraceptives, and in patients with paroxysmal nocturnal haemoglobinuria (Maroney & Mast; 2008). Experiments on genetically modified mice with TFPI gene disruption showed that they die prematurely in embryonic stage and before birth due to haemorrhagic and intravascular thrombi. Human embryos with TFPI deficiency may suffer a similar problem and this may explain why no cases with TFPI deficiency has been identified so far (Broze, 1998; Chan, 2001;

et al., 1995, Ehsan & Plumbley, 2002; Bereczky et al., 2010).

of cases are Type II (Bertina, 1997; Bereczky et al., 2010).

**3.4 Tissue Factor Pathway Inhibitor (TFPI) deficiency** 

Maroney & Mast; 2008).

**3.3 Protein S (PS) deficiency** 

thrombin in cases with AT deficiency may have a positive effect on an inhibitor of fibrinolysis called thrombin-activatable fibrinolysis inhibitor (TAFI), which may add to the hypercoagulable status in these patients, as will be explained later.

Hereditary AT deficiency has been found in 1-5 % of thrombotic cases, with a prevalence of one in 500-5000 in different populations (Tait et al., 1991; Koster et al., 1995a; Koeleman et al., 1997; Bertina, 1997; Laffan & Manning, 2002b; Ehsan & Plumbley, 2002; Dahlbäck, 2008; Patnaik & Moll, 2008). It has an autosomal dominant mode of inheritance, and it accounts for a 10-fold increased risk of developing VTE (Dahlbäck, 2008). AT deficiency maybe divided into two types: Type I (quantitative; lower amount) and Type II (qualitative; abnormal function). Type II AT deficiency is also subdivided into three subtypes based on the kind of abnormality in function it has: affecting inhibition of thrombin, affecting the binding to heparin, or affecting both. More than 80 genetic abnormalities (missense, nonsense, deletions) were reported to cause AT deficiency (Bertina, 1997; Hoffbrand et al., 2001; Ehsan & Plumbley, 2002; Dahlbäck, 2008). More than half of the patients with hereditary AT deficiency have been reported to suffer from VTE at an age less than 40 years (Finazzi et al., 1987; van Boven et al., 1996). No reports are present on cases of homozygous AT deficiency, suggesting it is incompatible with life to have complete absence of AT in the blood (Dahlbäck, 2008).

#### **3.2 Protein C (PC) deficiency**

PC and its active form APC inactivate clotting co-factors V and VIII and therefore downregulates the Coagulation process. Hence, any abnormality in PC may lead to continuous running of co-factors V and VIII causing VTE. Another method by which PC deficiency may cause VTE is through its interaction with the Fibrinolysis process. PC usually inhibits plasminogen activator inhibitor-1 (PAI-1), which is an inhibitor of tissue plasminogen activator (tPA) responsible for the presence of active plasmin (figure 2). Therefore, PC deficiency causes an impaired control over PAI-1, and this interferes with the normal function of the Fibrinolysis process, and hence may lead to accumulation of clots and eventually VTE.

Several cases of VTE were reported to have genetic deficiency of PC, which was first described in 1981 (Griffin et al, 1981). Hereditary PC deficiency has an autosomal dominant mode of inheritance, but many reports also claimed autosomal recessive mode (Mohanty et al., 1995; Ehsan & Plumbley, 2002; Bereczky et al., 2010). Almost 250 different genetic defects have been reported so far to be associated with PC deficiency (Bertina, 1997; D'Ursi et al., 2007; Bereczky et al., 2010). The prevalence of PC deficiency has been reported to be one in 200 to 16,000 normal individuals in different studies (Miletich et al., 1987; Tait et al., 1995; Mohanty et al., 1995; Koster et al., 1995b; Ehsan & Plumbley, 2002). The prevalence in patients with first episode of VTE is 2-5% (Bertina, 1997; Laffan & Manning, 2002b; Dahlbäck, 2008;). Heterozygous carriers of PC deficiency have 50% reduction in PC level, and they have an increased risk of developing thrombosis (Svensson & Dahlbäck, 1994; Hoffbrand et al., 2001). Homozygotes for PC deficiency may suffer from recurrent VTE episodes and from skin necrosis especially when treated with Warfarin, which is a vitamin K antagonist commonly used for treatment of VTE (Heeb et al., 1989; Svensson & Dahlbäck, 1994; Bennett, 1997; Hoffbrand et al., 2001; Dahlbäck, 2008). Infants with homozygous PC deficiency usually have fatal multiple microvascular thrombosis known as neonatal purpura fulminans (Ehsan & Plumbley, 2002; Dahlbäck, 2008). Two types of PC deficiency are present: Type I PC deficiency in which the level and function of PC are abnormal; and type II deficiency in which the level of PC is normal but the function is

