**3.7 Elevated clotting factors**

Several cases with VTE were found to be associated with elevated levels of clotting factors such as VIII, IX, XI, XII, fibrinogen and prothrombin. Elevated prothrombin is mostly associated with a genetic mutation in the prothrombin gene, which will be discussed in the next section. Elevated fibrinogen (hyperfibrinogenaemia) was found to promote faster fibrin formation and increased thrombus fibrin content, density, strength and stability. Hyperfibrinogenaemia was also found to have increased thrombolysis resistance, which explains more the association with VTE (Koster et al., 1995a; Poort et al, 1996; O'Donnell et al., 1997; Meijers et al., 2000; Kamphuisen et al., 2001; de Visser et al., 2001; Bertina et al., 2005; Machlus et al., 2011).

#### **3.8 Prothrombin G20210A mutation**

In 1996, Poort et al performed extensive DNA sequencing on the prothrombin gene located on chromosome 11 for patients with unexplained VTE. They discovered a single missense mutation (guanine to adenine; GA) at nucleotide position 20210 in the 3′ untranslated region of the prothrombin gene. Since the mutation is present outside the coding region for prothrombin, it does not affect the structure of the prothrombin molecule. However, the

HCII was first detected and isolated in the early 80s (Tollefsen & Blank, 1981, Tollefsen et al., 1982). It specifically inhibits thrombin with less affinity than AT and therefore it may be considered as a second line inhibitor of thrombin (Ehsan & Plumbley, 2002). A number of cases with HCII deficiency were reported to have VTE, but many cases remained asymptomatic (Ehsan & Plumbley, 2002; Laffan & Manning, 2002b). More studies are needed on larger number of cases to determine any significant effect of this defect in causing

As explained earlier, the main aim of the Coagulation system is to convert fibrinogen (clotting factor I) into fibrin clot. Fibrinogen is encoded by three genes on chromosome 4 (Acharya & Dimichele, 2008; Miesbach et al., 2010). Genetic abnormalities in the fibrinogen genes may lead to lower or no production of fibrinogen (quantitative defects), causing bleeding problems in patients. On the other hand, other genetic abnormalities may lead to the production of fibrinogen molecules with abnormal structure and/or function (qualitative defects). Such abnormalities may negatively affect the binding of fibrinogen with thrombin, the polymerization of fibrin molecules, or the fibrinolytic inactivation by plasmin. This is the condition known as "Dysfibrinogenaemia", which has an autosomal dominant or recessive mode of inheritance (Dahlbäck, 1995; Koeleman et al, 1997; Ehsan & Plumbley, 2002; Laffan & Manning, 2002b; Acharya & Dimichele; 2008). Dysfibrinogenaemia was first reported in 1965 (Beck et al., 1965). Around 60% of cases show no clinical manifestations, while 20% show bleeding problems and 20% show thrombosis (Ehsan & Plumbley, 2002; Miesbach et al., 2010). There are at least 15 different genetic defects affecting the fibrinogen gene that were associated with Dysfibrinogenaemia (Bertina, 1997; Miesbach et al., 2010;). Still, Dysfibrinogenaemia remains a very rare disorder (1% of VTE cases) and more cases should be studied to fully understand the disease (Manucci, 2000; Acharya &

Several cases with VTE were found to be associated with elevated levels of clotting factors such as VIII, IX, XI, XII, fibrinogen and prothrombin. Elevated prothrombin is mostly associated with a genetic mutation in the prothrombin gene, which will be discussed in the next section. Elevated fibrinogen (hyperfibrinogenaemia) was found to promote faster fibrin formation and increased thrombus fibrin content, density, strength and stability. Hyperfibrinogenaemia was also found to have increased thrombolysis resistance, which explains more the association with VTE (Koster et al., 1995a; Poort et al, 1996; O'Donnell et al., 1997; Meijers et al., 2000; Kamphuisen et al., 2001; de Visser et al., 2001; Bertina et al.,

In 1996, Poort et al performed extensive DNA sequencing on the prothrombin gene located on chromosome 11 for patients with unexplained VTE. They discovered a single missense mutation (guanine to adenine; GA) at nucleotide position 20210 in the 3′ untranslated region of the prothrombin gene. Since the mutation is present outside the coding region for prothrombin, it does not affect the structure of the prothrombin molecule. However, the

**3.5 Heparin Cofactor II (HCII) deficiency** 

VTE.

**3.6 Dysfibrinogenaemia** 

Dimichele; 2008).

**3.7 Elevated clotting factors** 

2005; Machlus et al., 2011).

