The 677C>T mutation in the MTHFR gene was very frequent in CR groups, particularly in the CR-Indians (Table 4). The mutant allele frequency of 0.822 and the prevalence of 70% homozygous subjects found in Guaymi Indians is the highest reported in the literature to date. For comparison, in Yupka Indians from western Venezuela an allele frequency of 0.45 (homozygosity 15%) was determined (Salazar-Sánchez et al., 1999; Vizcaino et al., 2001). In five Brazilian Amazonian tribes of Indians, Franco et al. (1998) found an allele frequency of 0.240 (homozygosity 7.8%). Arruda et al. (1998) described in Amazonian Tupy Indians a frequency of 0.114 (homozygosity 1.2%). It seems that intertribal heterogeneity exists, which might be caused by isolation of small subpopulations and a high degree of consanguinity, as well as by genetic drift (Zago et al., 1996). This might also be an explanation for the deviations from the Hardy-Weinberg equilibrium we found in Guaymi Indians and CR-Africans from Limon. The extremely high prevalence of the homozygous mutant allele (TT) in CR-Indians is remarkable, particularly considering that homozygosity has been discussed

as a risk factor for cardiovascular diseases (CVD) caused by mild homocysteinemia.

The relationship between homocysteine, folate, vitamin B12 levels and the MTHFR 677C>T was studied in Indians from Western Venezuela by Vizcaino et al. (2001). An elevated homocysteine level was detected in the homozygous TT group which could be partially explained by the folate deficiency found in this group. The implication for an increased risk of thrombotic events in the Yupka Indians from Venezuela does not appear to be relevant since the life expectancy in this population is relatively short and a high incidence of coronary complicactions has not been described (Diez-Ewald et al., 1999; Salazar-Sanchez et al., 2006). Clinical studies are necessary to determine the influence of this genotype on the prevalence of CVD in different ethnic groups and the relation with different life styles, e.g.

In CR-Africans from Limon, the TT genotype and T allele frequency were similar to that of Caucasians. The higher frequencies of the T allele in the Guanacaste CR-Africans can probably be explained by admixture with Indians in this area since the 16th/17 th century

The frequency of the D allele of the ACE polymorphism and the prevalence of the DD genotype were lower in CR-Indians than in CR-blood donors, CR-Africans or Germans (Table 4). Foy et al. (1996) reported a similar low frequency of the D allele in Pima Indians. CR-Africans had the same frequencies of the ACE DD genotype or D allele as Caucasians.

population the lowest frequency has been described (0.013) (Attie Castro et al., 2000).

the Chorotegas with Mesoamerican origin.

Zaire (Attie Castro et al., 2000).

folate level or vitamin B12 intake.

(Ibarra, 2001).

**2.7 ACE**

**2.6 MTHFR 677C>T**

This was also reported for individuals from Nigeria, Jamaica and the United States (Rotimi et al., 1996). However, it is still not clear and results are conflicting, whether there is an association between the I/D polymorphism of the ACE gene (Rotimi et al., 1996; Sagnella et al., 1999) and serum ACE activity in people of African descent (Bloem et al., 1996). All these results clearly suggest that ethnic origin should be carefully considered in association studies between ACE genotype and disease etiology.

Comparing the Indian tribes of this study, it appears that the Huetar Indians are a distinct group of CR-Indians. In Huetar Indians, the frequency of the FVL and ACE D-alleles was significantly higher and the R2-allele frequency of FV HR2 haplotype was lower than in the other tribes. These results are probably due to a higher degree of admixture with Europeans in the past, compared to other CR-Indian tribes (Barrantes et al., 1998; Ibarra, 2001). Concerning the frequencies of FV IVS16 and MTHFR polymorphisms, Chorotega Indians were different from Guatuso, Guaymi, Cabecar and Bribri, indicating their different origins. The similarity of the prevalence of the ACE polymorphism in the Chorotega and Bribri Indians is remarkable, but more markers should be studied before conclusions can be drawn. Note that in Chorotega Indians and CR-Africans from Guanacaste, ACE genotypes were not distributed as expected by the Hardy-Weinberg; the same although not significant tendency also occurred in Bribri Indians. One reason might be a higher degree of admixture with Caucasians, especially in the Chorotega, compared to the others tribes investigated in this study

In conclusion, the present study has shown that genetic polymporphisms may vary appreciably within and between racial groups. Clear differences in the prevalence of studied polymorphisms were found between the different subpopulations in Costa Rica. These results indicate the importance of considering more than one molecular risk factor for determination of the risk or predisposition for a given disease. It is necessary to analyze a panel of molecular factors to determine the genetic background of a subject or population in order to estimate the genetical predisposition to disease. It was demonstrated that the prevalence of some established risk factors was lower in CR Indians than in Caucasians (D allele of ACE) or absent (FVLeiden, FII 20210G>A polymorphisms). Concerning the FVII polymorphism the protective 77 genotype was frequent only in the Chorotegas Indians, but the protective homozygous Gln353 genotype was absent in all CR-Indian groups.

Knowledge of the prevalence of the studied polymorphisms in the Costa Rican populations provides the basis for follow studies of thrombosis in latinamerican populations.

#### **3. Acknowledgment**

This work was made possible by the support from the Faculty of Medicine, Ernst-Moritz-Arndt-University, Greifswald Germany; Deutscher Akademischer Austauschdienst (DAAD) and by Vicerrectoría de Investigación, University of Costa Rica (project No. 807-A3-903).

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**4**

*Serbia*

**Inherited Thrombophilia and**

**the Risk of Vascular Events**

Ivana Novaković, Dragana Cvetković and Nela Maksimović *Faculty of Medicine and Faculty of Biology, University of Belgrade, Belgrade,*

The definition of thrombophilia includes the impact of hereditary base in the tendency to develop thrombosis and its clinical manifestations. Prothrombotic phenotype results from the interaction of inherited disorders of coagulation and various "clinical" risk factors such as obesity, immobility, major and minor surgery, hormone therapy, malignancy, etc. According to the accepted multicausal model, inherited thrombophilia is a manifestation of mutual influence of gene-gene and gene-environmental factors. Inherited thrombophilia traits include a wide range of disorders. Deficits of some coagulation inhibitors are relatively rare, and their clinical significance is previously known. On the other hand, in genes which control the coagulation cascade, there are many variants that are widespread in the population. The practical significance of these variants, known as genetic polymorphisms, is different, and the subject of numerous epidemiological-genetic, clinical and health economic

In summary, the most common congenital disorders associated with thrombophilia are: a deficiency of antithrombin, protein C and protein S, variants of factor V Leiden and prothrombin 20210, and mild hyperhomocysteinemia (Table 1). Individually or in combination, these traits are present in about 40% of patients with venous thromboembolism (VTE), and in approximately the same percentage of women with disorders of pregnancy and puerperium, such as fetal loss, fetal growth restriction and

Deficiency of antithrombin, protein C and protein S are rare disorders, the total present in about 1% of the general population. It is considered that holders of these properties have a 5-8 times greater risk of VTE, and VTE among patients with their representation is about 10- 15%. They are usually associated with the action of environmental factors, and the first VTE

It is determined that a variant of coagulation factor V, designated as factor V Leiden, is basically a genetic polymorphism. This is a SNP polymorphism in the 506th codon, which triplet CGA for arginine replaces the CAA triplet for glutamine. The prevalence of factor V

studies (Novaković et al, 2010; Krcunović et al., 2010; Pavlović et al., 2011).

**1. Introduction**

preeclampsia (De Stefano et al, 2002).

**1.2 Factor V Leiden**

**1.1 Deficiency of natural coagulant inhibitors**

event occurs before 45 years of age (De Stefano et al, 2002).

polymorphism: relationships with gender, hypertension and impaired glucose metabolism. *J Hypertens* 1999; 17: 657-664.

## **Inherited Thrombophilia and the Risk of Vascular Events**

Ivana Novaković, Dragana Cvetković and Nela Maksimović *Faculty of Medicine and Faculty of Biology, University of Belgrade, Belgrade, Serbia*

#### **1. Introduction**

58 Thrombophilia

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Thompson EA, Neel JV, Smouse PE, Barrantes R Microevolution of the Chibcha-speaking

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Torroni A, Neel JV, Barrantes R, Schurr TG, Wallace DC. Mitochondrial DNA "clock" for the

Tug E, Aydin H, Kaplan E, Dogruer D. Frequency of genetic mutations associated with thromboembolism in the Western Black Sea Region. Intern Med. 2011;50(1):17-21. Vizcaino G, Diez-Ewald M, Herrmann FH, Schuster G, Perez-Resquejo JL. Relationships

Zago MA, Silva Jr WA, Tavella MH, Santos SEB, Guerreiro JF, Figueiredo MS

Zeerleder S, Schloesser M, Redondo M, Wuillemin WA, Engel W, Furlan M, B Lämmle.

Zito F, Drummond F, Bujac S, Esnouf P, Morrissey J, Humphries SE, Miller GJ.

Zivellin A, Griffin JH, Xu X, Pabinger I, Samama M, Conrad J, Brenner B, Eldor A, Seligsohn

Rica and Venezuela. *Thromb Haemost* 1999; 82 (suppl): 275.

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revealed by six VNTRs. *Human Hered* 1996; 46: 274-289.

XII deficiency. *Thromb Haemost* 1999; 82: 1240-1246.

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polymorphism: relationships with gender, hypertension and impaired glucose

Herrmann FH The prevalence of three molecular risk factors (G20210A, Prothrombin, C677T MTHFR, factor V Leiden) in various ethnic groups in Costa

FV R2 polymorphism (ex13), FV Dde-I polymorphism (Int 16). Risk factors for venous thrombosis? Results of a Pilot Study. 30th Hemophilia Symposium Hamburg 1999. Scharrer, Schramm (eds), Springer-Verlag, Berlin, 2001; 261-265. Thiel BA. Monografia de la población de la República de Costa Rica en el siglo XIX, 2d Ed.

peoples of lower Central America: rare genes in an Amerindian complex. *Am J Hum*

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Amerinds and its implications for timing their entry into North America. *Proc Natl*

between homocysteine, folate and vitamin B12 levels with the methylenetetrahydrofolate reductase polymorphism in Indians from Western

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Revaluation of the incidence of thromboembolic complications in congenital factor

Epidemiological and genetic associations of activated factor XII concentration with factor VII activity, fibrinopeptide A concentration, and risk of coronary heart

U. A single genetic origin for a common Caucasian risk factor for venous

The definition of thrombophilia includes the impact of hereditary base in the tendency to develop thrombosis and its clinical manifestations. Prothrombotic phenotype results from the interaction of inherited disorders of coagulation and various "clinical" risk factors such as obesity, immobility, major and minor surgery, hormone therapy, malignancy, etc. According to the accepted multicausal model, inherited thrombophilia is a manifestation of mutual influence of gene-gene and gene-environmental factors. Inherited thrombophilia traits include a wide range of disorders. Deficits of some coagulation inhibitors are relatively rare, and their clinical significance is previously known. On the other hand, in genes which control the coagulation cascade, there are many variants that are widespread in the population. The practical significance of these variants, known as genetic polymorphisms, is different, and the subject of numerous epidemiological-genetic, clinical and health economic studies (Novaković et al, 2010; Krcunović et al., 2010; Pavlović et al., 2011).

