Genomics and Proteomics in Venous

Genomics and Proteomics in Venous Thromboembolism: Building a Bridge toward a Rational Personalized Medicine Framework Giuseppe Lippi, M.D.,1 Massimo Franchini, M.D.,2 Martina Montagnana, M.D.,1 and Gian Cesare Guidi, M.D.1

ABSTRACT

Venous thromboembolism is a major health care problem worldwide and is sustained by a multifactorial pathogenesis where both congenital and acquired causes contribute. It is increasingly being highlighted that a reliable approach based on genomics and proteomics might be effective to construct a rational personalized medicine framework that can be applied in the preclinical, clinical, and therapeutic settings of venous thrombosis. The aim of this review is to provide a concise description of the current and future applications of genomics and proteomics in this challenging pathology. KEYWORDS: Diagnosis, genomics, proteomics, therapy, venous thromboembolism

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enous thrombosis is the process of clot (thrombus) formation within veins. Although this can occur in any venous system, the predominant clinical entity occurs in the vessels of the leg, giving rise to deep vein thrombosis (DVT), or in the lungs, resulting in a pulmonary embolus (PE).1 Collectively referred to as venous thromboembolism (VTE), DVT and PE are common disorders worldwide. The annual incidence of VTE among whites of European origin is 1 to 2 per 1000 up to the age of 40 years, and the incidence doubles each decade thereafter.2 Therefore, the global rates of VTE mask a considerable variation according to defined populations, such as the elderly people, rising from fewer than five cases per 100,000 children aged less than 15 years to 450 to 600 cases per 100,000 adults aged 80 years. For those aged 65 years and older, mortality due to pulmonary embolism in hospital and at 1 year is

21% and 39%, respectively, whereas in the under-40 years of age group the corresponding rates for VTE are 2% and < 10%.1 The causes of VTE may be either hereditary or acquired, and a risk factor for thrombosis can be identified in nearly 80% of patients, but usually more than one factor is at play in any given patient.1 It is increasingly being highlighted that a reliable clinical and diagnostic approach might be effective to unmask the most important genetic and environmental factors, allowing the construction of a rational personalized medicine framework that can be applied in both preclinical and clinical settings. Accordingly, the acknowledged number of inherited thrombophilic disorders for which genetic testing is available has increased enormously, and the test methods have changed from reliance on linkage to DNA sequencing for recognition of mutations as small as a single nucleotide.3

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Hot Topics I: A Potpourri of Current Issues and Controversies in Thrombosis and Hemostasis; Guest Editor, Emmanuel J. Favaloro, Ph.D., M.A.I.M.S. Semin Thromb Hemost 2007;33:759–770. Copyright # 2007 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. DOI 10.1055/s-2007-1000366. ISSN 0094-6176.

Sezione di Chimica Clinica, Dipartimento di Scienze Biomediche e Morfologiche, Universita` di Verona, Verona, Italy; 2Servizio di Immunoematologia e Trasfusione - Centro Emofilia, Azienda Ospedaliera di Verona, Verona, Italy. Address for correspondence and reprint requests: Prof. Giuseppe Lippi, M.D., Sezione di Chimica Clinica, Dipartimento di Scienze Morfologico-Biomediche, Ospedale Policlinico G.B. Rossi, Piazzale Scuro, 10, 37134 Verona, Italy (e-mail: [email protected]; [email protected]).

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Pharmacogenomics has evolved in parallel; emerging evidence attests that it might be useful for improvedquality dose management of warfarin, the most widely used anticoagulant drug. This review provides an overview of current and future applications of genetics and proteomics in VTE to help construct a rational personalized medicine framework.

GENOMICS IN THROMBOPHILIA Genetic Basis of Thrombophilia VTE is a multifactorial disease as confirmed by the frequent identification of one or more predisposing factors in affected patients. Although there is no a widely accepted definition for thrombophilia, this term is conventionally used to identify those hemostatic disorders, either genetic or acquired, that are likely to predispose to thrombosis.4 Most of the known genetic defects affect the function of the natural anticoagulant pathways (Table 1). Antithrombin (AT) is a single-chain glycoprotein belonging to the serine protease inhibitor (serpin) superfamily5 that functions as a natural anticoagulant by binding to and inactivating thrombin and the activated Table 1 Genetic Basis of Thrombophilia 1. Mutations in antithrombin gene 2. Mutations in protein C gene 3. Mutations in protein S gene 4. Factor V gene polymorphisms: o 1691G> A (factor V Leiden) o 4070A> G (factor V HR2 haplotype) 5. Prothrombin 20210G> A gene polymorphism 6. N5-methyltetrahydrofolate reductase (MTHFR) 677C> T gene polymorphism 7. Factor VII 10976G> A (Arg353Gln) and 10798C> T (Ala294Val) gene polymorphism 8. Factor XIII Val34Leu 9. b-Fibrinogen -455G> A gene polymorphism 10. a-Fibrinogen 2224G> A (Thr312Ala) gene polymorphism 11. Thrombin activatable fibrinolysis inhibitor (TAFI) -438A gene polymorphism 12. Tissue factor pathway inhibitor 536C> T gene polymorphism 13. Thrombomodulin 127G> A gene polymorphism 14. Angiotensin-converting enzyme (ACE) intron 16 insertion/deletion 15. Factor VII–activating protease 1601G> A gene polymorphism (FSAP Marburg I) 16. Plasminogen activator inhibitor type-1 (PAI-1) 1–675 insertion/deletion (5G/4G), 844A> G, and 11053T> G gene polymorphisms 17. Tissue plasminogen activator (tPA) intron h deletion/insertion 18. Apolipoprotein E2/E3/E4 gene polymorphism 19. Glycoprotein Ia 807C> T gene polymorphism