**3.8 Prothrombin G20210A mutation** 

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 explained later (Ehsan & Plumbley, 2002).

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

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

#### **3.9 Defects of fibrinolysis**

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

translated factor V molecule at amino acid residue number 506 (arginine to glutamine; CGACAA). Arginine 506 is an important cleavage site for APC. In other words, APC has to recognise arginine at position 506 of the factor V molecule in order to be able to inactivate factor V. This change in amino acid residue at position 506 of the mutant FVL molecule makes the FVL molecule "resistant" to the action of APC, and therefore the mutant FVL remains active. Mutant FVL was found to retain its coagulation function, and therefore the Coagulation process is not down-regulated by APC in regards to factor V. This explains why FVL leads to hypercoagulability and henceforth VTE (figure 5). Since then, the terms APC-R and FVL were linked together and used interchangeably. Several studies quickly followed that discovery and proved a positive association between FVL and VTE, showing that heterozygous carriers of the mutation are at higher risk of developing VTE by 10-fold while homozygous carriers have a much higher risk ratio reaching 140-fold (Dahlbäck et al., 1993; Zöller et al., 1994; Bertina et al., 1994; Hoagland et al., 1996; Dahlbäck, 1997; Faioni et al., 1997; Alderborn et al., 1997; Bontempo et al., 1997). Moreover, most homozygous cases were found to get at least one VTE event in their life time, and at an earlier time of their life

(Samama et al., 1996; Florell & Rodgers, 1997).

**I**

**Clotting time**

**Clotting time**

**> 60 sec**

*Ratio I:II > 2.0*

**Normal**

**APC-Resistance**

Fig. 4. APC-R test as developed originally by Dahlbäck et al., 1993 (prepared and drawn by

*Ratio I:II < 2.0*

**Plasma <sup>+</sup> APTT Reagent & CaCl2** **II**

**Clotting time**

**30 sec**

**< 60 sec**

**Plasma <sup>+</sup> APTT Reagent & CaCl2 <sup>+</sup> APC**

**Plasma <sup>+</sup> APTT Reagent & CaCl2 <sup>+</sup> APC**

the author).

linking plasminogen deficiency with VTE, and establishing Plasminogen Deficiency Registry databases may help to determine the prevalence and risk of this defect.

Another member of the Fibrinolysis process is tissue plasminogen activator (tPA) which is the main activator of plasmin in the Fibrinolysis process. Therefore, tPA deficiency may also lead to thrombosis. However, there is paucity in reports on cases with hereditary tPA deficiency to justify that (Patrassi et al., 1991; Brandt, 2002). The main inhibitor of tPA is the plasminogen activator inhibitor-1 (PAI-1). In this context, one should expect thrombosis to develop in cases having higher levels of PAI-1, rather than PAI-1 deficiency. This has been shown in different human cases and in transgenic mouse models. At least 5 polymorphisms were found in the PAI-1 gene, two of which were associated with thrombosis. In fact, this encouraged trials to use inhibitors of PAI-1 as anti-thrombotic treatments (Carmeliet et al., 1993; Huber, 2001; Wu & Zhao, 2002; Meltzer et al., 2010a; Jankun & Skrzypczak-Jankun, 2011). Trials were also conducted on inhibitors of another regulator of the Fibrinolysis process, namely Thrombin-Activatable Fibrinolysis Inhibitor (TAFI). TAFI, which was discovered in 1988, circulates as an inactive form, and is activated into its active form (TAFIa) by thrombin. TAFIa inhibits binding of plasmin to fibrinogen and therefore downregulates the Fibrinolysis process. AT deficiency, previously described, causes elevated levels of thrombin, and therefore elevated levels of TAFIa are also expected leading to lowering in the efficiency of fibrinolysis in removing clots. This is thought to be another pathophysiological pathway by which AT deficiency causes VTE. In addition, this may be an additive factor in increasing hypercoagulability in cases with Prothrombin G20210A mutation in which there is an elevated level of plasma prothrombin. However, studies on association between TAFI level and VTE gave inconsistent results. At least three genetic variations in the TAFI gene were identified, but linkage with risk of developing VTE is not very evident. Focus is now on developing inhibitors of TAFI as a possible anticoagulant therapy (Mosnier & Bouma, 2006; Bunnage & Owen, 2008; Meltzer et al., 2010a & b; Miljić et al., 2010).