In summary, the most common congenital disorders associated with thrombophilia are: a deficiency of antithrombin, protein C and protein S, variants of factor V Leiden and prothrombin 20210, and mild hyperhomocysteinemia (Table 1). Individually or in combination, these traits are present in about 40% of patients with venous thromboembolism (VTE), and in approximately the same percentage of women with disorders of pregnancy and puerperium, such as fetal loss, fetal growth restriction and preeclampsia (De Stefano et al, 2002).

#### **1.1 Deficiency of natural coagulant inhibitors**

Deficiency of antithrombin, protein C and protein S are rare disorders, the total present in about 1% of the general population. It is considered that holders of these properties have a 5-8 times greater risk of VTE, and VTE among patients with their representation is about 10- 15%. They are usually associated with the action of environmental factors, and the first VTE event occurs before 45 years of age (De Stefano et al, 2002).

#### **1.2 Factor V Leiden**

It is determined that a variant of coagulation factor V, designated as factor V Leiden, is basically a genetic polymorphism. This is a SNP polymorphism in the 506th codon, which triplet CGA for arginine replaces the CAA triplet for glutamine. The prevalence of factor V

Inherited Thrombophilia and the Risk of Vascular Events 61

The gene for prothrombin (coagulation factor II) also has a significant polymorphism 20210G> A. This polymorphism is located in the 3' untranslated region of gene, and is supposed to have a regulatory role. Its frequency in European populations is 1-5%, while it is very rare in people of African or Asiatic origin. 20210 allele leads to increased activity of

Meta analyzes show that the risk for the first thrombotic event increased 2-10 times in heterozygotes for factor V Leiden and 2-6 times in heterozygotes for allele 20210A. Combined heterozygous, possessing both polymorphisms, have as much as 20 times higher risk. These data are particularly important in situations that in themselves predispose thrombosis and thrombus-embolism, such as major orthopedic surgery or malignancy

In relation to thrombophilia, the importance of some other polymorphisms should be emphasized, such as polymorphism in the gene for methylene-tetrahidrofolat-reductase, MTHFR 677C> T. This polymorphism leads to substitution of alanine to valine at position 222 in the polypeptide, which causes thermolable form of the enzyme with decreased activity. T allele frequency is highest in Asian populations, lower in white European and American, and the lowest in African populations. In Europe, the incidence of TT homozygosity is 5-15%. People with 677TT and 677CT genotype have only 30% and 50% MTHFR enzymatic activity, respectively. MTHFR catalyzes the development of methyltetrahidrofolate, which is the main donor of methyl group in the remethylation of homocysteine to methionine. That is why the MTHFR 677 polymorphism associated with hyperhomocysteinemia, which is considered to be an independent risk factor for thrombophilia, and other disorders, such as atherosclerosis, neural tube defects, etc (Robertson et al, 2005, Wu et al, 2006; Simić-Ogrizović et al. 2006; Todorović et al. 2006;

 Patients with VTE, independently of the age of onset (</> 45 years), the circumstances of thrombosis (provoked /unprovoked), and the severity of the clinical manifestations. As a rule, patients with cancer may be excluded. Yet patients with hematologic

 Asymptomatic individuals who are first-degree relatives of a diagnosed carrier of a thrombophilic trait. This should be accompanied by accurate information and

Box 1. Candidates for screening for inherited thrombophilia (adapted from de Stefano et al,

contraceptives, hormone replacement therapy, or pregnancy.

neoplastic diseases and venous thromboembolism are potential candidates.

Women with complications of a pregnancy other than VTE:

prothrombin, and the hypercoagulation state (Robertson et al, 2005, Ho et al, 2006)

**1.3 Prothrombin G20210A**

(Robertson et al, 2005, Ho et al, 2006).

**1.4 Mild hyperhomocysteinemia**

Damnjanović et al. 2010 ).

counseling. Potential candidates:

2002 and Wu et al, 2006)

Leiden varies: is about 1-5% in North America, higher in North and Central Europe, while in the African and Asian population, this polymorphism is almost absent. It is estimated that this polymorphism originated 21- 24000 years ago. Under normal conditions, APC protein binds to factor V and cuts it into two inactive fragments. It is determined that the Leiden variant is resistant to APC protein, which prolongs the action of factor V. The result is a continuation of prothrombin activation and continuously maintain coagulation cascade (Robertson et al, 2005, Ho et al, 2006).

N/A, not readily available or unknown; VTE, venous thromboembolic event.

Table 1. Prevalence of major hypercoagulable states in different patient populations (after De Stefano et al, 2002).

Heterozygous for FVL have a 2-7 times greater risk of VTE event, while the homozygous risk increased even 40-80 times. The first event of VTE often occurs after 45 years of age. The risk is further multiplied in women during pregnancy, as well as due to the intake of oral contraceptives or hormone replacement therapy in menopause (Table 2). Thus, women heterozygous for factor V Leiden who take hormone preparations have 15.6 times higher risk of venous thrombosis, while pregnant women homozygous for this polymorphism show 34 times increased risk of thrombosis. The risk of spontaneous abortion is also increased (Robertson et al, 2005, Ho et al, 2006).

Table 2. Results for association of venous thromboembolism with oral contraceptive or hormone replacement use, Factor V Leiden, or both (after Wu et al, 2006).

#### **1.3 Prothrombin G20210A**

60 Thrombophilia

Leiden varies: is about 1-5% in North America, higher in North and Central Europe, while in the African and Asian population, this polymorphism is almost absent. It is estimated that this polymorphism originated 21- 24000 years ago. Under normal conditions, APC protein binds to factor V and cuts it into two inactive fragments. It is determined that the Leiden variant is resistant to APC protein, which prolongs the action of factor V. The result is a continuation of prothrombin activation and continuously maintain coagulation cascade

> **Patients with Single VTE (%)**

**Thrombophilic Families (%)**

**Population (%)**

Factor V Leiden 3-7 20 50 Prothrombin G20210A 1-3 6 18 Antithrombin deficiency 0.02 1 4-8 Protein C deficiency 0.2-0.4 3 6-8 Protein S deficiency N/A 1-2 3-13 Hyperhomocysteinemia 5-10 10-25 N/A

antibodies 0-7 5-15 N/A

Table 1. Prevalence of major hypercoagulable states in different patient populations (after

Heterozygous for FVL have a 2-7 times greater risk of VTE event, while the homozygous risk increased even 40-80 times. The first event of VTE often occurs after 45 years of age. The risk is further multiplied in women during pregnancy, as well as due to the intake of oral contraceptives or hormone replacement therapy in menopause (Table 2). Thus, women heterozygous for factor V Leiden who take hormone preparations have 15.6 times higher risk of venous thrombosis, while pregnant women homozygous for this polymorphism show 34 times increased risk of thrombosis. The risk of spontaneous abortion is also

**Number of % with factor in**

Oral contraceptive use 7 2530 61 29 2.0 (1.8 to 2.2) Factor V Leiden, no OC 6 1617 19 6 3.5 (2.5 to 4.9)

Factor V Leiden 6 1612 27 2 12 (7.9 to 19)

therapy 2 359 62 38 1.7 (1.4 to 2.1) Factor V Leiden, no HRT 2 221 21 7 3.1 (1.4 to 6.7) HRT and Factor V Leiden 2 218 27 3 9.2 (3.5 to 24) Table 2. Results for association of venous thromboembolism with oral contraceptive or

**women who Relative risk**

**VTE (95% CI)**

N/A, not readily available or unknown; VTE, venous thromboembolic event.

**Factor Studies Patients Had VTE Had no**

hormone replacement use, Factor V Leiden, or both (after Wu et al, 2006).

(Robertson et al, 2005, Ho et al, 2006).

Antiphospholipid

De Stefano et al, 2002).

Oral contraceptive and

Hormone replacement

**Hypercoagulable State General**

increased (Robertson et al, 2005, Ho et al, 2006).

The gene for prothrombin (coagulation factor II) also has a significant polymorphism 20210G> A. This polymorphism is located in the 3' untranslated region of gene, and is supposed to have a regulatory role. Its frequency in European populations is 1-5%, while it is very rare in people of African or Asiatic origin. 20210 allele leads to increased activity of prothrombin, and the hypercoagulation state (Robertson et al, 2005, Ho et al, 2006)

Meta analyzes show that the risk for the first thrombotic event increased 2-10 times in heterozygotes for factor V Leiden and 2-6 times in heterozygotes for allele 20210A. Combined heterozygous, possessing both polymorphisms, have as much as 20 times higher risk. These data are particularly important in situations that in themselves predispose thrombosis and thrombus-embolism, such as major orthopedic surgery or malignancy (Robertson et al, 2005, Ho et al, 2006).

#### **1.4 Mild hyperhomocysteinemia**

In relation to thrombophilia, the importance of some other polymorphisms should be emphasized, such as polymorphism in the gene for methylene-tetrahidrofolat-reductase, MTHFR 677C> T. This polymorphism leads to substitution of alanine to valine at position 222 in the polypeptide, which causes thermolable form of the enzyme with decreased activity. T allele frequency is highest in Asian populations, lower in white European and American, and the lowest in African populations. In Europe, the incidence of TT homozygosity is 5-15%. People with 677TT and 677CT genotype have only 30% and 50% MTHFR enzymatic activity, respectively. MTHFR catalyzes the development of methyltetrahidrofolate, which is the main donor of methyl group in the remethylation of homocysteine to methionine. That is why the MTHFR 677 polymorphism associated with hyperhomocysteinemia, which is considered to be an independent risk factor for thrombophilia, and other disorders, such as atherosclerosis, neural tube defects, etc (Robertson et al, 2005, Wu et al, 2006; Simić-Ogrizović et al. 2006; Todorović et al. 2006; Damnjanović et al. 2010 ).

	- one or more episodes of late fetal loss
	- two or more episodes of early fetal loss
	- Women with pre-eclampsia, fetal growth retardation or abruptio placentae
	- Asymptomatic women with a family history of VTE, before use of oral contraceptives, hormone replacement therapy, or pregnancy.