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coagulation factors IXa, Xa, XIa, and XIIa. The result of this activity is a reduction of both the production and half-life of thrombin. In addition to this active site responsible for coagulation factor inactivation, the AT molecule contains a heparin-binding site. When exogenous heparin or endogenous heparan sulfate binds to this site, the ability of antithrombin to inactivate the above-mentioned factors is greatly enhanced. As expected, any type of mutation that leads to a reduction of AT levels or to a decreased ability to interact with either the activated factors or heparin will result in an increased risk of thrombosis. Antithrombin deficiency is transmitted as an autosomal dominant trait, equally affecting males and females. Patients usually present with recurrent VTE during the second to third decade of life. The penetrance of this disease is high, as most affected family members experience a thrombotic event by the age of 45.6 AT deficiency is probably the most severe among the inherited thrombophilias, causing an up to 20-fold increased risk for thrombosis compared with that of individuals not carrying this mutation.7 However, the prevalence of this mutation in the general population is extremely low, being only 0.02%. Its prevalence among unselected patients with VTE is estimated at 1 to 3%. Currently, three different types of quantitative or qualitative AT deficiencies, caused by more than 250 different mutations, have been described.8 Type 1 deficiency is characterized by a reduction in functional activity and protein antigen. In type 2 deficiency, a low AT activity occurs in the presence of normal antigen levels. Finally, type 3 deficiency is associated with normal functional and antigenic AT levels and an impaired interaction between AT and heparin. Because no patient homozygous for type 1 or type 2 deficiencies has yet been identified, it can be suggested that a complete deficiency of AT is incompatible with survival. Patients homozygous for type 3 AT deficiency have a severe thrombotic phenotype.9 Protein C (PC) is a vitamin K–dependent glycoprotein normally synthesized in the liver. Under physiologic conditions, once activated by the thrombinthrombomodulin complex, it acts as an anticoagulant by proteolytic degradation of activated coagulant factors Va and VIIIa. Clearly, any mutation leading to a reduction of PC activity (currently, more than 150 different mutations affecting synthesis and function have been described) is able to increase the thrombotic risk.10 Inherited PC deficiency is transmitted as a dominant autosomal trait. Patients present with recurrent episodes of venous thrombosis, often before the age of 45. Homozygous individuals usually have a more severe clinical picture, not infrequently leading to neonatal purpura fulminans, a potentially fatal condition characterized by multiple thrombosis in small vessels leading to skin necrosis. The penetrance of the disease is less than that seen in AT deficiency; in fact, heterozygous

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individuals have a 10-fold increased risk for VTE compared with the general population.11 However, the disorder is quite rare with a prevalence in the population of only 0.2 to 0.4% and of 3 to 5% in patients selected for VTE. There are two different types of PC deficiency. Type 1 deficiency is a quantitative disorder characterized by parallel reduction of antigenic and functional levels of the protein, and type 2 deficiency is a qualitative defect being associated with low functional levels with preserved antigenic levels.8 Protein S (PS) is a vitamin K–dependent protein produced mainly by hepatocytes but also by endothelial cells and megakaryocytes. It functions as a cofactor of activated PC for the degradation of activated factors Va and VIIIa. Moreover, it is able to directly inactivate factors Va and Xa. PS circulates in plasma in equilibrium between an inactive form, bound to a carrier called C4bbinding protein (C4BP), and a free, functionally active form that accounts for 40% of the total plasma protein.8 The bioavailability of PS is closely linked to the concentration of C4BP, which acts as an important regulatory protein in the activated PC:PS inhibitor pathway. Familial PS deficiency is transmitted as an autosomal dominant trait and has a clinical presentation very similar to that observed in PC deficiency.12 Thus, heterozygous individuals experience early recurrent VTE episodes and sometimes warfarin-induced skin necrosis, and rare homozygotes exhibit a very severe clinical picture with neonatal purpura fulminans. The penetrance of the disease is also similar to that seen in PC deficiency, causing a nearly 10-fold increased risk of VTE in affected individuals compared with the normal population. The prevalence of PS deficiency in the general population is estimated at 0.03 to 0.1%, whereas it occurs in 1 to 3% of patients with venous thrombosis.13 There are three types of PS deficiency, caused by more than 130 different mutations. Type 1 deficiency is associated with reduced functional and antigenic levels. Type 2 deficiency is characterized by reduced functional but normal antigenic levels. Type 3 deficiency, caused by mutations that enhance the affinity of PS for C4BP, is characterized by reduced free protein levels.4 Activated protein C (APC) resistance was first described in 1994 by Dahlback and Hildebrand,14 who observed that plasma taken from many patients with venous thrombosis was resistant to the normal anticoagulant effect of APC. In fact, the activated partial thromboplastin time (APTT) of these patients was not prolonged after the addition of APC to their plasma. Subsequent studies15 demonstrated that 95% of these patients had a mutation in the factor V gene consisting of a single amino acid change (Arg506 to Gln) at one of the APC cleavage sites. This substitution makes factor V, called factor V Leiden (as it was initially characterized in Leiden, The Netherlands) resistant to the inactivation by APC. Factor V Leiden is a very common mutation: in