#### **3.10 Activated Protein C Resistance (APC-R) and Factor V Leiden Mutation (FVL)**

In 1993, Dahlbäck and his colleagues in Sweden were involved in studying patients with VTE. They added external APC to plasma of patients with VTE and recorded the effect of that on the Coagulation process. As explained earlier, APC inactivates co-factors V and VIII and therefore down-regulates the Coagulation process. Therefore, the addition of external APC should prolong the clotting time of the plasma under test. When they tried that on the plasma samples of VTE patients, they noticed that the expected prolongation effect did not happen in all cases (Figure 4). They discussed that there is a "resistance" to the action of APC, and therefore they called it "APC resistance or APC-R", a name which persisted until now. The team originally though that there must have been a yet unknown clotting co-factor that co-helps APC in inactivating factors V and VIII, and these patients showing APC-R should have had a deficiency in this yet-to-find co-factor. However, they could not find such a proposed co-factor. One year later, Bertina and his research team in the Netherlands could identify a missense point mutation in the factor V gene (guanine to adenine; GA) at nucleotide number 1691 of exon 10 of the factor V gene, only eleven nucleotides upstream to intron 10. This new mutation was termed Factor V Leiden mutation (FVL) after the Dutch city where they made their discovery in. This nucleotide change causes a change in the

linking plasminogen deficiency with VTE, and establishing Plasminogen Deficiency Registry

Another member of the Fibrinolysis process is tissue plasminogen activator (tPA) which is the main activator of plasmin in the Fibrinolysis process. Therefore, tPA deficiency may also lead to thrombosis. However, there is paucity in reports on cases with hereditary tPA deficiency to justify that (Patrassi et al., 1991; Brandt, 2002). The main inhibitor of tPA is the plasminogen activator inhibitor-1 (PAI-1). In this context, one should expect thrombosis to develop in cases having higher levels of PAI-1, rather than PAI-1 deficiency. This has been shown in different human cases and in transgenic mouse models. At least 5 polymorphisms were found in the PAI-1 gene, two of which were associated with thrombosis. In fact, this encouraged trials to use inhibitors of PAI-1 as anti-thrombotic treatments (Carmeliet et al., 1993; Huber, 2001; Wu & Zhao, 2002; Meltzer et al., 2010a; Jankun & Skrzypczak-Jankun, 2011). Trials were also conducted on inhibitors of another regulator of the Fibrinolysis process, namely Thrombin-Activatable Fibrinolysis Inhibitor (TAFI). TAFI, which was discovered in 1988, circulates as an inactive form, and is activated into its active form (TAFIa) by thrombin. TAFIa inhibits binding of plasmin to fibrinogen and therefore downregulates the Fibrinolysis process. AT deficiency, previously described, causes elevated levels of thrombin, and therefore elevated levels of TAFIa are also expected leading to lowering in the efficiency of fibrinolysis in removing clots. This is thought to be another pathophysiological pathway by which AT deficiency causes VTE. In addition, this may be an additive factor in increasing hypercoagulability in cases with Prothrombin G20210A mutation in which there is an elevated level of plasma prothrombin. However, studies on association between TAFI level and VTE gave inconsistent results. At least three genetic variations in the TAFI gene were identified, but linkage with risk of developing VTE is not very evident. Focus is now on developing inhibitors of TAFI as a possible anticoagulant therapy (Mosnier & Bouma, 2006; Bunnage & Owen, 2008; Meltzer et al., 2010a & b; Miljić et

**3.10 Activated Protein C Resistance (APC-R) and Factor V Leiden Mutation (FVL)**  In 1993, Dahlbäck and his colleagues in Sweden were involved in studying patients with VTE. They added external APC to plasma of patients with VTE and recorded the effect of that on the Coagulation process. As explained earlier, APC inactivates co-factors V and VIII and therefore down-regulates the Coagulation process. Therefore, the addition of external APC should prolong the clotting time of the plasma under test. When they tried that on the plasma samples of VTE patients, they noticed that the expected prolongation effect did not happen in all cases (Figure 4). They discussed that there is a "resistance" to the action of APC, and therefore they called it "APC resistance or APC-R", a name which persisted until now. The team originally though that there must have been a yet unknown clotting co-factor that co-helps APC in inactivating factors V and VIII, and these patients showing APC-R should have had a deficiency in this yet-to-find co-factor. However, they could not find such a proposed co-factor. One year later, Bertina and his research team in the Netherlands could identify a missense point mutation in the factor V gene (guanine to adenine; GA) at nucleotide number 1691 of exon 10 of the factor V gene, only eleven nucleotides upstream to intron 10. This new mutation was termed Factor V Leiden mutation (FVL) after the Dutch city where they made their discovery in. This nucleotide change causes a change in the

databases may help to determine the prevalence and risk of this defect.

al., 2010).