Box 1. Candidates for screening for inherited thrombophilia (adapted from de Stefano et al, 2002 and Wu et al, 2006)

Inherited Thrombophilia and the Risk of Vascular Events 63

Results of extensive cost-benefit studies do not support universal polymorphism screening prior to the introduction of hormone therapy, during pregnancy or after major orthopedic surgery. Instead, selective testing of patients with a history previous thrombo-embolism is recommended. To prevent spontaneous abortions in women with thrombophilia, low-dose aspirin and heparin is recommended. There is no universal agreement on the need for preventive therapy in elective orthopedic surgery (Robertson et al, 2005, Ho et al, 2006, Wu

**2. New approaches and research strategies in inherited thrombophilia**

Until recently, research aimed to elucidate the genetic component of the susceptibility to VTE was based on the traditional approach and employed primarily candidate gene studies; by this approach, the most significant and well-known polymorhisms of coagulation factors V and II (factor V Leiden and prothrombin 20210) were identified. Candidate genes are generally selected with respect to their known role in the traits and processes in interest. In relation to VTE, majority of studies have dealt with genes of coagulation/fibrinolysis

Thus, based on the known biology of clotting, Smith et al. (2007) investigated polymorphisms of 24 candidate genes coding for proteins affecting coagulation (factors II, V, VII, VIII, IX, X, XI, XII, XIIIa1, and XIIIb; fibrinogen α, β, and γ; and tissue factor), anticoagulation (antithrombin, proteins C and S, endothelial protein C receptor, thrombomodulin, and tissue factor pathway inhibitor [TFPI]), fibrinolysis (plasminogen and tissue-type plasminogen activator), and antifibrinolysis (PAI-1 and thrombin activatable

Although candidate gene studies have yielded some significant results, selection of appropriate candidates may be difficult, and new approaches had to be employed. One of these modern, widely used and successful strategies for identification of new genetic variants associated with susceptibility to multifactorial diseases is GWAS (genome wide association studies). GWAS is based on testing the association of a complex phenotype with large numbers of SNPs in large samples of patients. Recent use of this and other modern strategies resulted in detection of a number of new genetic variants potentially associated with VTE, but further research is needed to elucidate their significance as general risk

*ABO* locus variants were previously found to be associated with susceptibility to VTE: non-O blood group carriers were at higher risk, probably through higher levels of VWF and factor VIII, known risk factors for VTE (O'Donnell et al, 2002). This was confirmed by a recent study – Tregouet et al. (2009) conducted a GWAS on 317 000 SNPs in 453 VTE cases and 1327 controls, and found that 2 SNPs in ABO locus (rs505922 and rs657152) were significantly associated with VTE; carriers of O and A2 blood group were at lower

Within the *FGB/FGA/FGG* gene cluster (coding for fibrinogen beta, alpha and gamma), several SNPs were associated with VTE: rs867186 located between FGB and FGA (Tregouet et al, 2009), rs6050 (Thr312Ala) in FGA (Gohil et al, 2009) and rs2066865 located in the 3′UTR

Variation in gene encoding protein C (*PROC*) responsible for protein C deficiency was initially supposed to be strongly related to thrombosis, but this was not confirmed later (Dahlback, 2008). However, two polymorphisms within the promoter region (C/T at

et al, 2006).

cascade.

factors.

risk.

fibrinolysis inhibitor [TAFI]).

of the FGG gene (Uitte de Willige et al, 2005).

Box 2. Screening for inherited thrombophilia (first line panel) (adapted from de Stefano et al, 2002 and Wu et al, 2006)

Oral anticoagulant prophylaxis for as long as in patients with a normal genotype:

Oral anticoagulant prophylaxis for an indefinite duration:

Uncertain indications for oral anticoagulant prophylaxis of indefinite duration to be given on an individual basis (all genotypes):

Primary antithrombotic prophylaxis in asymptomatic relatives of proband patients with inherited thrombophilia:

in the case of: surgery, bed immobilization, plastering of the arms or of the legs, long air journeys (more than 4 hours)

in the puerperium (all genotypes)

throughout the whole pregnancy (severe thrombophilia; the indication for primary antithrombotic prophylaxis with low molecular weight heparin throughout the pregnancy in women with isolated heterozygosity for factor V Leiden or for the prothrombin G20210A is not yet certain).

Box 3. Secondary prophylaxis with oral anticoagulants after VTE in patients with inherited thrombophilia (adapted from de Stefano et al, 2002 and Wu et al, 2006)

Box 2. Screening for inherited thrombophilia (first line panel) (adapted from de Stefano et al,

 First unprovoked deep venous thrombosis with or without pulmonary embolism in patients with isolated heterozygosity for factor V Leiden or for the prothrombin G20210A or with

Two or more recurrent unprovoked episodes of deep venous thrombosis with or

 First unprovoked deep venous thrombosis with or without pulmonary embolism affecting individuals with severe thrombophilia (AT, PC, or PS deficiency,

 First life-threatening thrombotic episode (massive pulmonary embolism, cerebral venous thrombosis, splanchnic venous thrombosis), in particular if unprovoked (all

Uncertain indications for oral anticoagulant prophylaxis of indefinite duration to be given on an

Two or more recurrent provoked episodes of deep venous thrombosis with or without

Two or more recurrent unprovoked episodes of superficial thrombophlebitis

Two or more recurrent episodes of deep venous thrombosis with or without

 Diagnosis of severe thrombophilia in individuals with not recent occurrence of unprovoked deep venous thrombosis with or without pulmonary embolism. Primary antithrombotic prophylaxis in asymptomatic relatives of proband patients with

 Contraindication to oral contraception or hormone replacement therapy (in par ticular for women with severe thrombophilia, i.e. AT, PC, or PS deficiency,

Box 3. Secondary prophylaxis with oral anticoagulants after VTE in patients with inherited

in the case of: surgery, bed immobilization, plastering of the arms or of the legs, long

throughout the whole pregnancy (severe thrombophilia; the indication for primary antithrombotic prophylaxis with low molecular weight heparin throughout the pregnancy in women with isolated heterozygosity for factor V Leiden or for the

Oral anticoagulant prophylaxis for as long as in patients with a normal genotype:

First provoked deep venous thrombosis with or without pulmonary embolism (all

Antithrombin heparin cofactor activity (amidolytic method)

 Protein C (clotting or amidolytic method) Protein S (total and free antigen fractions)

APC-resistance plasma assay

moderate hyperhomocysteinemia.

genotypes).

individual basis (all genotypes):

inherited thrombophilia:

Prophylaxis:

pulmonary embolism

Oral anticoagulant prophylaxis for an indefinite duration:

without pulmonary embolism (all genotypes).

homozygosity for factor V Leiden, combined defects)

pulmonary embolism, of which only one unprovoked

homozygosity for factor V Leiden, combined defects)

thrombophilia (adapted from de Stefano et al, 2002 and Wu et al, 2006)

air journeys (more than 4 hours) in the puerperium (all genotypes)

prothrombin G20210A is not yet certain).

 Factor V Leiden Prothrombin G20210A

Homocysteine

genotypes).

2002 and Wu et al, 2006)

Results of extensive cost-benefit studies do not support universal polymorphism screening prior to the introduction of hormone therapy, during pregnancy or after major orthopedic surgery. Instead, selective testing of patients with a history previous thrombo-embolism is recommended. To prevent spontaneous abortions in women with thrombophilia, low-dose aspirin and heparin is recommended. There is no universal agreement on the need for preventive therapy in elective orthopedic surgery (Robertson et al, 2005, Ho et al, 2006, Wu et al, 2006).

### **2. New approaches and research strategies in inherited thrombophilia**

Until recently, research aimed to elucidate the genetic component of the susceptibility to VTE was based on the traditional approach and employed primarily candidate gene studies; by this approach, the most significant and well-known polymorhisms of coagulation factors V and II (factor V Leiden and prothrombin 20210) were identified. Candidate genes are generally selected with respect to their known role in the traits and processes in interest. In relation to VTE, majority of studies have dealt with genes of coagulation/fibrinolysis cascade.

Thus, based on the known biology of clotting, Smith et al. (2007) investigated polymorphisms of 24 candidate genes coding for proteins affecting coagulation (factors II, V, VII, VIII, IX, X, XI, XII, XIIIa1, and XIIIb; fibrinogen α, β, and γ; and tissue factor), anticoagulation (antithrombin, proteins C and S, endothelial protein C receptor, thrombomodulin, and tissue factor pathway inhibitor [TFPI]), fibrinolysis (plasminogen and tissue-type plasminogen activator), and antifibrinolysis (PAI-1 and thrombin activatable fibrinolysis inhibitor [TAFI]).

Although candidate gene studies have yielded some significant results, selection of appropriate candidates may be difficult, and new approaches had to be employed. One of these modern, widely used and successful strategies for identification of new genetic variants associated with susceptibility to multifactorial diseases is GWAS (genome wide association studies). GWAS is based on testing the association of a complex phenotype with large numbers of SNPs in large samples of patients. Recent use of this and other modern strategies resulted in detection of a number of new genetic variants potentially associated with VTE, but further research is needed to elucidate their significance as general risk factors.

*ABO* locus variants were previously found to be associated with susceptibility to VTE: non-O blood group carriers were at higher risk, probably through higher levels of VWF and factor VIII, known risk factors for VTE (O'Donnell et al, 2002). This was confirmed by a recent study – Tregouet et al. (2009) conducted a GWAS on 317 000 SNPs in 453 VTE cases and 1327 controls, and found that 2 SNPs in ABO locus (rs505922 and rs657152) were significantly associated with VTE; carriers of O and A2 blood group were at lower risk.

Within the *FGB/FGA/FGG* gene cluster (coding for fibrinogen beta, alpha and gamma), several SNPs were associated with VTE: rs867186 located between FGB and FGA (Tregouet et al, 2009), rs6050 (Thr312Ala) in FGA (Gohil et al, 2009) and rs2066865 located in the 3′UTR of the FGG gene (Uitte de Willige et al, 2005).