fact, it is present in 5% of healthy people of northern European origin and in 10 to 50% of patients presenting with a first episode of VTE. Patients heterozygous for factor V Leiden have a relatively low risk for thrombosis, about fivefold that of the general population. In fact, a large Italian study demonstrated that only 6% of carriers of factor V Leiden had VTE, mostly occurring during high-risk periods, such as after surgery.16 Conversely, homozygotes exhibit a very high risk for venous thrombosis, 80 times the normal risk.6 In 1996, Poort and colleagues found that 18% of selected patients with venous thrombosis and 1% of normal controls had a mutation at base 20210 of the prothrombin gene.17 Subsequent studies have clarified that the prevalence of the gene in the general population is between 2 and 5%.8 This mutation, which is apparently associated with increased basal levels of functionally normal prothrombin, causes a threefold increased risk in heterozygotes than in the normal healthy population. Thus, these individuals exhibit a relatively low thrombotic risk, and most of them will not experience a thrombotic episode by the age of 50 years. Homozygosity for prothrombin gene mutation is much rarer and causes a higher thrombotic risk.6 Homocysteine is an amino acid that is produced in the body by the metabolism of methionine. Homocysteine in turn is metabolized by two main pathways. The former involves the enzyme cystathionine bsynthase (CBS) and requires vitamin B6, the second involves the enzyme methionine synthase and requires both vitamin B12 and N5-methyltetrahydrofolate reductase (MTHFR). High levels of homocysteine have been associated with a higher risk for both venous and arterial thrombosis. Approximately 5% of the general population has higher than normal levels of homocysteine, and the prevalence among patients with venous thrombosis is 10%.18 Moreover, the presence of hyperhomocysteinemia is associated with a threefold increased risk for venous thrombosis compared with the normal population.11 Hyperhomocysteinemia can be congenital or acquired. Acquired forms are found in patients with dietary deficiencies of folate, vitamin B12, or vitamin B6, whereas inherited forms can be caused by mutations of the CBS or MTHFR genes. However, the most common hereditary abnormality associated with hyperhomocysteinemia is the substitution of cytosine by thymine at position 677 of the MTHFR gene. This variant makes the MTHFR gene product thermolabile, causes a 50% reduction in the activity of this enzyme, and is associated with mildly to moderately increased homocysteine levels only in homozygous individuals with concomitant inadequate dietary intake of folic acid. The relationship between this mutation, also present in homozygosis, and thrombosis is still controversial. Although some authors have reported a higher prevalence of the C677T genotype among patients with thrombosis,19 most studies agree that this mutation

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does not represent an independent risk factor for VTE.20 The most probable hypothesis is that the presence of the thermolabile MTHFR variant may predispose to thrombosis only when it is co-inherited with other genetic risk factors. Apart from congenital abnormalities of the plasmatic anticoagulant mechanisms, other conditions that at least in principle might be associated with an increased risk of VTE are congenital dysfibrinogenemia, which is characterized by the presence of abnormal fibrinogen in plasma, and congenital abnormalities of the fibrinolytic system. Congenital dysfibrinogenemia is a rare condition predisposing to both venous and arterial thrombosis. A definitive role of abnormalities of the fibrinolytic system in the pathogenesis of VTE has not been acknowledged, and they are therefore not routinely included in the laboratory investigation for thrombophilia.4 Some studies have demonstrated that factor VIII (FVIII) is an independent risk factor for venous thrombosis,21 and individuals with factor FVIII levels higher than 150 IU/dL might have a nearly fivefold increased risk of thrombosis.22 Increased FVIII levels are rather common in the general population, with a prevalence of 11%, whereas up to 25% of patients presenting with VTE have FVIII levels higher than normal.6 Studies have provided evidence for the familial inheritance of this trait, but the specific molecular characterization was not initially identified.23 Nevertheless recent findings suggest that high FVIII levels in VTE represent a complex trait where two paternally imprinted genes on chromosomes 5 and 11 interact in a multiplicative way influencing the levels of FVIII; only the combination of high-risk genotypes at both loci increases substantially the risk of VTE.24 Proteins encoded by the factor XIII and thrombin activatable fibrinolysis inhibitor (TAFI) genes are involved in stabilizing the fibrin clot and in making it more lysis resistant. Accordingly, the factor XIII 34Leu, TAFI-438A, -455G/A b-fibrinogen, and a-fibrinogen Thr312Ala alleles might be involved in VTE, and these polymorphisms might influence formation and fate of emboli and, accordingly, the risk of PE.25–27 The deletion/deletion (D/D) genotype of the angiotensin-converting enzyme (ACE) has also been purported to be a susceptibility marker of onset and recurrence of thrombosis in subjects apparently with no predisposing factors and traditional thrombophilic alterations, increasing the risk of VTE, especially postoperative thrombosis, and in subjects carrying additional thrombogenic conditions.28,29 Endothelial cell protein C receptor (EPCR) enhances the generation of APC by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloproteinase activity, is present in plasma. Emerging evidence attests that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the