translated factor V molecule at amino acid residue number 506 (arginine to glutamine; CGACAA). Arginine 506 is an important cleavage site for APC. In other words, APC has to recognise arginine at position 506 of the factor V molecule in order to be able to inactivate factor V. This change in amino acid residue at position 506 of the mutant FVL molecule makes the FVL molecule "resistant" to the action of APC, and therefore the mutant FVL remains active. Mutant FVL was found to retain its coagulation function, and therefore the Coagulation process is not down-regulated by APC in regards to factor V. This explains why FVL leads to hypercoagulability and henceforth VTE (figure 5). Since then, the terms APC-R and FVL were linked together and used interchangeably. Several studies quickly followed that discovery and proved a positive association between FVL and VTE, showing that heterozygous carriers of the mutation are at higher risk of developing VTE by 10-fold while homozygous carriers have a much higher risk ratio reaching 140-fold (Dahlbäck et al., 1993; Zöller et al., 1994; Bertina et al., 1994; Hoagland et al., 1996; Dahlbäck, 1997; Faioni et al., 1997; Alderborn et al., 1997; Bontempo et al., 1997). Moreover, most homozygous cases were found to get at least one VTE event in their life time, and at an earlier time of their life (Samama et al., 1996; Florell & Rodgers, 1997).

Fig. 4. APC-R test as developed originally by Dahlbäck et al., 1993 (prepared and drawn by the author).

Normal Population (%)

Sweden 41.5-50 7.5-11.4 Zöller et al., 1994; Bengtsson et al.,

Netherlands 21 2 Bertina et al., 1994; Beauchamp et al.,

Germany 30 7.1-12 Aschka et al, 1996; Schröder et al.,

Slovakia 29.5-37.0 4 Hudecek et al., 2003; Simkova et al.,

Spain 9.2-26.3 1.6-5.8 Olave et al., 1998; González Ordóñez

France 9-18 3.5-5.0 Leroyer et al., 1997; Mansourati et

Italy 9.0-42.8 2-13.1 Faioni et al., 1997; Simioni et al.,

Croatia 21.0-28.2 2.4-4.0 Coen et al., 2001; Cikes et al., 2004;

Greece 16.2-31.9 2.5-7.0 Rees et al., 1995; Lambropoulos et

USA 8.6 3.2-6.0 Ridker et al., 1997; Bontempo et al.,

Australia ---- 4-10.2 Aboud & Ma, 1997; Bennett et al.,


References

1994

1996

2004

Bengtsson et al., 1996

Stankovics et al., 1998

Hernández et al., 2007

et al., 2009

Jukic et al., 2009

1998

2003

et al., 1999; Vargas et al., 1999; Aznar et al., 2000; Ricart et al., 2006; García-

al., 2000; Meyer et al., 2001; Mazoyer

Bauder et al., 1997; Zabalegui et al.,

1997; Martinelli et al., 2004; Sottilotta et al., 2009; Gessoni et al., 2010

al., 1997; Antoniadi et al., 1999; Ioannou et al., 2000; Hatzaki et al.,

1997; Limdi et al., 2006

2001; Gibson et al., 2005;

1996; Alderborn et al., 1997

patients (%)

**European** UK ---- 1.74-5.6 Beauchamp et al., 1994;

Poland ---- 5 Herrmann et al, 1997

Belgium 22 3.3 Hainaut et al., 1997

Austria 26 ---- Melichart et al., 1996 Hungary 44 6.9 Nagy et al., 1997;

Serbia 29.9 5.8 Djordjevic et al., 2004 Azerbaijan ---- 14 Gurgey & Mesci, 1997


Yugoslavia 15.5 4.0 Mikovic et al., 2000 Slovenia 12.9 6.3 Bedencic et al., 2008

Israel ---- 4.3 Rosen et al., 1999 Brazil 20 2 Arruda et al., 1995 Table 1. Prevalence of FVL in Caucasian patients with VTE and healthy populations living

Country VTE

French/ Spanish Basques

Albania/ Kosovo

in European and non-European countries.

**Non-European** 

Fig. 5. Factor V molecule showing the site of amino acid 506 where FVL is present and how this leads to APC-R and VTE (prepared and drawn by the author).

The identification of APC-R/FVL and its high risk value have exploded a massive rush in researches to study this new disease, its prevalence and its relationship with VTE in almost every part of the world. First researches were conducted in Europe which concentrated on Caucasian populations. Results showed that FVL was present in a quite high percentage of patients with VTE (15-65%) and healthy subjects (1-15%). Other studies on Caucasians living in non-European countries, like the USA, Australia and Israel, revealed similar numbers (table 1). However, when studies started to appear in other ethnic groups and in other countries, FVL was astonishingly found to be very rare and in most occasions absent, like in Africans, South-East Asians, Chinese, Japanese, American Indians (native nations of America), Greenland Inuit (Eskimos) and native populations of Australia (table 2 and figure 6).