Variation in gene encoding protein C (*PROC*) responsible for protein C deficiency was initially supposed to be strongly related to thrombosis, but this was not confirmed later (Dahlback, 2008). However, two polymorphisms within the promoter region (C/T at

Inherited Thrombophilia and the Risk of Vascular Events 65

Nowadays, direct molecular detection of thrombophilia risk factors including F2 G20210A, factor V Leiden and MTHFR C677T mutations is offered by many clinical diagnostic laboratories. It is possible to discriminate between individuals heterozygous and homozygous for factor V Leiden on the basis of the degree of APCR in functional clottingbased assays. Testing for APC resistance (using an accurate assay) can be helpful in assessing for the presence of FVL, whether used initially as a ''screen'' or if used in conjunction with molecular testing. Therefore the APCR test serves as a phenotypic marker for FVL. A normal modified APCR test excludes the presence of FVL, but if abnormal, the FVL genotype should be confirmed by genetic analysis. Although the presence of PT G20210A is associated with increased levels of prothrombin in plasma, there is no phenotypic screening test for PT G20210A, and detection is always by genotyping. In contrast, there are no analogous functional assays for the detection of the PT G20210A mutation because there is no simple distinction between measurable PT levels in those with and without the mutation. Similarly, although the MTHFR thermolabile variant may be associated with increased levels of homocysteine, there is no other direct functional measure of any effect upon in vitro clotting assays. Detection of thrombophilias by functional testing is associated with a degree of uncertainty because interpretation of results can be influenced by the accuracy of normal ranges and the influence of other factors (both genetic and

Many different DNA-based methods have been described. In every laboratory method is chosen based on required sensitivity, necessary equipment, number of samples and economical capacity. In contrast to functional analyses results obtained by DNA based methods are essentially absolute, provided the requisite controls are in place, such that repeat testing is not required. Most of the methods described rely on DNA amplification by polymerase chain reaction (PCR). Genomic DNA is required for genetic analysis, but quantity and quality of DNA preparation may vary, depending on the requirements of the assay. Most laboratories routinely purify DNA for the analysis, but where only one or two single nucleotide polymorphisms (SNP) are to be evaluated, avoidance of time consuming DNA preparation can be of benefit. PCR-based methods rely on different tools to detect the genotype of the amplified alleles, such as RED, amplification-refractory mutation system (ARMS), enzyme-linked immunosorbent assay (ELISA)-based primer extension assay and fluorescence resonance energy transfer (FRET) assay. Multiplexing allows single procedure to detect more than one mutation. However, when electrophoresis is used, there may be many fragments present and care must be taken to ensure that interpretation of genotypes is clear-cut and not prone to error. Rare mutations at, or very close to the SNP site of interest may suggest different genotypes, depending on methodology. For example, rare silent mutations within the F5 gene (A1692C, G1689A and A1696G) and rare sequence variations at or near the prothrombin (F2) G20210A mutation (C20209T, A20207C, A20218G and C20221T). These will influence genetic analysis in a manner dependent upon the test system used. However, when these sequence variations are rare in the population their influence on

Original methods for detection of mutations are based on restriction fragment length polymorphism (RFLP). DNA fragment of interest is amplified by polymerase chain reaction

**3. Molecular testing of inherited thrombophilia**

environmental). Hence repeat testing is often required.

the assay may be considered negligible.

**3.1 Restriction enzyme digestion**

position -2405 and A/G at position -2418) were recently associated with increased risk of VT (Pomp et al. 2009). Carriers of CC/GG genotype, who showed the lowest protein C levels, were at higher risk of VT compared to carriers of the TT/AA genotype. Polymorphism at *CADM1,* encoding cell adhesion molecule 1, also expressed in endothelial cells, was recently identified as a probable risk factor for VTE in protein C-deficient kindreds (Hasstedt et al, 2009), but it is not clear whether it is a general risk factor also. In gene coding for protein C receptor (*PROCR* or *EPCR*) haplotype A3 (Gly219 allele of the Ser219Gly substitution) was associated with increased VTE risk (Qu et al, 2006; Tregouet et al, 2009), especially in highrisk groups of individuals, carriers of other mutations (Morange & Tregouet, 2010).

The association of gene *F12* (encoding coagulation factor FXII) variants with VTE has been subject to a long debate. Common *F12* variant has attracted much research interest, but the results were not unequivocal. *F12* C-46T variant (rs1801020) was reported to be associated with increased VTE risk (homozygous carriers of T allele compared to noncarriers; Tirado et al, 2004). This association was also observed in a number of other studies, e.g. in patients with cerebral VT (Reuner et al, 2008). However, some studies failed to confirm these findings, e.g. Grunbacher et al. (2005) found that F12 46-TT genotype was not associated with thrombosis risk, nor with age at first thrombosis. A metaanalysis by Johnson et al. (2011) led to conclusion that evidence for association of this *F12* variant with VTE was weak. By genomewide linkage analysis, Calafell et al. (2010) demonstrated that the F12 gene represents a quantitative trait locus (QTL) that influences factor XII levels, and showed that only the promoter -46C>T variant (rs1801020) accounted for the variance attributed to this QTL. The authors concluded that this variation is evolutionary neutral and that T allele appeared approximately 100,000 years ago, reaching high frequencies by genetic drift.

Buil et al. (2010) reported that a new locus, *C4BPB/C4BPA* (coding for C4-binding protein), is involved in susceptibility to VT through a still unknown, but protein S–independent mechanism. Bezemer et al. (2008) found SNPs significantly associated with VT in *CYP4V2/KLKB1/F11* gene cluster, as well as in the *GP6* and *SERPINC1* genes. Three SNPs were strongly associated with VT: rs13146272 in CYP4V2, rs2227589 in SERPINC1 and rs1613662 in GP6; 4 additional SNPs (in CYP4V2, KLKB1, and F11) were also associated with VT. The effect of CYP4V2 and GP6 loci polymorphisms (SNPs rs1613662 and rs13146272) was further confirmed by Tregouet et al. (2009), and the effect of F11 polymorphisms (SNPs rs2289252 and rs2036914) by Li et al. (2009). Recently, in a multi-stage multi-design study, Antoni et al. (2010) found evidence that *BAI3* locus (encoding brain specific angiogenesis inhibitor 3) is associated with early-onset VTE; rs9363864-AA genotype was associated with a lower risk for VTE and low levels of FVIII and VWF. Three variants were recently found to be associated with activated partial thromboplastin time: *F12* (rs2731672), *KNG1* (rs710446), and *HRG* (rs9898) (Houlihan et al. 2010); *KNG1* Ile581Thr was confirmed as a risk factor for VT (Morange et al., 2011).

Morange et al. (2010) in a follow-up study found another new locus involved in susceptibility to VT that was not a part of commonly studied coagulation/fibrinolysis pathway: *HIVEP1* (coding for human immunodeficiency virus type 1 enhancer-binding protein). Allele rs169713C was associated with an increased risk for VT.

Future research in relation to genetic basis of VTE is aimed to explore other possibilities as well (Morange & Tregouet 2010), such as the effects of CNVs (copy number variations) or changes in epigenetic mechanisms (e.g. DNA methylation patterns).

position -2405 and A/G at position -2418) were recently associated with increased risk of VT (Pomp et al. 2009). Carriers of CC/GG genotype, who showed the lowest protein C levels, were at higher risk of VT compared to carriers of the TT/AA genotype. Polymorphism at *CADM1,* encoding cell adhesion molecule 1, also expressed in endothelial cells, was recently identified as a probable risk factor for VTE in protein C-deficient kindreds (Hasstedt et al, 2009), but it is not clear whether it is a general risk factor also. In gene coding for protein C receptor (*PROCR* or *EPCR*) haplotype A3 (Gly219 allele of the Ser219Gly substitution) was associated with increased VTE risk (Qu et al, 2006; Tregouet et al, 2009), especially in high-

The association of gene *F12* (encoding coagulation factor FXII) variants with VTE has been subject to a long debate. Common *F12* variant has attracted much research interest, but the results were not unequivocal. *F12* C-46T variant (rs1801020) was reported to be associated with increased VTE risk (homozygous carriers of T allele compared to noncarriers; Tirado et al, 2004). This association was also observed in a number of other studies, e.g. in patients with cerebral VT (Reuner et al, 2008). However, some studies failed to confirm these findings, e.g. Grunbacher et al. (2005) found that F12 46-TT genotype was not associated with thrombosis risk, nor with age at first thrombosis. A metaanalysis by Johnson et al. (2011) led to conclusion that evidence for association of this *F12* variant with VTE was weak. By genomewide linkage analysis, Calafell et al. (2010) demonstrated that the F12 gene represents a quantitative trait locus (QTL) that influences factor XII levels, and showed that only the promoter -46C>T variant (rs1801020) accounted for the variance attributed to this QTL. The authors concluded that this variation is evolutionary neutral and that T allele appeared approximately 100,000 years

Buil et al. (2010) reported that a new locus, *C4BPB/C4BPA* (coding for C4-binding protein), is involved in susceptibility to VT through a still unknown, but protein S–independent mechanism. Bezemer et al. (2008) found SNPs significantly associated with VT in *CYP4V2/KLKB1/F11* gene cluster, as well as in the *GP6* and *SERPINC1* genes. Three SNPs were strongly associated with VT: rs13146272 in CYP4V2, rs2227589 in SERPINC1 and rs1613662 in GP6; 4 additional SNPs (in CYP4V2, KLKB1, and F11) were also associated with VT. The effect of CYP4V2 and GP6 loci polymorphisms (SNPs rs1613662 and rs13146272) was further confirmed by Tregouet et al. (2009), and the effect of F11 polymorphisms (SNPs rs2289252 and rs2036914) by Li et al. (2009). Recently, in a multi-stage multi-design study, Antoni et al. (2010) found evidence that *BAI3* locus (encoding brain specific angiogenesis inhibitor 3) is associated with early-onset VTE; rs9363864-AA genotype was associated with a lower risk for VTE and low levels of FVIII and VWF. Three variants were recently found to be associated with activated partial thromboplastin time: *F12* (rs2731672), *KNG1* (rs710446), and *HRG* (rs9898) (Houlihan et al. 2010); *KNG1* Ile581Thr was confirmed as a risk factor for

Morange et al. (2010) in a follow-up study found another new locus involved in susceptibility to VT that was not a part of commonly studied coagulation/fibrinolysis pathway: *HIVEP1* (coding for human immunodeficiency virus type 1 enhancer-binding

Future research in relation to genetic basis of VTE is aimed to explore other possibilities as well (Morange & Tregouet 2010), such as the effects of CNVs (copy number variations) or

protein). Allele rs169713C was associated with an increased risk for VT.

changes in epigenetic mechanisms (e.g. DNA methylation patterns).

risk groups of individuals, carriers of other mutations (Morange & Tregouet, 2010).

ago, reaching high frequencies by genetic drift.

VT (Morange et al., 2011).

#### **3. Molecular testing of inherited thrombophilia**

Nowadays, direct molecular detection of thrombophilia risk factors including F2 G20210A, factor V Leiden and MTHFR C677T mutations is offered by many clinical diagnostic laboratories. It is possible to discriminate between individuals heterozygous and homozygous for factor V Leiden on the basis of the degree of APCR in functional clottingbased assays. Testing for APC resistance (using an accurate assay) can be helpful in assessing for the presence of FVL, whether used initially as a ''screen'' or if used in conjunction with molecular testing. Therefore the APCR test serves as a phenotypic marker for FVL. A normal modified APCR test excludes the presence of FVL, but if abnormal, the FVL genotype should be confirmed by genetic analysis. Although the presence of PT G20210A is associated with increased levels of prothrombin in plasma, there is no phenotypic screening test for PT G20210A, and detection is always by genotyping. In contrast, there are no analogous functional assays for the detection of the PT G20210A mutation because there is no simple distinction between measurable PT levels in those with and without the mutation. Similarly, although the MTHFR thermolabile variant may be associated with increased levels of homocysteine, there is no other direct functional measure of any effect upon in vitro clotting assays. Detection of thrombophilias by functional testing is associated with a degree of uncertainty because interpretation of results can be influenced by the accuracy of normal ranges and the influence of other factors (both genetic and environmental). Hence repeat testing is often required.