thrombin in cases with AT deficiency may have a positive effect on an inhibitor of fibrinolysis called thrombin-activatable fibrinolysis inhibitor (TAFI), which may add to the

Hereditary AT deficiency has been found in 1-5 % of thrombotic cases, with a prevalence of one in 500-5000 in different populations (Tait et al., 1991; Koster et al., 1995a; Koeleman et al., 1997; Bertina, 1997; Laffan & Manning, 2002b; Ehsan & Plumbley, 2002; Dahlbäck, 2008; Patnaik & Moll, 2008). It has an autosomal dominant mode of inheritance, and it accounts for a 10-fold increased risk of developing VTE (Dahlbäck, 2008). AT deficiency maybe divided into two types: Type I (quantitative; lower amount) and Type II (qualitative; abnormal function). Type II AT deficiency is also subdivided into three subtypes based on the kind of abnormality in function it has: affecting inhibition of thrombin, affecting the binding to heparin, or affecting both. More than 80 genetic abnormalities (missense, nonsense, deletions) were reported to cause AT deficiency (Bertina, 1997; Hoffbrand et al., 2001; Ehsan & Plumbley, 2002; Dahlbäck, 2008). More than half of the patients with hereditary AT deficiency have been reported to suffer from VTE at an age less than 40 years (Finazzi et al., 1987; van Boven et al., 1996). No reports are present on cases of homozygous AT deficiency, suggesting it is incompatible with life to have complete absence of AT in the

PC and its active form APC inactivate clotting co-factors V and VIII and therefore downregulates the Coagulation process. Hence, any abnormality in PC may lead to continuous running of co-factors V and VIII causing VTE. Another method by which PC deficiency may cause VTE is through its interaction with the Fibrinolysis process. PC usually inhibits plasminogen activator inhibitor-1 (PAI-1), which is an inhibitor of tissue plasminogen activator (tPA) responsible for the presence of active plasmin (figure 2). Therefore, PC deficiency causes an impaired control over PAI-1, and this interferes with the normal function of the Fibrinolysis

Several cases of VTE were reported to have genetic deficiency of PC, which was first described in 1981 (Griffin et al, 1981). Hereditary PC deficiency has an autosomal dominant mode of inheritance, but many reports also claimed autosomal recessive mode (Mohanty et al., 1995; Ehsan & Plumbley, 2002; Bereczky et al., 2010). Almost 250 different genetic defects have been reported so far to be associated with PC deficiency (Bertina, 1997; D'Ursi et al., 2007; Bereczky et al., 2010). The prevalence of PC deficiency has been reported to be one in 200 to 16,000 normal individuals in different studies (Miletich et al., 1987; Tait et al., 1995; Mohanty et al., 1995; Koster et al., 1995b; Ehsan & Plumbley, 2002). The prevalence in patients with first episode of VTE is 2-5% (Bertina, 1997; Laffan & Manning, 2002b; Dahlbäck, 2008;). Heterozygous carriers of PC deficiency have 50% reduction in PC level, and they have an increased risk of developing thrombosis (Svensson & Dahlbäck, 1994; Hoffbrand et al., 2001). Homozygotes for PC deficiency may suffer from recurrent VTE episodes and from skin necrosis especially when treated with Warfarin, which is a vitamin K antagonist commonly used for treatment of VTE (Heeb et al., 1989; Svensson & Dahlbäck, 1994; Bennett, 1997; Hoffbrand et al., 2001; Dahlbäck, 2008). Infants with homozygous PC deficiency usually have fatal multiple microvascular thrombosis known as neonatal purpura fulminans (Ehsan & Plumbley, 2002; Dahlbäck, 2008). Two types of PC deficiency are present: Type I PC deficiency in which the level and function of PC are abnormal; and type II deficiency in which the level of PC is normal but the function is

process, and hence may lead to accumulation of clots and eventually VTE.

hypercoagulable status in these patients, as will be explained later.

blood (Dahlbäck, 2008).