Fig. 6. Prevalence of FVL worldwide in different ethnic groups (prepared and drawn by the author).

14 Venous Thrombosis – Principles and Practice

Fig. 5. Factor V molecule showing the site of amino acid 506 where FVL is present and how

The identification of APC-R/FVL and its high risk value have exploded a massive rush in researches to study this new disease, its prevalence and its relationship with VTE in almost every part of the world. First researches were conducted in Europe which concentrated on Caucasian populations. Results showed that FVL was present in a quite high percentage of patients with VTE (15-65%) and healthy subjects (1-15%). Other studies on Caucasians living in non-European countries, like the USA, Australia and Israel, revealed similar numbers (table 1). However, when studies started to appear in other ethnic groups and in other countries, FVL was astonishingly found to be very rare and in most occasions absent, like in Africans, South-East Asians, Chinese, Japanese, American Indians (native nations of America), Greenland Inuit

Fig. 6. Prevalence of FVL worldwide in different ethnic groups (prepared and drawn by the

this leads to APC-R and VTE (prepared and drawn by the author).

(Eskimos) and native populations of Australia (table 2 and figure 6).

author).


Table 1. Prevalence of FVL in Caucasian patients with VTE and healthy populations living in European and non-European countries.

Fig. 7. Molecular studies exploring the origin of FVL (prepared and drawn by the author).


Table 2. Prevalence of FVL in non-Caucasian patients with VTE and healthy populations in different parts of the world.

Finding FVL to be mostly confined to Caucasian populations have brought speculations that FVL might have occurred as a single event in one European Caucasian ancestor, and the current carriers of the mutation descended from that ancestor. To prove a single origin of FVL, several molecular studies were conducted on different single nucleotides polymorphisms (SNPs) in the factor V gene trying to identify any association of FVL with such SNPs. At least 9 SNPs were found to be always associated with FVL. These studies included carriers and non-carriers of FVL, as well factor V gene in chimpanzees to elucidate when FVL might have first appeared (figure 7). These studies, combined with the distribution of FVL worldwide and the anthropological knowledge on movement of Mankind in the far past suggested that FVL should have occurred after the separation between Caucasoid (who settled in Europe) and Mongoloid (who moved to East Asia) populations, which is estimated to occur around 32,000 years ago (figure 8). This means that FVL is less than 32 thousand years old and this is why it is present in Caucasians only. The next question is: where in Europe has it happened? Since 1997 Castoldi et al suggested that FVL probably occurred outside Europe. It was observed that FVL was very rare in the

**Asians** Japan 0 0 Fujimura et al., 1995; Zama et al.,

Korea 0 ---- Kim et al., 1998

Indonesia ---- 0 Pepe et al., 1997 Malaysia 0.5 ---- Lim et al., 1999

Normal Population (%)

China 0 0 Pepe et al., 1997; Ho et al., 1999

India 3 1.3 Herrmann et al., 1997; Ghosh et al.,


Singapore 5 ---- Lim et al., 1999; Lee, 2002

Pakistan 1.25 ---- Nasiruddin et al., 2005 USA ---- 0 Gregg et al., 1997

Ethiopia ---- 0 Pepe et al., 1997;

USA 1.4 0.9 Gregg et al., 1997;

Ecuador ---- 0 Pepe et al., 1997 Venezuela ---- 4.4 Vizcaino et al., 2000

Venezuela ---- 1.25 Vizcaino et al., 2000 USA ---- 0 Gregg et al., 1997

Australia ---- 0 Bennett et al., 2001;

Table 2. Prevalence of FVL in non-Caucasian patients with VTE and healthy populations in

Finding FVL to be mostly confined to Caucasian populations have brought speculations that FVL might have occurred as a single event in one European Caucasian ancestor, and the current carriers of the mutation descended from that ancestor. To prove a single origin of FVL, several molecular studies were conducted on different single nucleotides polymorphisms (SNPs) in the factor V gene trying to identify any association of FVL with such SNPs. At least 9 SNPs were found to be always associated with FVL. These studies included carriers and non-carriers of FVL, as well factor V gene in chimpanzees to elucidate when FVL might have first appeared (figure 7). These studies, combined with the distribution of FVL worldwide and the anthropological knowledge on movement of Mankind in the far past suggested that FVL should have occurred after the separation between Caucasoid (who settled in Europe) and Mongoloid (who moved to East Asia) populations, which is estimated to occur around 32,000 years ago (figure 8). This means that FVL is less than 32 thousand years old and this is why it is present in Caucasians only. The next question is: where in Europe has it happened? Since 1997 Castoldi et al suggested that FVL probably occurred outside Europe. It was observed that FVL was very rare in the