Many different DNA-based methods have been described. In every laboratory method is chosen based on required sensitivity, necessary equipment, number of samples and economical capacity. In contrast to functional analyses results obtained by DNA based methods are essentially absolute, provided the requisite controls are in place, such that repeat testing is not required. Most of the methods described rely on DNA amplification by polymerase chain reaction (PCR). Genomic DNA is required for genetic analysis, but quantity and quality of DNA preparation may vary, depending on the requirements of the assay. Most laboratories routinely purify DNA for the analysis, but where only one or two single nucleotide polymorphisms (SNP) are to be evaluated, avoidance of time consuming DNA preparation can be of benefit. PCR-based methods rely on different tools to detect the genotype of the amplified alleles, such as RED, amplification-refractory mutation system (ARMS), enzyme-linked immunosorbent assay (ELISA)-based primer extension assay and fluorescence resonance energy transfer (FRET) assay. Multiplexing allows single procedure to detect more than one mutation. However, when electrophoresis is used, there may be many fragments present and care must be taken to ensure that interpretation of genotypes is clear-cut and not prone to error. Rare mutations at, or very close to the SNP site of interest may suggest different genotypes, depending on methodology. For example, rare silent mutations within the F5 gene (A1692C, G1689A and A1696G) and rare sequence variations at or near the prothrombin (F2) G20210A mutation (C20209T, A20207C, A20218G and C20221T). These will influence genetic analysis in a manner dependent upon the test system used. However, when these sequence variations are rare in the population their influence on the assay may be considered negligible.

#### **3.1 Restriction enzyme digestion**

Original methods for detection of mutations are based on restriction fragment length polymorphism (RFLP). DNA fragment of interest is amplified by polymerase chain reaction

Inherited Thrombophilia and the Risk of Vascular Events 67

In order to simplify the procedure, increase the efficiency and reduce the costs by eliminating the use of restriction enzymes new methods have been developed. One of them is ARMS based on allele specific amplification of DNA sample. Standard procedure is performed in two separate reactions and with three different primers. In each reaction one of the primers is common (used in both reactions) and the other one is allele specific with 3'end complementary either to wild type allele or mutant allele. Results are obtained by analysis of PCR products on agarose or polyacrylamide gels. The presence or absence of PCR products represents the presence or absence of target alleles. Usually control primers are amplified in the same reaction with allele specific primers to ensure that the PCR reaction is working properly. Multiplex ARMS assay is also widely in use and it allows the analysis of two or more mutations. The Stagen kits for FVL and PT G20210A (Stago, Asnieres, France) amplify ß-2 microglobulin and an unconnected region of F5 (or F2) genes as controls, to ensure efficiency of the PCR reactions. Although this method is simple, efficient and cost effective the specificity of amplification relies highly on careful primer design, careful titration of primer concentrations, and stringent temperature cycling

The other simple and fast method which avoids the need for restriction enzyme digestion is a single-strand conformation polymorphism (SSCP). It relies on the fact that denatured DNA molecules show different migration patterns on electrophoresis gels even when they differ in only one base. After PCR amplification DNA fragment of interest is denatured to single strands and analyzed by gel electrophoresis. Different alleles will form different 3D conformation and different migration patterns will be observed on gels. The advantage of this method is that it may identify rare mutations, such as the silent A1692C transition in the F5 gene, which is falsely identified as FVL when MnlI restriction enzyme is used (Keeney et al., 1999). These authors also described an SSCP multiplex assay to identify FVL and PT G20210A mutations in whole blood samples. Care must be taken on the interpretation of SSCP, as mutations may cause relatively small changes in electrophoretic mobility Cooper &

ELISA-based methods generally hybridize a biotinylated PCR product to oligonucleotide bound to microtitre plate wells. Streptavidin–horseradish peroxidase conjugate then binds

Fig. 3. Polyacrilamide gel electrophoresis of PCR-RFLP products for the MTHFR polymorphism Lane 1- molecular size marker; lane2,3,7,9-heterozygous patients; lane4,6,8,10-normal homozygous patients; lane 5-mutant homozygous patient

**3.2 Amplification refractory mutation system (ARMS)**

parameters (McGlennen, 2002).

Rezende (2007).

**3.3 Single strand conformational polymorphism**

**3.4 Enzyme-linked immunosorbent assays**

(PCR) and than subjected to digestion by specific restriction enzymes whose restriction site is either created or abolished by the presence of mutation. Fragments of different sizes, generated after digestion, are separated by agarose or polyacrylamide gel electrophoresis and visualized under ultraviolet light after staining with ethidium bromide. The number of bands allows the discrimination of possible genotypes. For the FVL mutation restriction enzyme MnlI is used. The fragment generated by PCR contains one restriction enzyme site which serves as a positive internal control. It ensures that the restriction enzyme has been added, and that the restriction digestion has occurred. The second restriction site for MnlI is created by FVL mutation (Bertina, 1994). In the method described for detection of the PT G20210A in the presence of the mutant allele an artificial HindIII restriction site had to be created by the mutagenic amplification primer (Poort et al, 1996). Improvement of the original method provides additional restriction site which serves as a control as in the case of FVL detection (Pecheniuk, 2000). The MTHFR C677T mutation is also easily detected by PCR and RFLP analysis since the mutation creates restriction enzyme site for HinfI enzyme (Frosst, 1995). The use of multiplex PCR prior to RFLP and the use of whole blood instead of isolated DNA largely improved the original RFLP analyses (Gomez, 1998).

Fig. 1. Polyacrylamide gel electrophoresis of PCR-RFLP products for the FII polymorphism. Lane 1-molecular size marker; lane 2,3,6-normal patient; lane 4,5- heterozygous patient; lane7-heterozygous control; lane 8-normal control; lane 9-blank control

Fig. 2. Polyacrilamide gel elelctrophoresis of PCR-RFLP products for the FVL polymorphism Lane 1-molecular size marker; lane 2-normal control; lane 3-hetrozygous control; lane 5-homozygous mutant control, lane 6,7-heterozygous patients, lane 8-blank control

(PCR) and than subjected to digestion by specific restriction enzymes whose restriction site is either created or abolished by the presence of mutation. Fragments of different sizes, generated after digestion, are separated by agarose or polyacrylamide gel electrophoresis and visualized under ultraviolet light after staining with ethidium bromide. The number of bands allows the discrimination of possible genotypes. For the FVL mutation restriction enzyme MnlI is used. The fragment generated by PCR contains one restriction enzyme site which serves as a positive internal control. It ensures that the restriction enzyme has been added, and that the restriction digestion has occurred. The second restriction site for MnlI is created by FVL mutation (Bertina, 1994). In the method described for detection of the PT G20210A in the presence of the mutant allele an artificial HindIII restriction site had to be created by the mutagenic amplification primer (Poort et al, 1996). Improvement of the original method provides additional restriction site which serves as a control as in the case of FVL detection (Pecheniuk, 2000). The MTHFR C677T mutation is also easily detected by PCR and RFLP analysis since the mutation creates restriction enzyme site for HinfI enzyme (Frosst, 1995). The use of multiplex PCR prior to RFLP and the use of whole blood instead of

isolated DNA largely improved the original RFLP analyses (Gomez, 1998).

lane7-heterozygous control; lane 8-normal control; lane 9-blank control

Fig. 1. Polyacrylamide gel electrophoresis of PCR-RFLP products for the FII polymorphism. Lane 1-molecular size marker; lane 2,3,6-normal patient; lane 4,5- heterozygous patient;

Fig. 2. Polyacrilamide gel elelctrophoresis of PCR-RFLP products for the FVL polymorphism

Lane 1-molecular size marker; lane 2-normal control; lane 3-hetrozygous control; lane 5-homozygous mutant control, lane 6,7-heterozygous patients, lane 8-blank control

Fig. 3. Polyacrilamide gel electrophoresis of PCR-RFLP products for the MTHFR polymorphism Lane 1- molecular size marker; lane2,3,7,9-heterozygous patients; lane4,6,8,10-normal homozygous patients; lane 5-mutant homozygous patient

#### **3.2 Amplification refractory mutation system (ARMS)**

In order to simplify the procedure, increase the efficiency and reduce the costs by eliminating the use of restriction enzymes new methods have been developed. One of them is ARMS based on allele specific amplification of DNA sample. Standard procedure is performed in two separate reactions and with three different primers. In each reaction one of the primers is common (used in both reactions) and the other one is allele specific with 3'end complementary either to wild type allele or mutant allele. Results are obtained by analysis of PCR products on agarose or polyacrylamide gels. The presence or absence of PCR products represents the presence or absence of target alleles. Usually control primers are amplified in the same reaction with allele specific primers to ensure that the PCR reaction is working properly. Multiplex ARMS assay is also widely in use and it allows the analysis of two or more mutations. The Stagen kits for FVL and PT G20210A (Stago, Asnieres, France) amplify ß-2 microglobulin and an unconnected region of F5 (or F2) genes as controls, to ensure efficiency of the PCR reactions. Although this method is simple, efficient and cost effective the specificity of amplification relies highly on careful primer design, careful titration of primer concentrations, and stringent temperature cycling parameters (McGlennen, 2002).

#### **3.3 Single strand conformational polymorphism**

The other simple and fast method which avoids the need for restriction enzyme digestion is a single-strand conformation polymorphism (SSCP). It relies on the fact that denatured DNA molecules show different migration patterns on electrophoresis gels even when they differ in only one base. After PCR amplification DNA fragment of interest is denatured to single strands and analyzed by gel electrophoresis. Different alleles will form different 3D conformation and different migration patterns will be observed on gels. The advantage of this method is that it may identify rare mutations, such as the silent A1692C transition in the F5 gene, which is falsely identified as FVL when MnlI restriction enzyme is used (Keeney et al., 1999). These authors also described an SSCP multiplex assay to identify FVL and PT G20210A mutations in whole blood samples. Care must be taken on the interpretation of SSCP, as mutations may cause relatively small changes in electrophoretic mobility Cooper & Rezende (2007).