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4678CC genotype have higher APC levels and lower risk of VTE.30 Additional gene polymorphisms have been implicated in influencing susceptibility to thromboembolic diseases, such as tissue factor pathway inhibitor 536C> T, thrombomodulin 127G> A, factor VII–activating protease 1601G> A (FSAP Marburg I), plasminogen activator inhibitor 1–675 insertion/ deletion (5G/4G), tissue plasminogen activator intron h deletion/insertion, apolipoprotein E2/E3/E4, and glycoprotein Ia 807C> T.31 Although the role of these less conventional polymorphisms in the pathogenesis of thrombosis merit further scrutiny, the current contradictory or missing information in the relationship with the clinics do not currently support their wide inclusion within the thrombophilia testing.32

Genetic Testing Genetic testing was originally developed nearly 20 years ago, and its original applications were limited to counseling and prenatal diagnosis of a few hereditary diseases. Technological advances and continuous identification of a variety of genes responsible for many hereditary diseases have enormously amplified its development and diffusion from basic research to clinical laboratories. In the 21st century, genetic testing has become a nearly irreplaceable tool for the diagnostic approach to hereditary diseases. Besides the pivotal role for diagnosing monogenic disorders, genetic testing has a cost-effective impact also on multifactorial, multigenic pathologies, such as cardiovascular disease and VTE,33,34 in that it allows the construction of personalized frameworks including risk stratification for primary or secondary prophylaxis, duration of anticoagulant therapy, and family studies. Basically, the rationale to perform genetic analysis for VTE comes from the high prevalence of some specific inherited factors, especially the FV Leiden mutation and the G20210A polymorphism, and for the significant risk conferred by the combination of additional less-frequent genetic defects.35 Therefore, appropriate use of genetic tests, under reasonable conditions and according to the clinical setting (screening or diagnosis), offers the best etiologic contribution to determine the thrombotic disease, enabling construction of a highly specific and individual risk profile, which would finally improve the personalized prediction.34 It is now certain that genetic testing will become routine and, in time, it will be used massively in both hospital and community medicine. To face up to the exponential increase in demand, the clinical laboratories should have new high-speed, powerful, and economic equipment. Simple and fast genotyping methods for identification of a variety of selected thrombophilic mutations based on polymerase chain reaction (PCR) technology are already commercially available. Techniques involving gel analysis have been historically used to detect the presence or


absence of restriction sites, electrophoretic mobility shifts, as in single-strand conformation polymorphism or denaturing gradient gel electrophoresis, and product formation in allele-specific amplification.36 The recently developed multiplex allele-specific amplification (ASA-PCR) technology represents a valid alternative to standard protocols such as restriction fragment length polymorphism-PCR, allele-specific oligonucleotide hybridization, non-PCR oligonucleotide cleavage technology, and real-time PCR, especially when a simultaneous determination of the multiple genetic mutations is required.34 DNA microchips, taking advantage of colorimetric or fluorescence detection (including energy transfer), are currently under development. These promising devices allow qualitative and quantitative interpretation of results, providing a reliable solution to the predictable increase of requests for genetic testing by high throughput and walkaway performance, which is always useful in a clinical laboratory.37 Oligonucleotide microarray analysis, in which hundreds or thousands of genes and mutations can be tested in parallel, offers tremendous promise for more accurate, sensitive, and efficient genetic testing. Although inherited factors predisposing to VTE now represent daily genetic investigations, there is, however, a need for a rationale intervention in the general organization of such investigations, which should be rationalized in accordance with reliable guidelines, along with constant training of general practitioners and education of consumers to the appropriate use of genetic testing.38 Clinical information, including the medical and family history and the findings of the physical examination, is vital for the selection of appropriate diagnostic tests, as well as for the interpretation of test results. Because of the enormous number of potential thrombophilic mutations and the associated costs for detection, there is now consolidated evidence that there is no indication for indiscriminate genetic screening in either the general population or in asymptomatic women before prescribing oral contraceptives. According to the American College of Medical Genetics, screening for genetic risk of thrombosis should hence be performed in patients who had experienced a previous episode of VTE at age less than 50 years, particularly whenever they had a family history of VTE.39 Genetic testing might be also useful in highly selected thrombosis-prone families, as the risk is significantly higher in affected family members compared with those family members without the identifiable thrombophilic defect. The identification of the same defect in family members would allow avoidance of high-risk situations or facilitate targeted prophylaxis at times of unavoidable high risk. Thrombophilia testing should be based on the phenotype for AT, PC, and PS, on both the phenotype and genotype (FV Leiden) for APC resistance, and on the genotype (G20210A mutation) for hyperprothrombinemia.40