**Amerindians** Ecuador ---- 0 Pepe et al., 1997

**Eskimos** Greenland ---- 0 De Maat et al., 1996

References

Kodaira et al., 1997; Ro et al., 1999

2001; Mishra & Bedi, 2010

Abdulkadir et al., 1997

Limdi et al., 2006

Erber et al., 2004

1996;

VTE patients (%)

Country/

**Africans/ Black**

**Indigenous Australians** 

Sub-

Sahara

different parts of the world.

Ethnic groups

Fig. 7. Molecular studies exploring the origin of FVL (prepared and drawn by the author).

cases having both FVL and haemophilia, in which increased clotting due to FVL compensates for the bleeding tendency due to haemophilia, and hence better survival chances (Yan & Nelson, 2004; Franchini & Lippi, 2010). In addition, recent studies showed that FVL decreased risk of developing severe sepsis from infections to some extent, again another privilege to

> Normal Population (%)

Lebanon 9.9-70.6 13.6-18.7 Irani-Hakime et al., 2000; Irani-Hakime et

Syria ---- 13.6 Irani-Hakime et al., 2000; Dashti et al., 2010

Jordan 23.9-25.7 10.5-27.2 Awidi et al., 1999; Eid & Rihani, 2004; Eid &

Turkey 21-30.8 4.6-9.8 Ozbek & Tangün, 1996; Gurgey & Mesci,

Cyprus ---- 13.4 Angelopoulou et al., 2000

Table 3. Prevalence of FVL in patients with VTE and normal populations in different East

Fig. 9. Map of the Mediterranean area showing decrease in prevalence of FVL as moving from East Mediterranean to North and West Europe. This made many believe that the origin of FVL is somewhere in the Eastern Mediterranean region (prepared and drawn by the author).

References

al., 2001; Taher et al., 2001; Tamim et al., 2002; Finan et al., 2002; Almawi et al., 2005; Bouaziz-Borgi et al., 2006; Isma'eel et al.,

Rosen et al., 1999; Dashti et al., 2010;

Shubeilat, 2005; Nusier et al., 2007; Obeidat et al., 2009; Al-Sweedan et al., 2009; Dashti

1997; Gurgey et al., 2001; Atasay et al., 2003; Irdem et al., 2005; Kalkanli et al., 2006; Kabukcu et al., 2007; Celiker et al., 2009; Oguzulgen et al., 2009; Diz-Kucukkaya et

2006a &b; Zahed et al., 2006

Hussein et al., 2010

et al., 2010

al., 2010

think of. That was also proved in animal models (Kerlin et al., 2003).

VTE patients (%)

& outside Israel) ---- 11.7-27.2

Country/

*East* 

*Mediterranean*

Ethnic groups

Palestine (inside

Mediterranean countries.

Fig. 8. Map showing movement of humans in the far past from Africa to the Middle East (March out-of-Africa), then to Europe and Asia. FVL event is thought to occur earlier than 32,000 years ago after the divergence of Caucasoid and Mongoloid populations (prepared and drawn by the author).

French and Spanish Basque populations, who are thought to be the oldest ethnic groups in Europe. This suggested FVL to occur outside Europe first (Bauder et al., 1997; Zabalegui et al., 1998). The prevalence of FVL in Eastern Mediterranean countries have shown the highest numbers in the world (table 3), bringing speculations that FVL might have occurred somewhere there and then spread to Europe (Castoldi et al., 1997; Bauder et al., 1997; Zabalegui et al., 1998; Irani-Hakime et al., 2000; Taher et al., 2001; Lucotte & Mercier et al., 2001, Dashti & Jadaon, 2011). Lucotte & Mercier (2001) proposed that FVL expanded in Europe during the Neolithic period, from a probable Anatolian center of origin in Turkey, which has occurred around 10,000 years ago. This may explain the highest prevalence of FVL in East Mediterranean countries, with noticeable gradual decrease in prevalence when radiating away from this region towards Europe and other parts of the world (Figure 9). Our research team in Kuwait has found that Arabs from Eastern Mediterranean countries who carry FVL also carried the same SNPs found to be associated with FVL in European carriers of the mutation (Jadaon et al., 2011). This gives another confirmation that FVL had most probably occurred as a single event in the past in the Eastern Mediterranean region. The large number of cases having FVL in our days suggests that FVL should have an