#### **3.4 Enzyme-linked immunosorbent assays**

ELISA-based methods generally hybridize a biotinylated PCR product to oligonucleotide bound to microtitre plate wells. Streptavidin–horseradish peroxidase conjugate then binds

Inherited Thrombophilia and the Risk of Vascular Events 69

PCR amplification, an endpoint plate reading is performed using real time PCR system. Allelic discrimination is achieved by measuring of the fluorescence values based on the

The gold standard method of mutation detection (screening) is bidirectional sequencing. The advantage of this method is that unusual mutations which can cause analytical anomalies can be easily detected. Variation of the standard dideoxy sequencing is Pyrosequencing (Biotage AB, Uppsala, Sweden) which simplifies further the analysis and makes it possible to analyze a large number of PCR products quickly and with minimal

DNA microarray technology provides rapid simultaneous testing for large number of single point mutations. It is a method of choice for laboratories with large number of test requests (Schrijver, 2003). Custom designed oligonucleotide sequences complementary to the normal DNA sequence and known SNP are attached to a chip. The DNA sample screened for mutation is amplified by PCR, fluorescently labeled and hybridized with the oligonucleotides in the microarray. Computer analysis of the color pattern of the microarray generated after hybridization allows rapid automated mutation testing (Turnpeny & Ellard, 2007). Several commercial platforms which include factor V Leiden, PT G20210A and MTHFR C677T mutation are available. However, they are still expensive for most of the laboratories. Low-density arrays with 10 to 20 markers could be a good alternative. In a single test all thrombophilic markers identified through the human genome project could be

This assay, unlike most of the other DNA-based genotyping methods does not employ PCR. Analysis is performed with genomic DNA. The Invader technology is based on the generation of a fluorescent signal in the reaction solution following the cleavage of synthetic oligonucleotide probe assembled with a so called invader probe and the DNA template that contains either the normal or mutation nucleotide (McGlennen, 2002). The analysis is

The main advantages of the Invader assay are that it does not employ the PCR reaction, therefore, failures resulting from contamination are less likely to occur (Cooper & Rezende, 2007). There is only a requirement for heating block and a fluorometer, but no dedicated apparatus is required for this assay. Genotypes can be obtained reliably directly from genomic DNA. It suffers from two limitations. First it requires relatively large amounts of target DNA. Second, only one SNP can be genotyped per reaction (Olivier, 2005). Modifying of the technology to accommodate multiple SNPs and large numbers of samples

This work was supported by Ministry of Science, Republic of Serbia (Grant No. 175091).

performed in microtiter well and the signal is detected by fluorescent plate reader.

simultaneously will significantly improve this assay .

signals from each well (Spector, 2005).

**3.7 Direct sequencing of DNA**

**3.8 DNA microarray technology**

analysed (McGlennen, 2002).

**3.9 Invader assay**

**4. Acknowledgement**

effort.

to the bound amplicon and finally, buffered hydrogen peroxide with chromophore detects the bound amplicon. Comparison of colour density generated by reactions with wild-type and mutant oligonucleotide probes determines the genotype. A variation of this test is a multiplex assay, where two separate PCR reactions utilize a common biotinylated primer and reverse primers specific for wild-type or the mutant allele, and PCR products hybridize onto an oligonucleotide probe in microtitre plate wells. The former assay design may be more robust, as a single amplification reaction amplifies DNA irrespective of genotype. An ELISA assay for FVL using a reverse allele-specific oligonucleotide hybridization was described by Kowalski et al. (2000). ELISA-based methods require the use of a thermocycler, but other specialist genotyping apparatus is not required. In comparison to gel technology, chemicals used are less hazardous. The StripA Assay (ViennaLab, Vienna, Austria) is an ELISA, which utilizes the reverse hybridization principle, binding reactants to a membrane strip rather than to microtitre plate wells. The target sequence of DNA is amplified in a single reaction which produces a biotin-labelled amplicon. The amplicon is selectively hybridized to wild-type and mutant oligonucleotide detector bands on an individual membrane strip, and detected by means of streptavidin–alkaline phosphatase with colourforming substrate. Washing and hybridization steps can be automated. Control and test bands are examined by eye or scanned to determine genotype. StripA kits are available for the detection of FVL and PT G20210A mutations and as a multiplex assay.

#### **3.5 Real-time PCR**

Real time high resolution melting curve analysis (HRM) employs a new class of fluorescent dyes that intercalate with double-stranded DNA. The intercalating dye is incorporated in PCR reaction and the products are then heated to separate the two strands. Fluorescence levels decrease as the DNA strands dissociate and this melting profile depends on the PCR product size and sequence. HRM appears to be very sensitive and can be used for high throughput mutation screening. Melting temperatures of wild type and mutant allele differ and genotype can be easily interpreted. Whilst Tm values can be extremely useful and reliable in detecting wild-type and common mutations, they cannot be relied upon to characterize unusual mutations. Instrument is relatively expensive and prone to contamination. A multiplex assay for FVL and PT was developed by Van den Bergh et al. (2000). The authors considered their assay to be robust, reliable with high discrimination power. Interpretation of results is based on the presence or absence of peaks with a Tm that is specific for the wild-type or mutant allele. It helps to identify additional sequence variations. This approach is simple allowing the result to be obtained in approximately two hours. Although the instrument is relatively expensive and the method is prone to contamination it is robust and reliable for identification of mutation of interest.

#### **3.6 Mutation detection using hydrolysis probes**

Some systems use only a single fluorescently labeled oligonucleotide probe (TaqMan hydrolysis probes) that provide very sensitive and specific detection of DNA. For genotyping this technology uses a PCR primer pair and two allele specific hydrolysis probes, one designed to detect the mutated allele and the other wild-type allele. Both allelespecific hydrolysis probes have a quencher dye and different reporter dyes attached. During the extension phase of PCR amplification the probe is hydrolyzed by Taq polymerase, reporter and quencher dyes are separated which causes the increase in fluorescence. After PCR amplification, an endpoint plate reading is performed using real time PCR system. Allelic discrimination is achieved by measuring of the fluorescence values based on the signals from each well (Spector, 2005).

#### **3.7 Direct sequencing of DNA**

68 Thrombophilia

to the bound amplicon and finally, buffered hydrogen peroxide with chromophore detects the bound amplicon. Comparison of colour density generated by reactions with wild-type and mutant oligonucleotide probes determines the genotype. A variation of this test is a multiplex assay, where two separate PCR reactions utilize a common biotinylated primer and reverse primers specific for wild-type or the mutant allele, and PCR products hybridize onto an oligonucleotide probe in microtitre plate wells. The former assay design may be more robust, as a single amplification reaction amplifies DNA irrespective of genotype. An ELISA assay for FVL using a reverse allele-specific oligonucleotide hybridization was described by Kowalski et al. (2000). ELISA-based methods require the use of a thermocycler, but other specialist genotyping apparatus is not required. In comparison to gel technology, chemicals used are less hazardous. The StripA Assay (ViennaLab, Vienna, Austria) is an ELISA, which utilizes the reverse hybridization principle, binding reactants to a membrane strip rather than to microtitre plate wells. The target sequence of DNA is amplified in a single reaction which produces a biotin-labelled amplicon. The amplicon is selectively hybridized to wild-type and mutant oligonucleotide detector bands on an individual membrane strip, and detected by means of streptavidin–alkaline phosphatase with colourforming substrate. Washing and hybridization steps can be automated. Control and test bands are examined by eye or scanned to determine genotype. StripA kits are available for

the detection of FVL and PT G20210A mutations and as a multiplex assay.

contamination it is robust and reliable for identification of mutation of interest.

Some systems use only a single fluorescently labeled oligonucleotide probe (TaqMan hydrolysis probes) that provide very sensitive and specific detection of DNA. For genotyping this technology uses a PCR primer pair and two allele specific hydrolysis probes, one designed to detect the mutated allele and the other wild-type allele. Both allelespecific hydrolysis probes have a quencher dye and different reporter dyes attached. During the extension phase of PCR amplification the probe is hydrolyzed by Taq polymerase, reporter and quencher dyes are separated which causes the increase in fluorescence. After

**3.6 Mutation detection using hydrolysis probes**

Real time high resolution melting curve analysis (HRM) employs a new class of fluorescent dyes that intercalate with double-stranded DNA. The intercalating dye is incorporated in PCR reaction and the products are then heated to separate the two strands. Fluorescence levels decrease as the DNA strands dissociate and this melting profile depends on the PCR product size and sequence. HRM appears to be very sensitive and can be used for high throughput mutation screening. Melting temperatures of wild type and mutant allele differ and genotype can be easily interpreted. Whilst Tm values can be extremely useful and reliable in detecting wild-type and common mutations, they cannot be relied upon to characterize unusual mutations. Instrument is relatively expensive and prone to contamination. A multiplex assay for FVL and PT was developed by Van den Bergh et al. (2000). The authors considered their assay to be robust, reliable with high discrimination power. Interpretation of results is based on the presence or absence of peaks with a Tm that is specific for the wild-type or mutant allele. It helps to identify additional sequence variations. This approach is simple allowing the result to be obtained in approximately two hours. Although the instrument is relatively expensive and the method is prone to

**3.5 Real-time PCR**

The gold standard method of mutation detection (screening) is bidirectional sequencing. The advantage of this method is that unusual mutations which can cause analytical anomalies can be easily detected. Variation of the standard dideoxy sequencing is Pyrosequencing (Biotage AB, Uppsala, Sweden) which simplifies further the analysis and makes it possible to analyze a large number of PCR products quickly and with minimal effort.

#### **3.8 DNA microarray technology**

DNA microarray technology provides rapid simultaneous testing for large number of single point mutations. It is a method of choice for laboratories with large number of test requests (Schrijver, 2003). Custom designed oligonucleotide sequences complementary to the normal DNA sequence and known SNP are attached to a chip. The DNA sample screened for mutation is amplified by PCR, fluorescently labeled and hybridized with the oligonucleotides in the microarray. Computer analysis of the color pattern of the microarray generated after hybridization allows rapid automated mutation testing (Turnpeny & Ellard, 2007). Several commercial platforms which include factor V Leiden, PT G20210A and MTHFR C677T mutation are available. However, they are still expensive for most of the laboratories. Low-density arrays with 10 to 20 markers could be a good alternative. In a single test all thrombophilic markers identified through the human genome project could be analysed (McGlennen, 2002).

#### **3.9 Invader assay**

This assay, unlike most of the other DNA-based genotyping methods does not employ PCR. Analysis is performed with genomic DNA. The Invader technology is based on the generation of a fluorescent signal in the reaction solution following the cleavage of synthetic oligonucleotide probe assembled with a so called invader probe and the DNA template that contains either the normal or mutation nucleotide (McGlennen, 2002). The analysis is performed in microtiter well and the signal is detected by fluorescent plate reader.

The main advantages of the Invader assay are that it does not employ the PCR reaction, therefore, failures resulting from contamination are less likely to occur (Cooper & Rezende, 2007). There is only a requirement for heating block and a fluorometer, but no dedicated apparatus is required for this assay. Genotypes can be obtained reliably directly from genomic DNA. It suffers from two limitations. First it requires relatively large amounts of target DNA. Second, only one SNP can be genotyped per reaction (Olivier, 2005). Modifying of the technology to accommodate multiple SNPs and large numbers of samples simultaneously will significantly improve this assay .

#### **4. Acknowledgement**

This work was supported by Ministry of Science, Republic of Serbia (Grant No. 175091).

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Catanese, J., Young, B.A., Reitsma, P.H., Devlin, J.J., Rosendaal, F.R. (2008). Gene variants associated with deep vein thrombosis. *JAMA* 299: 1306–1314.

Cambien, F., Lathrop, M., Zeller, T., Alessi, M.C., Rodriguez de Cordoba, S., Münzel, T., Wild, P., Fontcuberta, J., Gagnon, F., Emmerich, J., Almasy, L., Blankenberg, S., Soria, J.M., Morange, P.E. (2010). C4BPB/C4BPA is a new susceptibility locus for venous thrombosis with unknown protein S– independent mechanism: results from genome-wide association and gene expression analyses followed by case-control studies. *Blood* 115(23): 4644–4650.