Phenotype determination is much easier, even with simple instrumentation, but it is more difficult to standardize and the results may be variable.4 Indeed, the identification of specific mutations in the AT, PS, or PC genes would enable construction of highly specific risk profiles dependent on the severity of the defect. However, it would be prohibitive to undertake DNA sequencing of the whole gene, making this approach unsustainable for clinical laboratories, limiting these peculiar analyses to highly specialized centers.4,41,42 Most of the tests used to investigate thrombophilia require considerable experience and skill in the interpretation of their results. Hence, specialized coagulation laboratories are more suitable for this purpose because clinicians must be aware that an appropriate counseling is labor intensive but essential, in that results of genetic testing might even produce psychological distress. The risk of false reassurance if the result is negative or the potential for loss of hope if it is positive should also be taken into consideration.3

From Genomics to Proteomics VTE is not readily explained on the basis of simple Mendelian patterns of inheritance. The genetic basis of a multifactorial disease is that a genetically susceptible individual may or may not develop the disease depending on the interaction of several risk factors. Much of the phenotypic variance accounting for the different susceptibility to diseases among individuals cannot be attributed to single-gene effects, and other sources of variance should be taken into account, including multigene effects, environmental influences, noise, and epigenetic effects.43 Accordingly, the progress in recognizing a variety of inherited factors implicated in VTE poses the question of how to integrate these data into clinical perspective and diagnosis, in that at the same apparent level of genetic risk, two individuals would not necessarily have the same probability of developing VTE. Therefore, although genetic testing enables construction of detailed models developed to explain polygenic variation, this would finally represent nothing more than a probability. The case of the FV Leiden is also emblematic, in that R506Q substitution accounts for more than 90% cases of APC resistance, but such resistance may also be associated with pregnancy, oral contraceptive use, lupus anticoagulant, elevated FVIII levels, and rarely mutations other than FV Leiden. Therefore, whereas APC resistance may occur in the absence of FV Leiden, the converse is not always true.44 Additional evidence of a lack of genotype-to-phenotype translation is observed in the newborn plasma, which shows more rapid thrombin formation and enhanced sensitivity to APC compared with adult plasma, suggesting the existence of balancing mechanisms such as lower level of factor V in FV Leiden heterozygous newborns.45 Taken together,

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these paradigmatic evidences highlight a general need for extending the analysis beyond the genotype to accurately assess thrombotic risk. Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS), in which protein samples are prefractionated on a chemically selective metal chip surface (in this particular case, a negatively charged surface) before matrix-assisted laser desorption/ionization (MALDI) or SELDI ProteinChip technology, are increasingly used in obtaining the quantitative profiles of tissue proteomes, particularly plasma proteomes.46 Regardless of inherent shortcomings of these promising technologies (noise and mass resolution in some instruments), as well as more general pitfalls in biomarker research (strength of association with pathology), the proteomic approach is gaining growing attention in thrombosis and hemostasis. The clinical usefulness of SELDI-TOF technology in conjunction with a bioinformatics tool has already been acknowledged in specific circumstances, as patients with inherited thrombophilic disorders who suffer from thrombotic events might display significant differences in low-molecular-weight proteomics profile compared with those who remain disease-free.47 Proteomic analysis can also be used to provide molecular characterization and phenotypical expression of a variety of prothrombotic mutations, such as in the case of the prothrombin G20210A polymorphism, where two-dimensional gel electrophoresis and electrospray ionization (ESI) tandem MS revealed that this mutation confers greater stability to the protein through increased glycosylation.48 There is emerging and convincing evidence that the plasma proteomic approach might be also useful for detecting the thrombophilic state in the wider, genetically disparate, population in which the predisposing factors may be acquired as well as genetic.49 Changes in individual plasma profiles can be used as markers of current or future disease, including VTE, even when these differences remain within the range for healthy individuals. Proteomics might be even more clinically useful than genomics. The incomplete correlation between genotype and phenotype is frequently explained by interindividual protein expression levels and posttranscriptional mechanisms that control the rate of synthesis and the half-life of proteins.50 Proteomic analysis would hence reveal protein expression patterns that might be more stringently associated with disease, overcoming the problem of the incomplete translation of the genotype into a prothrombotic phenotype, an approach already proven useful for predicting human atherosclerotic plaque progression.51

PHARMACOGENOMICS IN ANTICOAGULANT THERAPY Anticoagulant therapy is pivotal in VTE. The therapeutical approach is conventionally categorized within

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two phases. Sudden anticoagulation is given immediately after diagnosis to minimize the risk of thrombus extension and PE. Up to one third of VTE patients present with symptomatic PE, which, if untreated, is associated with a fatality rate of 20 to 25%. However, such risk can be reduced to less than 1.5% with an appropriate anticoagulant treatment.2 Extended anticoagulation is designed to prevent recurrent VTE and reduce the consequences of postphlebitic syndrome, especially in patients with extensive iliofemoral involvement.52 Although emerging anticoagulants such as long-acting synthetic pentasaccharides, antagonists of activated factor X, and thrombin53 are already in advanced development and have the potential to streamline the future VTE treatment, the traditional approach based on oral coumarins and parenteral heparins remains the mainstay of therapy. A variety of indications, therapeutic goals, and recommended durations of therapy exist for the use of conventional anticoagulants. The optimal duration of anticoagulation mainly depends on the nature of the thrombotic trigger. In patients with a temporary prothrombotic risk factor such as surgery, immobilization, or trauma, a relatively short duration of oral anticoagulation (3 to 6 months) is generally recommended. Patients with idiopathic VTE require a considerably longer duration of anticoagulation (at least 6 months), whereas long-term to indefinite anticoagulation might be required in patients with high risk of stroke, persistent or paroxysmal atrial fibrillation or flutter, coronary heart disease, and valvular heart diseases.54–56 Traditionally, oral anticoagulants (warfarin being the most widely used) have been the treatment of choice for long-term prophylaxis and therapy of VTE, regardless of the notoriously narrow therapeutic range. A high-quality therapy management of the patient is essential to achieve and maintain therapeutic efficacy. In fact, the 10-fold interpatient variability in the doses required to attain therapeutic responses means that even little variations in dosing may result in hemorrhagic or thrombotic complications.

