Genetic polymorphisms associated with venous and arterial thrombosis; an overview. (Advances in the Science of Pathology).
The hemostatic process is an orchestrated balance of prothrombotic and antithrombotic factors in the vasculature. The prothrombotic mechanisms of coagulation activation and platelet adhesion/aggregation are balanced by naturally occurring anticoagulants, endothelial cells, and the fibrinolytic system (Figure 1). After vascular injury, the hemostatic system is activated with initiation of the coagulation cascade largely due to exposure of tissue factor, leading to the formation of the serine protease thrombin, with the subsequent the conversion of fibrinogen to fibrin monomer, and the polymerization of fibrin. (4) Simultaneously, platelets adhere to exposed extracellular matrix proteins, undergo activation by thrombin and other agonists, and aggregate to form a platelet-fibrin thrombus. This prothrombotic process is regulated by natural anticoagulants, such as antithrombin, activated protein C (APC) and its cofactor (protein S), as well as tissue factor pathway inhibitor. The fibrinolytic system is concomitantly activated by plasminogen activators released from endothelial cells, leading to the production of the active enzyme plasmin and eventual degradation of the fibrin clot. (5)
[FIGURE 1 OMITTED]
A tendency toward thrombosis could be due to abnormalities of the vasculature, the blood components, or rheology (the Virchow triad). Endothelial cells normally have an antithrombotic effect, largely due to membrane-bound thrombomodulin, which is responsible for activating protein C. (6) Other antithrombotic endothelial functions include platelet inhibition due to release of prostacyclin and nitrous oxide. Endothelial causes for a thrombotic tendency could be genetic dysregulation of the antithrombotic mechanisms or endothelial denudation with exposure of prothrombotic plaque and extracellular matrix proteins, as occurs with atherosclerosis. Additionally, changes in blood flow may cause thrombosis due to stasis, but may also affect endothelial gene expression. (7)
Possible genetic dysfunction of the blood components could be due to loss-of-function mutations of the natural anticoagulants, gain-of-function mutations of the procoagulant proteins, abnormalities leading to decreased fibrinolytic function, or platelet functional abnormalities associated with increased activation (Figure 2). In addition to genetic causes, there are many recognized acquired risk factors, as indicated in Table 1. As mechanistic studies have progressed, the pathophysiologic mechanism for thrombosis with many of the acquired risk factors has been associated with perturbations in procoagulant factors, the vasculature, or blood flow.
[FIGURE 2 OMITTED]
Recognition of familial tendencies for thrombosis initially led to a search for genetic abnormalities in the coagulation system. The genetic abnormalities first associated with thrombophilia were mutations leading to loss of function of natural anticoagulants, such as antithrombin, protein C, or protein S. (8-10) These disorders are rare, and heterozygous individuals have an increased risk for venous thromboembolism. Numerous different mutations result in deficiency or dysfunction of these proteins, thus making genetic testing difficult. For example, more than 70 different protein C mutations have been described. (11) Of patients with thrombophilia, genetic deficiencies of the natural anticoagulants will be found in less than 20% of cases. Deficiency of the profibrinolytic factor, plasminogen, has been associated with thrombophilia, but this association is less clear than that of the natural anticoagulants. (12)
The search to explain thrombophilia in patients for whom no cause could be found led to sequencing of the genes of many of the procoagulant proteins. This process in turn led to the description of relatively common single nucleotide polymorphisms in many of these genes. However, unlike the loss-of-function mutations described for the natural anticoagulants, the functional implications of many of these polymorphisms were not always clear. Without a clear dysfunctional mutation, the clinical implications of these mutations have been studied by performing population studies, often comparing patients with thrombosis with healthy control subjects (case-control studies). The prevalence of the polymorphism in the patient population compared with that in the control population is used statistically to calculate the relative thrombotic risk of the genetic alteration.
Through many of these population studies, it has become clear that thrombotic vascular disease is a multifactorial and multigenic process. Individuals possessing more than 1 congenital risk factor or 1 congenital risk factor plus an acquired risk factor are at greater risk for thrombosis than those with only 1 known risk factor. (13) Studies have found an increased relative risk of combined genetic and acquired risk factors in patients taking oral contraceptives, (14) undergoing hormone replacement therapy, (15) who have had surgery, (16) and with the presence of the lupus anticoagulant. (17)
Genetic polymorphisms with either a positive or negative association with thrombosis and arterial vascular disease have been found in many of the procoagulant proteins, including factor V, prothrombin, fibrinogen, factor VII, factor XI, and factor XIII (Figure 3). Additionally, polymorphisms of platelet glycoproteins, fibrinolytic proteins, and enzymes in the transstrlfttration pathway leading to high homocysteine levels have been described in association with thrombotic vascular disease. In general, risk factors that favor venous thrombosis are abnormalities of the natural anticoagulant pathways, while polymorphisms of the procoagulant proteins and transsulfuration pathway are associated variably with either venous or arterial disease, and platelet glycoprotein polymorphisms are associated predominantly with arterial disease. These polymorphisms and their implications form the body of this review and are summarized in Table 2. In the following sections on each of the polymorphisms, the evidence for clinical associations with either venous or arterial thrombosis will be discussed. As more and more polymorphisms are elucidated, the true multigenic scope of thrombosis will emerge, but the clinical implications of individual polymorphisms will grow increasingly complex.