M., Souto, J. C., Blangero, J., Fontcuberta, J., Soria, J. M. (2010). Sequence variation and genetic evolution at the human F12 locus: mapping quantitative trait nucleotides that influence FXII plasma levels. *Hum Molec Genet* 19: 517-525, ISSN

the prothrombin G20210A mutations. *International Journal of Laboratory Hematology*

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Inherited Thrombophilia and the Risk of Vascular Events 73

Reuner, K.H., Jenetzky, E., Aleu, A., Litfin, F., Mellado, P., Kloss, M., Juttler, E., Grau, A.J.,

Robertson, L., Wu, O., Langhorne, P., Twaddle, S., Clark, P., Lowe, G.D.O., et al. (2005).

Schrijver, I. (2003). Diagnostic Single Nucleotide Polymorphism Analysis of Factor V Leiden

Simić-Ogrizović S., Stosović M., Novaković I., Pejanović S., Jemcov T., Radović M.,

Spector, E. (2005). Technical standards and guidelines: Venous thromboembolism (Factor V

Tirado, I., Soria, J.M., Mateo, J., Oliver, A., Souto, J.C., Santamaria, A., Felices, R., Borrell,

Todorovic Z., Džoljić E., Novaković I., Mirković D., Stojanović R., Nešić Z., Krajinović M.,

Tregouet, D.A., Heath, S., Saut, N., Biron-Andreani, C., Schved, J.F., Pernod, G., Galan, P.,

Turnpeny, P. & Ellard, S. (2007). *Emery's Elements of Medical Genetics* (13), Elsevier,

Uitte de Willige, S., De Visser, M.C., Houwing-Duistermaat, J.J., Rosendaal, F.R., Vos, H.L. &

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and Prothrombin 20210G>A. *American Journal of Clinical Pathology* 119 (4): 490-496.

Djukanovic Lj. (2006). Fuzzy role of hyperhomocysteinemia in hemodialysis

Lumley, T., Bis, J.C., Wiggins, K.L., Rosendaal, F.R. (2007). Association of genetic variations with nonfatal venous thrombosis in postmenopausal women. *JAMA*

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Drouet, L., Zelenika, D., Juhan-Vague, I., Alessi, M.C., Tiret, L., Lathrop, M., Emmerich, J. & Morange, P.E. (2009). Common susceptibility alleles are unlikely to contribute as strongly as the FV and ABO loci to VTE risk: results from a GWAS

Bertina, R.M. (2005). Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma' levels.

Morange, P.E., Tregouet, D.A. (2010). Deciphering the molecular basis of venous

Morange, P.E., Bezemer, I., Saut, N., Bare, L., Burgos, G., Brocheton, J., Durand, H., Biron-

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Novaković, I., Maksimović, N., Cvetković, S., Cvetković, D. (2010). Gene polymorphisms as

O'Donnell, J., Boulton, F.E., Manning, R.A. & Laffan, M.A. (2002). Amount of H antigen

Olivier M. (2005). The Invader assay for SNP genotyping. *Mutation Research*, 573 (12): 103-

Pavlović A., Pekmezović T., Obrenović R., Novaković I., Tomić G., Mijajlović M., Šternić

Pecheniuk, N. (2000). DNA technology for detection of common genetic variants that

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Poort , S., Rosendaal F.R., Reitsma, P.H., Bertina, R.M. (1996). A common genetic variation in

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**5**

**APC Resistance**

*Cardiovascular Research Institute Maastricht, Maastricht University*

In order to secure the continued supply of oxygen to the tissues, the integrity of the vascular system is of prime importance for survival. Several systems therefore exist that safeguard the circulatory system and that include emergency- and repair systems. As a protective system, primary haemostasis and blood coagulation function in concert in order to prevent extensive loss of blood from the organism in the event of vascular damage (Dahlbäck, 2000). Not only humoral and cellular factors limit the loss of blood, a first-line-of-defense is formed by the physiological process of vasoconstriction which is initiated upon a breach of vascular

Being capable of converting the fluid-like medium blood into a gel-like blood clot within a very short span of time implies that the haemostatic system incorporates the intrinsic dangerous capacity of damaging the very system it is intended to protect. This is the reason why the haemostatic system is subject to strict regulation by several anticoagulant mechanisms, which together prevent the excessive or inappropriate deposition of blood

Rather than being a dormant system that only responds to changes in the vasculature, it is generally accepted that the haemostatic response, as it occurs in healthy individuals, is instead the result of the upregulation of ongoing coagulation reactions. Under normal conditions, these coagulation reactions, as well as anticoagulation reactions proceed continuously at a low level and pro- and anticoagulant reactions balance each other in a dynamic equilibrium. The temporal upregulation of only procoagulant reactions will shift

The maintenance of both pro- and anticoagulant reactions at a low but vigilant level, ensures that the haemostatic system is able to generate a swift response when needed, which can be achieved through up-regulation of either the pro- or anticoagulant processes. The initiation of procoagulant processes implicitly instigates the initiation of anticoagulation responses, as will be detailed below, by the activation of the proteolytic protein C anticoagulant pathway. The coupling of pro-and anticoagulant responses is of vital importance since whenever procoagulant reactions are not controlled by anticoagulant mechanisms, or when anticoagulant processes are defective, the formation of thrombin will become excessive and

Despite considerable progress in their diagnosis and treatment, ischemic heart disease and cerebrovascular disease, remain to be the major cause of mortality worldwide, with over 80% of cardiovascular disease deaths now taking place in low-and middle-income countries

the balance to favor a procoagulant response, (Dahlbäck & Stenflo, 1994).

**1. Introduction**

integrity.

clots within the vascular system.

the risk for thrombosis will increase.

Gerry A.F. Nicolaes

*The Netherlands*

## **APC Resistance**

Gerry A.F. Nicolaes

*Cardiovascular Research Institute Maastricht, Maastricht University The Netherlands*

#### **1. Introduction**

74 Thrombophilia

van den Bergh, F., van Oeveren-Dybicz, A., Bon, M. (2000). Rapid Single-Tube

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Screening for thrombophilia in high-risk situations: systematic review and costeffectiveness analysis. The Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) study. *Health Technology Assessment* 10: 1114

> In order to secure the continued supply of oxygen to the tissues, the integrity of the vascular system is of prime importance for survival. Several systems therefore exist that safeguard the circulatory system and that include emergency- and repair systems. As a protective system, primary haemostasis and blood coagulation function in concert in order to prevent extensive loss of blood from the organism in the event of vascular damage (Dahlbäck, 2000). Not only humoral and cellular factors limit the loss of blood, a first-line-of-defense is formed by the physiological process of vasoconstriction which is initiated upon a breach of vascular integrity.

> Being capable of converting the fluid-like medium blood into a gel-like blood clot within a very short span of time implies that the haemostatic system incorporates the intrinsic dangerous capacity of damaging the very system it is intended to protect. This is the reason why the haemostatic system is subject to strict regulation by several anticoagulant mechanisms, which together prevent the excessive or inappropriate deposition of blood clots within the vascular system.

> Rather than being a dormant system that only responds to changes in the vasculature, it is generally accepted that the haemostatic response, as it occurs in healthy individuals, is instead the result of the upregulation of ongoing coagulation reactions. Under normal conditions, these coagulation reactions, as well as anticoagulation reactions proceed continuously at a low level and pro- and anticoagulant reactions balance each other in a dynamic equilibrium. The temporal upregulation of only procoagulant reactions will shift the balance to favor a procoagulant response, (Dahlbäck & Stenflo, 1994).

> The maintenance of both pro- and anticoagulant reactions at a low but vigilant level, ensures that the haemostatic system is able to generate a swift response when needed, which can be achieved through up-regulation of either the pro- or anticoagulant processes. The initiation of procoagulant processes implicitly instigates the initiation of anticoagulation responses, as will be detailed below, by the activation of the proteolytic protein C anticoagulant pathway. The coupling of pro-and anticoagulant responses is of vital importance since whenever procoagulant reactions are not controlled by anticoagulant mechanisms, or when anticoagulant processes are defective, the formation of thrombin will become excessive and the risk for thrombosis will increase.

> Despite considerable progress in their diagnosis and treatment, ischemic heart disease and cerebrovascular disease, remain to be the major cause of mortality worldwide, with over 80% of cardiovascular disease deaths now taking place in low-and middle-income countries

APC Resistance 77

acquired risk factors, use of oral contraceptives, pregnancy/puerperium, the presence of antiphospholipid antibodies, immobilization, surgery, malignancies and trauma are amongst the most studied. Furthermore, age and sex are recognized as independent

In this chapter resistance to APC, the central protein of the protein C anticoagulant pathway, will be discussed, both for genetically determined forms of APC resistance and acquired types of APC resistance. Particular attention will be given to the molecular events that occur during the APC-catalyzed down-regulation of thrombin formation in normal individuals, since knowledge of these processes is pivotal in our understanding of the causative role of

During homeostasis several anticoagulant systems balance the procoagulant forces that act in human blood, thereby preventing excessive platelet and fibrin depositions. A major inhibitory buffer is formed by the circulating inhibitors, which include antithrombin, alpha-2-macroglobulin, antitrypsin or tissue factor pathway inhibitor (TFPI) which provide the necessary negative feedback to procoagulant forces. These circulating inhibitors target the activated forms of the serine proteases from the coagulation cascade like thrombin and FXa and act by complex formation with their target proteases, resulting in a loss of proteolytic

Key regulators in the amplification of thrombin formation are however the non-enzymatic cofactor proteins factor V (FV) and factor VIII (FVIII) and by virtue of the absolute requirement of their activity for normal thrombogenesis, they are obvious targets for the attenuation of thrombin formation. Given that these molecules lack proteolytic activity, they cannot be inactivated via complex formation but instead they are targeted by proteolytic inactivation, resulting in disintegration of the protein structure and concomittant loss of cofactor activity. The main proteolytic enzyme responsible for the inactivation of activated

Low levels of activated protein C circulate, but for a sufficient anticoagulant response to occur, an upregulation of protein C is required through a specific set of molecular events, which together are described as the protein C anticoagulant pathway. The main player in this pathway is protein C. Protein C is a vitamin K dependent zymogen belonging to the class of chymotrypsin-like serine proteases (Stenflo, 1976). Being a zymogen, protein C circulates at a plasma concentration of ~65 nM in humans as an inactive pro-enzyme which requires processing in order to obtain its enzymatic activity. A pivotal role in the initiation of the protein C pathway is the formation of a complex between thrombin and thrombomodulin (Fuentes-Prior et al, 2000), see Fig. 1 below. Note that it is thrombin, the very enzyme that ultimately needs to be downregulated, that initiates its own attenuation. Thrombomodulin is a transmembrane protein that is present on the undamaged endothelium, in particular the endothelium of the smaller blood vessels. The membranebound thrombomodulin is able to capture thrombin by binding to the exosite I of thrombin upon which the thrombin-thrombomodulin complex is formed. The exosite I of thrombin is primarily involved in procoagulant interactions of the enzyme, namely in the recognition and activation of fibrinogen, FV and FVIII (Lane & Caso, 1989). After binding of thrombomodulin to the thrombin exosite I the procoagulant properties of any thrombin molecules that have migrated from a site of ongoing coagulation and are transported into

contributing factors in the pathogenesis of venous thrombosis (Bertina, 2001).