The Biochemical Basis of Warfarin Therapy The two key enzymes in the pharmacological activity of warfarin are cytochrome P450 (CYP) 2C9 and the C1 subunit of the vitamin K 2,3-epoxide reductase complex (VKORC1). The superfamily of cytochrome P450 enzymes (CYPs), a distinctive enzyme system by which the human body defends itself against toxic compounds, is the subject of a complex regulation process involving various mechanisms on the levels of expression and activity.57 CYPs enzymes are predominately involved in phase 1 metabolism of xenobiotics. As only six isoenzymes are responsible for 90% of known oxidative


drug metabolism, several frequently prescribed drugs share the CYP-mediated metabolic pathways.58 Human CYPs metabolize substrate molecules of different size and shape, which implies a large, highly flexible substrate binding cavity. The common anticoagulant drug warfarin is metabolized by CYP2C9 to form 7-hydroxywarfarin (71%), 6-hydroxywarfarin (22%), and 4hydroxywarfarin (7%).59 The substrate binding sites of CYPs consist of two elements: a large, mobile binding cavity connected to the protein surface via substrateinduced channels, and a narrow, rigid, and hydrophobic funnel connecting the binding cavity and the active-site heme. Substrate specicity and region selectivity is a result of a delicate balance between different states of the substrate-enzyme complex.60 Similar to many other CYPs,61 CYP2C9 is responsible for the metabolism of at least 16% of known drugs.62 The active site of CYP2C9 possesses at least two major substrate binding sites, a pi-stacking site for aromatic rings and an ionic binding site for organic anions.63 An additional electrostatic binding site also appears to contribute to the orientation of coumarin analogues in the CYP2C9 active site by interacting with the C2-carbonyl group of the coumarin nucleus. Particularly, two important amino acids implicated in the link seem to be arginines 97 and 108.64 Accordingly, in addition to the warfarin molecule in the crystal structure, a second warfarin molecule could be accommodated in the binding cavity of CYP2C9.65 The binding of the former S-warfarin molecule creates a high-affinity site for a second ligand, which may help to explain the prevalence of drug-drug interactions involving this and other mammalian P450s.66 The vitamin K–dependent gamma-carboxylation system in the endoplasmic reticulum (ER) membrane responsible for gamma-carboxyglutamic acid modification of vitamin K–dependent proteins includes gammacarboxylase and vitamin K 2,3-epoxide reductase (VKOR).67 The subunit C1 of the VKOR (VKORC1) is pivotal in the vitamin K cycle, a cofactor required for the activation of vitamin K–dependent clotting factors.68 VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria, and Archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues.69 The human VKORC1 gene maps to chromosome 16 and consists of three exons encoding a 163-amino-acid integral ER membrane protein with three or four predicted transmembrane a-helices.70 Rost et al71 originally cloned the VKORC1 gene, which encodes a protein of 163 amino acids with a calculated relative molecular mass of 18 kDa. Northern blot analysis of fetal and adult human tissues detected a single 1.0-kb transcript, providing no evidence for alternative splicing. Highest expression was seen in fetal and adult liver, followed by fetal heart, kidney, and lung, adult heart, and pancreas. Immuno-

fluorescence experiments demonstrated VKORC1 expression in the ER. Only recently, after the successful cloning of the VKORC1 subunit of the putative VKOR lipid-protein enzyme complex, it became clear that thiols are involved in the catalytic mechanism of vitamin K reduction by VKOR and that warfarin appears to bind to a thiol redox center in VKOR.67 The most plausible model for the mechanism of inhibition of VKOR by warfarin came from work by Fasco et al72 and Wallin et al,73 who demonstrated that warfarin binds to an oxidized thiol redox center and prevents reduction of the center by an unknown electron donor. The identity of the physiologic electron donor has not been established.