[FIGURE 3 OMITTED]
POLYMORPHISMS IN THE PROTEIN C PATHWAY
Factor V Leiden and Activated Protein C Resistance
Activated factor V (factor Va) is a cofactor protein in the prothrombinase complex that, together with the serine protease factor Xa, is responsible for conversion of prothrombin to the active enzyme thrombin. Activated protein C regulates the functionality of the complex by proteolytic degradation of factor Va at amino acids Arg506, Arg306, and Arg679 (Figure 4). (18) Factor Va also serves as a cofactor in the degradation of factor VIIIa by APC. Patients resistant to the activity of APC were described by Dahlback in 1993, (19) and the molecular basis for this defect was shown to be a point mutation in the factor V gene located on chromosome 1 (1691G [right arrow] A), which was called factor V Leiden (FVL). (20) The FVL mutation results in an arginine-glutamine substitution at amino acid 506 (Arg506Gln), the site of the first molecular cleavage of factor Va by APC. This substitution results in diminished APC cleavage of factor Va and continued formation of thrombin by the prothrombinase complex (ie, APC resistance). Approximately 95% of individuals with functional APC resistance have the FVL mutation, which can be detected by a polymerase chain reaction amplification of a 223-base pair (bp) fragment after MnlI cleavage, (21) but newer assays by invader probe, Third Wave technology, and Light Cycler technology have also been developed (Figure 5). A screening assay based on the ability of APC to prolong activated partial thromboplastin clotting times is available with several modifications, including predilution with factor V-deficient plasma. (22) After the addition of APC, the clotting time in patients with FVL does not increase as much as in patients with wild-type factor V. The assay result is usually reported as a ratio of the clotting time with APC versus the clotting time without added APC.
[FIGURES 4 & 5 OMITTED]
The FVL mutation is fairly common in the white population, with a frequency of 2% to 15%, but it is uncommon in African blacks and Asians. (23,24) The mutation is detected in up to 40% of patients with venous thrombosis. (20) Factor V Leiden alone imparts approximately an eightfold increased risk for venous thrombosis in heterozygotes and an 80-fold increase in homozygotes. (2,11) Clinical studies have shown FVL to be a risk factor for deep venous thrombosis, pulmonary embolism, cerebral vein thrombosis, and superficial thrombophlebitis. (2) An association with arterial thrombosis is not clear. Several family studies have shown a strong interaction of the FVL mutation with other inherited thrombophilia syndromes, such as protein C and protein S deficiencies. (25,26) Patients heterozygous for both FVL and another condition have been shown to be more likely to experience a thrombotic event. In women taking oral contraceptives, the presence of the FVL mutation has been shown to further increase the risk of thrombosis. (27)
Other low-frequency factor V mutations have been described, such as factor V Cambridge (Arg306Thr) (28) and factor V Hong Kong (Arg306Gly). (29) These mutations may also result in APC resistance, but the clinical association with venous thrombosis is less clear. Sequencing of exon 13 of the factor V gene has revealed a 4070A [right arrow] G mutation (His199Arg) associated with several other polymorphisms, known as the HR2 haplotype. (30,31) Patients with this haplotype may have a relative increase in the more thrombogenic and glycosylated factor V isoform (FV1). (31) It is not clear whether the HR2 haplotype is a general risk factor for thrombosis, but it appears to increase the risk of venous thrombosis in patients who also have FVL. (32) These low-frequency mutations will be detected by the functional APC resistance assay, but will not be detected in molecular genetic assays for FVL. Thus, the APC resistance assay should be used for screening, followed by specific assays for FVL.