APC resistance in the occurrence of thromboembolic disease.

activity and clearance of the active protease from circulation.

FV (FVa) and FVIII (FVIIIa) is the activated form of protein C.

**2. Protein C anticoagulant pathway**

and occurring almost equally in men and women (Global Health Observatory (GHO), World Health Organisation [WHO], 2008).

Thromboembolic disease contributes to this overall mortality, as it is implicated in many manifestations of cardiovascular disease. The clinical manifestation of a thrombus is that of the pathological presence of an occlusion in a blood vessel or in the heart, which causes an obstruction of the physiological flow of blood through the circulatory system. Such occlusions can occur in either the venous or arterial part of the vessel tree and the condition is consequently classified as either venous or arterial thrombosis. Despite the fact that in both venous and arterial thrombosis the normal haemostatic balance is disturbed, the two types of thrombosis are considered distinct disease states that each have their own particular molecular pathology and underlying risk factors (Rosendaal, 1999, 2005).

The proper functioning of the anticoagulant protein C pathway is undisputedly required to prevent the occurrence of thrombosis, and in particular venous thromboses, as is most strikingly illustrated for protein C deficiency. Newborns who are homozygous protein C deficient can develop the life-threatening condition of acute onset "purpura fulminans" (Dreyfus et al, 1991, 1995) which can be treated by replacement therapy employing purified protein C concentrates.

Resistance to activated protein C (APC), or APC resistance, the subject of this chapter, has been implicated mostly in pathogenesis of venous thrombosis, despite the fact that the causative FVLeiden mutation, which is present in a majority of cases, has been related to myocardial infarction and/or overall coronary disease (Rallidis et al.2003; Segev et al., 2005; Ye et al., 2006). Various clinical studies have been performed to identify the contribution of the anticoagulant protein C pathway to arterial thromboses and stroke and these have not been conclusive in providing clear evidence for the contribution of the protein C pathway to arterial thrombosis (e.g. Folsom, 1999; Hankey, 2001; Boekholdt, 2007). That there is a link between the hemostatic system and arterial thrombosis or atherosclerosis appears clear as it has been established that many complex diseases, such as atherosclerosis, show an extensive crosstalk between inflammation and coagulation, as was recently reviewed by Borisoff and coworkers (Borisoff et al, 2011).

Venous thrombosis (or venous thromboembolism, VTE) is a multifactorial disease (Seligsohn&Zivelin, 1997; Rosendaal, 1999) that affects one in 10,000 individuals under the age of 40 years annually and one in 1,000 individuals over 75 years of age, causing significant morbidity and mortality (Salzman & Hirsh, 1994; Anderson et al., 1991; Nordstrom et al. 1992). The multifactorial character of VTE implies that for the disease to occur often several circumstantial and genetic factors occur simultaneously. Together these factors are capable of tipping the natural haemostatic balance between pro- and anticoagulant forces. Important to note is that environmental and acquired factors are able to modulate the existing genetic risk factors for thrombosis in an individual and these acquired factors, which may be very diverse by nature, are consequently directly involved in the pathogenesis of thrombosis. Thrombosis will develop then when the combined risk factors interact such that the threshold for thrombosis is surpassed. Upon passing of the threshold, the anticoagulant mechanisms are no longer able to counteract the procoagulant forces. Consequently a thrombotic event can occur (Rosendaal, 1999).

Of the inherited risk factors for venous thrombotic disease, most are found in the protein C anticoagulant system which will be detailed below. Examples of these are resistance to activated protein C (APC) caused by the FVLeiden mutation (FV R506Q) and the deficiencies of the cofactor, protein S, and deficiency of protein C itself (Segers et al., 2007). Of the

and occurring almost equally in men and women (Global Health Observatory (GHO),

Thromboembolic disease contributes to this overall mortality, as it is implicated in many manifestations of cardiovascular disease. The clinical manifestation of a thrombus is that of the pathological presence of an occlusion in a blood vessel or in the heart, which causes an obstruction of the physiological flow of blood through the circulatory system. Such occlusions can occur in either the venous or arterial part of the vessel tree and the condition is consequently classified as either venous or arterial thrombosis. Despite the fact that in both venous and arterial thrombosis the normal haemostatic balance is disturbed, the two types of thrombosis are considered distinct disease states that each have their own particular

The proper functioning of the anticoagulant protein C pathway is undisputedly required to prevent the occurrence of thrombosis, and in particular venous thromboses, as is most strikingly illustrated for protein C deficiency. Newborns who are homozygous protein C deficient can develop the life-threatening condition of acute onset "purpura fulminans" (Dreyfus et al, 1991, 1995) which can be treated by replacement therapy employing purified

Resistance to activated protein C (APC), or APC resistance, the subject of this chapter, has been implicated mostly in pathogenesis of venous thrombosis, despite the fact that the causative FVLeiden mutation, which is present in a majority of cases, has been related to myocardial infarction and/or overall coronary disease (Rallidis et al.2003; Segev et al., 2005; Ye et al., 2006). Various clinical studies have been performed to identify the contribution of the anticoagulant protein C pathway to arterial thromboses and stroke and these have not been conclusive in providing clear evidence for the contribution of the protein C pathway to arterial thrombosis (e.g. Folsom, 1999; Hankey, 2001; Boekholdt, 2007). That there is a link between the hemostatic system and arterial thrombosis or atherosclerosis appears clear as it has been established that many complex diseases, such as atherosclerosis, show an extensive crosstalk between inflammation and coagulation, as was recently reviewed by Borisoff and

Venous thrombosis (or venous thromboembolism, VTE) is a multifactorial disease (Seligsohn&Zivelin, 1997; Rosendaal, 1999) that affects one in 10,000 individuals under the age of 40 years annually and one in 1,000 individuals over 75 years of age, causing significant morbidity and mortality (Salzman & Hirsh, 1994; Anderson et al., 1991; Nordstrom et al. 1992). The multifactorial character of VTE implies that for the disease to occur often several circumstantial and genetic factors occur simultaneously. Together these factors are capable of tipping the natural haemostatic balance between pro- and anticoagulant forces. Important to note is that environmental and acquired factors are able to modulate the existing genetic risk factors for thrombosis in an individual and these acquired factors, which may be very diverse by nature, are consequently directly involved in the pathogenesis of thrombosis. Thrombosis will develop then when the combined risk factors interact such that the threshold for thrombosis is surpassed. Upon passing of the threshold, the anticoagulant mechanisms are no longer able to counteract the procoagulant

Of the inherited risk factors for venous thrombotic disease, most are found in the protein C anticoagulant system which will be detailed below. Examples of these are resistance to activated protein C (APC) caused by the FVLeiden mutation (FV R506Q) and the deficiencies of the cofactor, protein S, and deficiency of protein C itself (Segers et al., 2007). Of the

forces. Consequently a thrombotic event can occur (Rosendaal, 1999).

molecular pathology and underlying risk factors (Rosendaal, 1999, 2005).

World Health Organisation [WHO], 2008).

protein C concentrates.

coworkers (Borisoff et al, 2011).

acquired risk factors, use of oral contraceptives, pregnancy/puerperium, the presence of antiphospholipid antibodies, immobilization, surgery, malignancies and trauma are amongst the most studied. Furthermore, age and sex are recognized as independent contributing factors in the pathogenesis of venous thrombosis (Bertina, 2001).

In this chapter resistance to APC, the central protein of the protein C anticoagulant pathway, will be discussed, both for genetically determined forms of APC resistance and acquired types of APC resistance. Particular attention will be given to the molecular events that occur during the APC-catalyzed down-regulation of thrombin formation in normal individuals, since knowledge of these processes is pivotal in our understanding of the causative role of APC resistance in the occurrence of thromboembolic disease.

#### **2. Protein C anticoagulant pathway**

During homeostasis several anticoagulant systems balance the procoagulant forces that act in human blood, thereby preventing excessive platelet and fibrin depositions. A major inhibitory buffer is formed by the circulating inhibitors, which include antithrombin, alpha-2-macroglobulin, antitrypsin or tissue factor pathway inhibitor (TFPI) which provide the necessary negative feedback to procoagulant forces. These circulating inhibitors target the activated forms of the serine proteases from the coagulation cascade like thrombin and FXa and act by complex formation with their target proteases, resulting in a loss of proteolytic activity and clearance of the active protease from circulation.

Key regulators in the amplification of thrombin formation are however the non-enzymatic cofactor proteins factor V (FV) and factor VIII (FVIII) and by virtue of the absolute requirement of their activity for normal thrombogenesis, they are obvious targets for the attenuation of thrombin formation. Given that these molecules lack proteolytic activity, they cannot be inactivated via complex formation but instead they are targeted by proteolytic inactivation, resulting in disintegration of the protein structure and concomittant loss of cofactor activity. The main proteolytic enzyme responsible for the inactivation of activated FV (FVa) and FVIII (FVIIIa) is the activated form of protein C.

Low levels of activated protein C circulate, but for a sufficient anticoagulant response to occur, an upregulation of protein C is required through a specific set of molecular events, which together are described as the protein C anticoagulant pathway. The main player in this pathway is protein C. Protein C is a vitamin K dependent zymogen belonging to the class of chymotrypsin-like serine proteases (Stenflo, 1976). Being a zymogen, protein C circulates at a plasma concentration of ~65 nM in humans as an inactive pro-enzyme which requires processing in order to obtain its enzymatic activity. A pivotal role in the initiation of the protein C pathway is the formation of a complex between thrombin and thrombomodulin (Fuentes-Prior et al, 2000), see Fig. 1 below. Note that it is thrombin, the very enzyme that ultimately needs to be downregulated, that initiates its own attenuation. Thrombomodulin is a transmembrane protein that is present on the undamaged endothelium, in particular the endothelium of the smaller blood vessels. The membranebound thrombomodulin is able to capture thrombin by binding to the exosite I of thrombin upon which the thrombin-thrombomodulin complex is formed. The exosite I of thrombin is primarily involved in procoagulant interactions of the enzyme, namely in the recognition and activation of fibrinogen, FV and FVIII (Lane & Caso, 1989). After binding of thrombomodulin to the thrombin exosite I the procoagulant properties of any thrombin molecules that have migrated from a site of ongoing coagulation and are transported into

APC Resistance 79

pathways. In the APC-catalyzed inactivation of FVIIIa (see also below) FV acts in synergy with protein S as a cofactor to APC (Shen & Dahlbäck, 1994; Varadi et al, 1995). Somewhat conflicting results were obtained whether or not FV is also a cofactor in the inactivation of FVa. Though extremely difficult to study in purified reaction systems, this issue was recently addressed (Cramer et al, 2010) and it was concluded that FV expresses activity in