Genetic Factors in Warfarin Dose Prediction Pharmacogenomics is the branch of pharmaceutics that deals with the influence of genetic variation on drug response in patients by correlating gene expression or single-nucleotide polymorphisms (SNPs) with a drug’s efficacy or toxicity. By doing so, pharmacogenomics aims to develop rational means to optimize drug therapy, with respect to the patients’ genotype, to ensure maximum efficacy with minimal adverse effects. About 30 genes contribute to therapeutic effects of warfarin, and genetic polymorphisms in these genes may contribute to modulate its anticoagulant activity. In contrast with monogenic pharmacogenetic traits, warfarin drug response is a polygenic trait, and development of diagnostic tools predictive of adverse reactions to warfarin requires a novel approach.74 Genetic polymorphisms have been described for many phase I and phase II drug-metabolizing enzymes including several cytochromes P450, N-acetyltransferases, and thiopurine S-methyltransferase. A strong association was found between interindividual variability in the anticoagulant effect of warfarin and genetic polymorphisms in six genes including vitamin VKOR, CYP2C9, PROC, EPHX1, GGCX, and ORM1. However, the strongest predictors appeared to be VKORC1, the warfarin target enzyme, and CYP2C9, the warfarin metabolic enzyme.74 Because VKORC1 recycles reduced vitamin K, which is essential for the posttranslational gammacarboxylation of vitamin K–dependent clotting factors II (prothrombin), VII, IX, and X,75 different mutations in the VKORC1 gene result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2).71 Several genetic polymorphisms in the VKORC1 gene have been identified so far, including -4931T> C, -4451C> A, -2659G> C, -1877A> G, -1639G> A, 497C> G, 1173C> T. The VKORC1 -1639A allele accounts for low dosage requirements of most patients.76,77 SNPs in the C1 subunit of VKOR correlate with coumarin sensitivity, and patients known to be homozygous for a common VKORC1 promoter polymorphism, -1639G> A

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(also designated as VKOR 3673, haplotype A, or haplotype*2), should be started on lower coumarin doses than genotype GG patients.78 Recently, three main haplotypes of VKORC1 (*2, *3, and *4) have been observed, which explain most of the genetic variability in warfarin dose among whites. Patients with VKORC1*2 haplotype seem to require much lower warfarin doses than do other patients.79 Loebstein et al also suggested that VKORC1 Asp36Tyr polymorphism might be a new marker of the high end of the warfarin dosing range.80 The gene coding for the CYP2C9 carries numerous inherited polymorphisms.81 Those coding for R144C (*2) and I359L (*3) amino acid substitutions have both significant functional effects and appreciable high population frequencies, and their in vivo consequences have been studied in humans with regard to drug metabolism.82 In a large prospective study83 of warfarin genetic dose-determinants, carriage of a single or double CYP2C9 variant reduced warfarin dose 18 to 72% and of a VKORC1 variant by 65%. Patients with either of two common variants, CYP2C9*2 or CYP2C9*3, metabolize coumarins slowly and are twice as likely to have a laboratory or clinical adverse event, unless their initial coumarin doses are reduced. Accordingly, the incidence of bleeding complications in CYP2C9*2 and CYP2C9*3 carriers is significantly higher than that in noncarriers and interacted with the presence of local bleeding sources.81 It has recently been demonstrated that genotyping both VKORC1 and CYP2C9 in conjunction with some patients’ physical characteristics will help estimate warfarin dose more precisely and thus improving the efficiency of the dosage titration process. Basically, VKORC1 and CYP2C9 genotypes along with age, sex, and body weight account for 61% of the variance in warfarin daily maintenance dose.76,84 Taken together, this evidence supports the hypothesis that construction of dense genetic maps based on SNPs for VKORC1 and CYP2C9 are important instruments to dissect polygenic traits of drug response and, in combination with appropriate nongenetic factors, might help to define a warfarin dose-response phenotype. Apolipoprotein E (APOE) genotype is unlikely to have a clinically significant effect on warfarin dose requirements.85 According to a different viewpoint, being a carrier of a combination of polymorphisms of VKORC1 and CYP2C9, rather than of one of these polymorphisms, is associated with severe overanticoagulation. Moreover, the time to achieve stability is mainly associated with the CYP2C9 genotype.86

Nongenetic Factors in Warfarin Dose Prediction Although genetic insights into VKORC1 and CYP2C9 polymorphisms might be helpful in the challenging

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endeavor to personalize warfarin therapy, there are some drawbacks that should be considered. First, the contributions of both VKORC1 and CYP2C9 gene polymorphisms account for nearly 27% and 22% of the variability of maintenance dosage, respectively.76,84 Accordingly, the overall variability of warfarin dosage explained by these two genes approaches nearly 50%, provided that other nongenetic factors are maintained fairly stable throughout the titration period. However, several synthetic preservative substances contained in foods, such as benzethonium chloride (BTC), are potent inhibitor of CYP2C9 activity in vitro, producing unpredictable effects of warfarin compliance.87 It has also been demonstrated that CYP2C9 genotyping may not be useful in African Americans or as a marker of long-term over-anticoagulation once a stable dose is reached.88 A variety of additional physiologic, environmental, and pharmacologic factors might influence the individual compliance to the therapy, regardless of the identified genetic polymorphisms.55 These basically include drugs, diet, and comorbidity. Drugs such as cholestyramine can reduce the anticoagulant effect of warfarin by reducing its absorption. Moreover, the binding of a former drug molecule creates a high-affinity site for a second ligand on the CYP2C9 molecule, creating a substantial drug-drug interaction.66 This model explains why many drugs potentiate the anticoagulant effect of warfarin by inhibiting its clearance through stereoselective or nonselective pathways.55 Drugs may also influence the pharmacodynamics of warfarin by either inhibiting the synthesis or increasing the clearance of vitamin K–dependent coagulation factors or by interfering with other pathways of hemostasis. Subjects receiving long-term warfarin therapy are highly sensitive to fluctuating levels of dietary vitamin K. Vitamin K is predominately derived from phylloquinones in plant material, which act through the warfarin-insensitive pathway. An increased intake of dietary vitamin K that reduces the anticoagulant response to warfarin occurs in patients consuming green vegetables or vitamin K–containing supplements, whereas reduced dietary vitamin K intake potentiates the effect of warfarin in patients who have been treated with antibiotics and intravenous fluids without vitamin K supplementation and who have states of fat malabsorption.55 Finally, hepatic dysfunction potentiates the response to warfarin through the impaired synthesis of coagulation factors, whereas a variety of hypermetabolic states produced by fever or hyperthyroidism increase warfarin responsiveness, probably by increasing the catabolism of vitamin K–dependent coagulation.55 Because important fluctuations in the pharmacological treatment, vitamin K intake, and health status might occur with frequency in patients on warfarin therapy, drug titration should be performed more frequently when any potentially interfering factor is added