evolutionary survival advantage explaining its persistence until now. For example, Man had faced many challenges since long time in the past, like fighting wild animals, diseases or each other which should have caused a lot of injuries and fatal bleeding incidences. Increased clotting tendency due to FVL might have an advantageous effect in such occasions giving carriers of FVL some privilege. Women with FVL have higher clotting tendency which may be advantageous in preventing fatal blood loss during menstruation and childbirth. Less blood loss also improves the haemoglobin level in these women creating better life expectancy (Lindqvist et al., 2001; Lindqvist & Dahlbäck, 2008; Franchini & Lippi, 2011). This is also true in

Fig. 8. Map showing movement of humans in the far past from Africa to the Middle East (March out-of-Africa), then to Europe and Asia. FVL event is thought to occur earlier than 32,000 years ago after the divergence of Caucasoid and Mongoloid populations (prepared

French and Spanish Basque populations, who are thought to be the oldest ethnic groups in Europe. This suggested FVL to occur outside Europe first (Bauder et al., 1997; Zabalegui et al., 1998). The prevalence of FVL in Eastern Mediterranean countries have shown the highest numbers in the world (table 3), bringing speculations that FVL might have occurred somewhere there and then spread to Europe (Castoldi et al., 1997; Bauder et al., 1997; Zabalegui et al., 1998; Irani-Hakime et al., 2000; Taher et al., 2001; Lucotte & Mercier et al., 2001, Dashti & Jadaon, 2011). Lucotte & Mercier (2001) proposed that FVL expanded in Europe during the Neolithic period, from a probable Anatolian center of origin in Turkey, which has occurred around 10,000 years ago. This may explain the highest prevalence of FVL in East Mediterranean countries, with noticeable gradual decrease in prevalence when radiating away from this region towards Europe and other parts of the world (Figure 9). Our research team in Kuwait has found that Arabs from Eastern Mediterranean countries who carry FVL also carried the same SNPs found to be associated with FVL in European carriers of the mutation (Jadaon et al., 2011). This gives another confirmation that FVL had most probably occurred as a single event in the past in the Eastern Mediterranean region. The large number of cases having FVL in our days suggests that FVL should have an evolutionary survival advantage explaining its persistence until now. For example, Man had faced many challenges since long time in the past, like fighting wild animals, diseases or each other which should have caused a lot of injuries and fatal bleeding incidences. Increased clotting tendency due to FVL might have an advantageous effect in such occasions giving carriers of FVL some privilege. Women with FVL have higher clotting tendency which may be advantageous in preventing fatal blood loss during menstruation and childbirth. Less blood loss also improves the haemoglobin level in these women creating better life expectancy (Lindqvist et al., 2001; Lindqvist & Dahlbäck, 2008; Franchini & Lippi, 2011). This is also true in

and drawn by the author).

cases having both FVL and haemophilia, in which increased clotting due to FVL compensates for the bleeding tendency due to haemophilia, and hence better survival chances (Yan & Nelson, 2004; Franchini & Lippi, 2010). In addition, recent studies showed that FVL decreased risk of developing severe sepsis from infections to some extent, again another privilege to think of. That was also proved in animal models (Kerlin et al., 2003).


Table 3. Prevalence of FVL in patients with VTE and normal populations in different East Mediterranean countries.

Fig. 9. Map of the Mediterranean area showing decrease in prevalence of FVL as moving from East Mediterranean to North and West Europe. This made many believe that the origin of FVL is somewhere in the Eastern Mediterranean region (prepared and drawn by the author).

VTE is still not clear, the involvement of phospholipids in the Coagulation process and its regulators may give a possible explanation. In fact, there are *in vitro* evidences that LA lead to inhibition of APC, AT and fibrinolysis, and increase in expression of TF. In addition, LA were shown to directly contribute to hypercoagulability in animal models (Roubey, 1994; Kinev & Roubey, 2008; Farmer-Boatwright & Roubey, 2009). The prevalence of LA was reported to be up to 20% of VTE cases, with up to 10-fold increased risk of developing VTE (Ghosh et al., 2001; Galli et al., 2002; Ehsan & Plumbley, 2002; Jadaon & Dashti, 2005b;