**3.2 Protein C (PC) deficiency** 

defective. Type I is more common and has been found to be present in 1 to 14 % of cases having recurrent thrombosis. Type II is present in 10-15% of PC deficiency cases (Mohanty et al., 1995, Ehsan & Plumbley, 2002; Bereczky et al., 2010).

#### **3.3 Protein S (PS) deficiency**

PS acts as a co-factor in the process of inactivation of clotting co-factors V and VIII by APC, enhancing the process by 10-fold (ten Kate & van der Meer, 2008). PS has a very high affinity towards complement 4b binding protein (C4bBP). PS bound to C4bBP becomes inactive, and only free PS is active. Normally, the concentration of PS is more than C4bBP, and therefore only 60% of PS is present in an inactive form bound to C4bBP, while 40% remain as free active PS (Simmonds et al, 1998; Ehsan & Plumbley, 2002; Laffan & Manning, 2002b; Dahlbäck, 2008). First cases with hereditary PS deficiency were reported in 1984 (Comp & Esmon, 1984; Comp et al, 1984). Hereditary PS deficiency is an autosomal dominant disorder that has been associated with a 3- to 11-fold increased risk of venous thrombosis (Svensson and Dahlbäck, 1994; Hoffbrand et al, 2001; Ehsan & Plumbley, 2002; ten Kate & van der Meer, 2008; Bereczky et al., 2010). Similar to PC deficiency, homozygous cases with PS deficiency have tendency towards developing neonatal purpura fulminans and Warfarin-associated skin necrosis (Hoffbrand et al, 2001; Ehsan & Plumbley, 2002). In addition, PS deficiency has been linked to foetal loss (ten Kate & van der Meer, 2008). More than 200 genetic abnormalities in the PS gene were identified to cause PS deficiency, half of which were missense mutations and one-fifth were deletions or insertions (Bertina, 1997; ten Kate & van der Meer, 2008; Bereczky et al., 2010). The prevalence of PS deficiency is 0.03-2% in the general population and 1-13% in patients with VTE (Lane et al., 1996; Bertina, 1997; Seligsohn & Lubetsky, 2001; Dykes et al., 2001; Ehsan & Plumbley, 2002; Beauchamp et al., 2004; ten Kate & van der Meer, 2008; Bereczky et al., 2010). There are three types of hereditary PS deficiency. In Type I, total and free PS levels are lower than normal. Type II PS deficiency is the dysfunctional type of PS deficiency, in which the level of PS remains normal. A third type (Type III) is characterized by a mild deficiency in PS, and this is reflected in lower free PS level (Ehsan & Plumbley, 2002; ten Kate & van der Meer, 2008; Bereczky et al., 2010). Type I and III are the quantitative types of PS deficiency while Type II is the qualitative type. Certain sources refer to Type II as Type IIb and Type III as Type IIa (Ehsan & Plumbley, 2002). The majority of hereditary PS deficiency are Type I while 5-15% of cases are Type II (Bertina, 1997; Bereczky et al., 2010).

#### **3.4 Tissue Factor Pathway Inhibitor (TFPI) deficiency**

TFPI is a protease that inhibits TF-VIIa complex in the presence of factor Xa, thereby regulating the Extrinsic pathway of coagulation. Only 10% of TFPI is present as a free active form in the blood while the majority is in combination with lipoproteins. Deficiency in TFPI may lead to a hypercoagulable state and hence VTE (Novotny et al., 1989; Novotny, 1994; Samama et al, 1996; Cella et al, 1997; Ehsan & Plumbley, 2002). TFPI decreased activity was noticed to contribute in developing thrombosis in women using oral contraceptives, and in patients with paroxysmal nocturnal haemoglobinuria (Maroney & Mast; 2008). Experiments on genetically modified mice with TFPI gene disruption showed that they die prematurely in embryonic stage and before birth due to haemorrhagic and intravascular thrombi. Human embryos with TFPI deficiency may suffer a similar problem and this may explain why no cases with TFPI deficiency has been identified so far (Broze, 1998; Chan, 2001; Maroney & Mast; 2008).