Thrombomodulin and the Endothelial Cell Protein C Receptor
Thrombomodulin is an intrinsic membrane glycoprotein localized to the luminal surface of endothelial cells that acts as a thrombin receptor and facilitates the activation of protein C (Figure 4). Due to the important role of thrombomodulin in the protein C pathway, genetic abnormalities of thrombomodulin leading to decreased functionality should theoretically be associated with thrombosis. Several mutations of the thrombomodulin gene resulting in amino acid point mutations have been described in patients and families with venous thromboembolic disease, (33) but the significance of these findings awaits larger studies. A C/T dimorphism at nucleotide 1418, resulting in an Ala455Val substitution was found to be associated with premature myocardial infarction, (34) but this dimorphism is not thought to be associated with venous thrombosis. (35) A search for polymorphisms in the thrombomodulin promoter region found only a single G [right arrow] A mutation at nucleotide -33 that had a weak association with venous thrombosis. (36)
An endothelial cell surface receptor for protein C described on large vessel endothelium is thought to localize protein C to the endothelial surface and facilitate its activation by the thrombin/thrombomodulin complex. (37) An insertion mutation in exon 3 that may have an association with venous thrombosis has been reported, but further studies are required. (38)
POLYMORPHISMS OF PROCOAGULANT PROTEINS
Thrombin plays a central role in hemostasis, in that it converts fibrinogen to fibrin, but it also activates factors V, VIII, XI, and XIII. Thrombin is also a potent activator of platelets. Owing to the central role of thrombin, increased prothrombin levels are a likely risk factor for venous thrombosis. In studying patients from families with unexplained thrombophilia, Poort et al (39) identified a G [right arrow] A transition at nucleotide 20210 in the 3' untranslated portion of the prothrombin gene in 5 of 28 patients. The mutation can be detected by polymerase chain reaction, as a new HindIII site is introduced by the base transition (Figure 6). The mutation is associated with increased prothrombin levels, with heterozygotes having a level about 50% higher than unaffected individuals. (11,39) However, increased prothrombin levels are not necessarily associated with the prothrombin 20210G [right arrow] A mutation. It is not clear whether the 20210G [right arrow] A mutation is responsible for the increased prothrombin levels in these patients.
[FIGURE 6 OMITTED]
The prothrombin 20210G [right arrow] A mutation is seen in only 1% to 2% of the normal population, but is detected in up to 20% of individuals with venous thrombosis. (40,41) Prothrombin 20210G [right arrow] A results in elevated prothrombin levels and a twofold to fivefold increased risk of venous thrombosis. (11,39) The prothrombin 20210G [right arrow] A mutation is also associated with a further increased risk of venous thrombosis in patients taking oral contraceptives, and for individuals taking oral contraceptives with both FVL and prothrombin G [right arrow] A mutations, the risk is multiplicative. (27) Whether the prothrombin mutation is associated with arterial thrombosis is not clear, but it may increase the risk for myocardial infarction in women. (42) Some studies have also associated the prothrombin mutation with a risk for thrombotic stroke. (43) The mechanism associated with the increased thrombotic risk is not clear, as some investigators have found increased prothrombin F1+2 cleavage peptide levels in the plasma, evidence of increased thrombin formation, (44) while others have only shown increased endogenous thrombin potential. (45)
Fibrinogen mutations leading to hypofibrinogenemia or dysfibrinogenemia are usually clinically silent or associated with a bleeding diathesis. However, several rare dysfibrinogens are associated with a hypercoagulable state and venous thrombosis. (46) Two such dysfibrinogens are Dusart (Paris V) and Chapel Hill III, which have thrombin-clottable fibrinogen that is resistant to degradation by plasmin. (47) Fibrinogen Oslo I has a short thrombin time, fast polymerization, and increased platelet aggregation. Fibrinogen New York I has a B[beta]39-72 deletion that has defective tissue plasminogen activator binding to fibrin with decreased potentiation of plasminogen activation. (48)
Fibrinogen levels are controlled by both genetic (49) and environmental factors, such as the acute phase response and inflammatory disorders. Increased fibrinogen levels have been associated predominantly with arterial disease, such as myocardial infarction and stroke, where they have been shown to be an independent risk factor, but elevated fibrinogen levels may be associated with venous thrombosis as well. (50,51)
Fibrinogen is a dimer of 3 polypeptide chains: A[alpha], B[beta], and [gamma]. There are several genetic polymorphisms of the fibrinogen gene that lead to increased fibrinogen levels, but as these are generally in uncoded regions, the fibrinogen produced is structurally normal. These include the Bcl-1 allele in the 3' region of the [beta] chain and the G [right arrow] A polymorphism at nucleotide -455 in the 5' promoter region of the B[beta] gene. (52) A G [right arrow] A polymorphism at nucleotide 448 codes for an arginine-to-lysine substitution at 13 amino acids from the C-terminal end of the B[beta] chain. (53) A C [right arrow] T polymorphism at nucleotide -148 in the 5' promoter region of the B[beta] gene is not associated with elevated fibrinogen concentrations. (54) The increased fibrinogen levels seen with some of these polymorphisms is variable with gender and smoking history. (53,55,56)
While increased fibrinogen levels have been associated with vascular disease, the association of the fibrinogen genetic polymorphisms with vascular disease is less clear. The Bcl-1 allele may be associated with carotid atherosclerosis, myocardial infarction, and venous thrombosis. (56,57) The -455G [right arrow] A polymorphism has been associated with coronary artery disease in some, (55) but not all studies. (58,59) The -148C [right arrow] T polymorphism, not accompanied by increased fibrinogen levels, has been associated nonetheless with an increased risk of carotid atherosclerosis. (54) A polymorphism in the coding region of the [alpha] chain (Thr312Ala), which leads to an amino acid substitution near the factor XIII cross-linking site, may be associated with increased poststroke mortality and pulmonary embolism. (60)
Factor VII Polymorphisms
Several polymorphisms in the factor VII gene are associated with decreased factor VII levels, which may be protective against thrombosis. These include an arginine-glutamine mutation at amino acid 353 (Arg353Gln) of factor VII and an H7H7 polymorphism in the hypervariable region 4 of intron 7. (61) The presence of both of these polymorphisms, together with low-normal factor VII levels was found to have a decreased risk for arterial thrombosis. (61) Recently, a novel polymorphism in intron 1a of the factor VII gene (+73G [right arrow] A) was described that may be in linkage disequilibrium with the 353 Gin allele. (62) The presence of both 73A and 353 Gin had the lowest factor VII levels and the lowest risk of myocardial infarction compared to individuals without the mutation. Further studies are needed to clarify the interaction of the various factor VII polymorphisms in the risk for vascular disease.