Pregnancy and delivery (and post-delivery up to 6 weeks after childbirth) account for more than 6-fold increased risk of developing VTE. This may be due to changes in the Coagulation and Fibrinolysis processes happening during these events, which include increased levels of most clotting factors and their activators, with reduced fibrinolysis and PS level. Women taking oral contraceptives or receiving hormone replacement therapy were also found to have higher risk of developing VTE, which may again be attributed to increased clotting factors and decreased AT and tPA (Rosing et al., 1997; Hoffbrand et al.,

VTE are common complications of major trauma like in surgery, fractures and blood transfusion, especially in elder and obese patients. VTE was also noticed to be secondary to other diseases like liver and kidney diseases, myeloproliferative disorders, disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP) and haemolytic uraemic syndrome (HUS) (Hoffbrand et al., 2001; Ehsan & Plumbley, 2002). Furthermore, patients with cancers of the gastrointestinal and urogenital tracts and the lungs were reported to have higher risk of VTE. This may be due to activation of clotting factors by the cancerous cells themselves or the chemotherapy these patients usually receive which affects the liver and vitamin K metabolism, or predisposes to DIC (Dahlbäck, 1995; Florell & Rodgers, 1997; Hoffbrand et al., 2001; Ehsan & Plumbley, 2002; Anderson & Weitz, 2010; Vossen et al., 2011). Therefore, such diseases affect the Coagulation and Fibrinolysis processes in favour of developing VTE. Also, VTE in such diseases, especially those involving chronic inflammation, maybe due to increased C4bBP which captures more PS and lowers the availability of the active free PS and therefore may cause hypercoagulation. VTE was also present in many cases challenged with prolonged immobilization like being confined to bed due to major illnesses or post-operation, or after long airplane journey. The latter attracted a lot of public attention and was referred to in the media as "traveller's thrombosis" or "economy-class syndrome" (Hoffbrand et al., 2001; O'Keeffe & Baglin, 2003;

All genetic and acquired risk factors discussed above are usually present as a "single" defect in a patient. However, many cases with VTE were reported to have more than one genetic/acquired risk factor at the same time, which may account for a higher risk of developing VTE (Jadaon & Dashti, 2005a & b). For example, several studies reported a

Farmer-Boatwright & Roubey, 2009; Anderson & Weitz, 2010).

2001; Ehsan & Plumbley, 2002; Chan, 2010; Anderson & Weitz, 2010).

**5. Combined genetic and acquired risk factors for VTE** 

**4.2 Pregnancy, childbirth and hormone therapy** 

**4.3 Other acquired risk factors** 

DeHart, 2003; Bhatia et al., 2009).

Other mutations in the factor V gene were later identified, including Factor V Cambridge (Arg306Thr) (Williamson et al., 1998), Factor V Hong Kong (Arg306Gly) (Chan et al., 1998), Factor V Liverpool (Ile359Thr) (Mumford et al, 2003), and Factor V Kuwait (His1254Arg) (Jadaon et al., 2006). The number of cases reported to have these mutations were small and the relationship with VTE could not be well established like FVL, although many of these cases were reported in patients with VTE. Another mutation in the factor V gene called HR2 Haplotype (His1299Arg) was identified in 1996, which is discussed separately in the following section.

### **3.11 HR2 haplotype**

In 1996, a new missense mutation in exon 13 of factor V gene (A4070G) was identified (Lunghi et al., 1996). This mutation leads to an amino acid change, replacing histidine with arginine at amino acid residue position 1299 (His1299Arg). The new mutation was first assigned the name R2 polymorphism because of the use of the restriction enzyme Rsa I in the test used to detect it. Later on, R2 polymorphism was found to be in tight association with at least 12 polymorphisms in the factor V gene. Therefore, these SNPs were collectively called HR2 (Haplotype R2) (Bernardi et al., 1997; Castoldi et al., 2000). Several studies in different parts of the world gave the prevalence of HR2 to be 9.5%–15.2% in VTE cases and 5.8%–10.4% in healthy controls, with an increased risk by at least 2.5-fold (Bernardi et al., 1997; Alhenc-Gelas et al., 1999; Castoldi et al., 2000; Pecheniuk et al., 2001; Margaglione et al., 2002; Castaman et al., 2003; Faioni et al., 2004; Jadaon & Dashti, 2005a). The exact mechanism by which HR2 haplotype increases the risk for development of VTE is still not that clear. However, Castoldi et al (2000) have studied the two isoforms of factor V, V1 and V2, in cases with or without HR2. V1 is 7-fold more thrombogenic than V2. They showed that V1 was present in cases with HR2 more than in cases without the haplotype. This may give a possible explanation for the hypercoagulation and increased risk of developing VTE in carriers of HR2. However, more studies may be needed before understanding the real mechanism involved in that.