Prothrombin G20210A mutation was found to be associated with elevated levels of plasma prothrombin (elevation by one-third above normal; 133%), and therefore accounts for hypercoagulability and an increased risk of developing VTE (2 to 4-fold) (Poort et al, 1996; Bertina, 1997; Koeleman et al, 1997; Hillarp et al, 1997; Alhenc-Gelas et al. 1997; Hoffbrand et al., 2001; Ehsan & Plumbley, 2002; Laffan & Manning, 2002b; Dahlbäck, 2008). In fact, it has been demonstrated that prothrombin levels more than 115% have 2-fold increased risk of developing VTE (Poort et al, 1996). A study by Ceelie et al (2004) has proven that Prothrombin G20210A mutation leads to increased mRNA and protein expression. Another point worth mentioning here is that increased prothrombin levels may lead to an increase in the inhibitor of fibrinolysis called TAFI. This increase in TAFI disturbs the Fibrinolysis process and therefore may add to the hypercoagulable status in these patients, as will be

Several studies reported the prevalence of Prothrombin G20210A mutation to be 1-4% in healthy populations and 6-8% in patients with VTE. However, that was true when populations of Caucasian origin were studied. The Prothrombin G20210A mutation was very rare or absent in populations of East Asia and Africa, and in native populations of America and Australia (Franco et al., 1998; Dilley et al., 1998; Lin et al., 1998; Isshiki et al., 1998; Ruiz-Argüelles et al., 1999; Angelopoulou et al, 2000; Ghosh et al., 2001; Ruiz-Argüelles, 2001; Bennett et al, 2001; Lee, 2002; El-Karaksy et al, 2004; Eid & Rihani, 2004; Erber et al, 2004; Gibson et al., 2005; Dahlbäck, 2008). This brought speculations that Prothrombin G20210A mutation might have occurred as a single event in a single Caucasian ancestor. This hypothesis was strengthened by a molecular study that estimated the

Another mutation in the prothrombin gene was later discovered in 2002 at a neighbour position to the Prothrombin G20210A mutation, namely Prothrombin C20209T mutation. Unlike the Prothrombin G20210A mutation, this newer mutation was found in non-Caucasians in addition to Caucasians (Warshawsky et al, 2002; Arya, 2005; Danckwardt et

Fibrinolysis is the process responsible for the removal of intravascular clots. Therefore, one may expect that defects in this process can provide an environment suitable for the development of thrombosis. However, there is yet no final or definite proof of that in spite of the fact that reduced fibrinolysis efficacy (hypofibrinolysis) was observed in many patients with VTE with higher risk values (Laffan & Manning, 2002b; Lisman et al., 2005; Meltzer et al., 2008). For example, defects in plasminogen may cause defective fibrinolysis and impaired removal of fibrin clots, and hence might lead to accumulation of thrombi. There are two types of hereditary plasminogen deficiency: Type I hypoplasminogenaemia (quantitative) and Type II dysplasminogenaemia (qualitative), which are caused by many mutations and thought to be inherited as autosomal dominant defects. Hypoplasminogenaemia is associated with abnormal fibrin removal during wound healing, leading to pseudomembrane diseases in the mucous membranes, while dysplasminogenaemia is probably only a silent polymorphism without clinical manifestations (Aoki et al., 1978; Song et al., 2003; Schuster et al., 2007; Mehta & Shapiro, 2008; Klammt et al., 2011). At the same time, hereditary plasminogen deficiency was found in 2-8% of patients with thrombosis (Aoki et al., 1978; Dolan et al., 1988; Heijboer et al., 1990; Brandt, 2002; Song et al., 2003). Thus, more studied maybe needed before definitely

occurrence of the mutation around 24 thousand years ago (Zivelin et al., 2006).

al, 2006). Still, clear-cut association with VTE has to be established.

explained later (Ehsan & Plumbley, 2002).

**3.9 Defects of fibrinolysis** 