or withdrawn, regardless of the VKORC1 and CYP2C9 polymorphisms. Therefore, although the individualization of therapy based on genetic factors has the great potential to improve efficiency and safety of the dosage titration process, it should be clearly acknowledged that the genetic variability explains a large fraction but not all of the interindividual variation in warfarin dosage. Statistical models for warfarin dose calculation should hence incorporate genetic as well as ethnic, clinical, pharmacological, and environmental factors to be really cost-effective and safe.

CONCLUSION Concepts of genetic testing are changing with the proliferation of tests based on an ever better knowledge of diseases. The greatest public health benefits are forecast to be derived from detecting common polygenic susceptibilities to blockbuster killers, such as cardiovascular disease, diabetes, and VTE. Because thrombosis is often the final pathway in a variety of systemic disorders, it is not surprising that considerable interest is rapidly increasing on this subject. Thrombophilic defects are common, they can inform preventive measures, such as prescribing (prophylactic anticoagulants in high-risk situations) and lifestyle (avoid risk situation avoidance), and they can be managed in mainstream primary and secondary care. These aspects render thrombophilia a good proxy or paradigm for testing for common susceptibilities that may become an increasing part of everyday health care, putting the clinicians under pressure to initiate thrombophilia testing on an ever-growing number of patients. Because the correct intensity of an anticoagulation regimen and a steady therapeutic range are the two mainstays of warfarin therapy, the progress in pharmacogenetics should enable everyone to have appropriate qualitative and quantitative treatment according to their genetic makeup, which should improve both efficacy and safety. In an ideal situation, genetic testing is less invasive, less expensive, and more accurate than other analyses, though it is never going to replace clinical medicine.3,89 Moreover, it is also labor intensive, and clinical laboratories may not be able to recover the costs of developing tests for rare inherited disorders. Therefore, use of genetic analysis should be discretionary and regulated. The most cost-effective approach is to start with testing for the most common inherited disorders, such as factor V Leiden and the prothrombin 20210G> A gene polymorphism. The investigation of additional monogenic thrombophilic mutations should be reserved to highly selected patients. Because of the presence of several disease-causing mutations, such as those occurring in the AT, PS, and PC genes, DNA sequencing for recognition of mutations as small as a single nucleotide would be informative but still unaf-

fordable to clinical laboratories that already perform phenotype testing. In the very near future, construction of dense genetic maps based on SNPs for a variety of genes is expected to become a powerful aid to assist disease prediction and dissect polygenic traits of drug response. However, because of the potential implications of results of genetic analyses, laboratories must undertake stringent internal quality control measures and participate in external quality assurance (QA) programs.90 Provided that extensive counseling is ensured to maximize the benefit and minimize the risks, results of genetic testing combined with ethnic, clinical, environmental, and psychological factors would hence definitely assist the clinical decision making, building a solid bridge toward personalized medicine. In the very near future, proteomics, which evaluates changes in protein expression, posttranslational modifications, protein interactions, protein structure, and splice variants, would allow construction of highly informative protein profiles in health and disease, assisting prediction and diagnosis of disease and identifying targets for new therapeutic agents.

ABBREVIATIONS ACE angiotensin-converting enzyme APC activated protein C APOE apolipoprotein E APTT activated partial thromboplastin time ASA allele-specific amplification AT antithrombin BTC benzethonium chloride CBS cystathionine b-synthase C4BP C4b-binding protein CYP cytochrome P450 DD deletion/deletion DVT deep vein thrombosis EPCR endothelial cell protein C receptor ER endoplasmic reticulum ESI electrospray ionization FSAP factor VII–activating protease 1601G> A FVIII factor VIII MALDI matrix-assisted laser desorption/ionization MS mass spectrometry MTHFR N5-methyltetrahydrofolate reductase PAI-1 plasminogen activator inhibitor type-1 PC protein C PCR polymerase chain reaction PE pulmonary embolus PS protein S QA quality assurance SELDI surface-enhanced laser desorption/ ionization SNP single-nucleotide polymorphism TAFI thrombin activatable fibrinolysis inhibitor TOF time-of-flight

767

768


tPA VKOR VKORC VTE WR

tissue plasminogen activator vitamin K 2,3-epoxide reductase vitamin K 2,3-epoxide reductase complex venous thromboembolism warfarin resistance

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