Elevated Factor VIII Levels
Factor VIII is an important cofactor in the activation of factor X by the tenase complex. Elevated factor VIII could be potentially prothrombotic by increasing stability of the tenase complex or by conferring a relative resistance to APC degradation. (63) Elevated levels of factor VIII were associated with venous thrombosis in the Leiden Thrombophilia Study, (64) and the association has also been observed subsequently. (65) While factor VIII levels have been known to increase with an acute phase response, the role of elevated factor VIII as a thrombotic risk factor has been shown to be independent of this effect. (66) Factor VIII levels are known to vary depending on blood group type, and familial clustering of elevated factor VIII levels has been detected, so a genetic association is likely, but no firm genetic mechanisms have yet been illuminated. (2,3,67)
Factor XI is a coagulation protein in the intrinsic coagulation pathway that is responsible for activating factor IX (Figure 3). An elevated level of factor XI has recently been reported to be a risk factor for venous thrombosis. (68) A factor XI level above the 90th percentile was found to impart the increased risk, independent of other known thrombotic risk factors. Genetic associations with the elevated factor XI levels are postulated, but to our knowledge none have been reported to date.
Factor XIII Polymorphisms
Factor XIII is a thrombin-activatable protein responsible for cross-linking the [gamma] chains of fibrin monomer and stabilizing the fibrin clot. Factor XIII is a tetramer consisting of 2 catalytic subunits (A subunit) and 2 nonenzymatic subunits (B subunit). Several polymorphisms have been detected in the A subunit, including one which results in the substitution of leucine for valine at amino acid 34 (Val34Leu), only 3 amino acids away from the thrombin cleavage site. (69) This mutation has been associated with increased factor XIII activation by thrombin and increased cross-linking activity. (70) The Val34Leu mutation paradoxically has been associated with a decreased risk of venous thrombosis. (71) Another study confirmed the association, finding only the Leu/Leu genotype to be protective, (72) but subsequent studies have not confirmed this association. (73,74) This polymorphism has been shown to be protective against myocardial infarction, (75) but is also associated with cerebral hemorrhage. (76)
METHYLENE TETRAHYDROFOLATE REDUCTASE (MTHFR) POLYMORPHISMS AND HYPERHOMOCYSTINEMIA
A high plasma homocysteine level has been shown to be an independent risk factor for venous thrombosis, as well as arterial vascular disease. (77,78) Nutritional deficiencies of folic acid, vitamin [B.sub.12], or vitamin [B.sub.6] are associated with hyperhomocystinemia, due to their role in homocysteine metabolism (Figure 7). (77) Mutations of the cystathionine [beta]-synthase enzyme, when homozygous, result in homocystinuria with extremely elevated homocysteine levels and both thrombosis and atherosclerosis, together with other connective tissue and skeletal abnormalities. (79) An 833T [right arrow] C mutation in the cystathionine [beta]-synthase recently has been described with a mild phenotype except for familial thrombophilia. (80) However, even individuals who are heterozygous for the cystathionine [beta]-synthase mutation, with slightly elevated homocysteine levels, may be at risk for vascular disease. (81)
[FIGURE 7 OMITTED]
Other candidates to explain high homocysteine levels would be genetic abnormalities of methionine synthase or 5,10-methelentetrahydrofolate reductase (MTHFR). A thermolabile variant of MTHFR due to a C [right arrow] T missense mutation at nucleotide 677 (677C [right arrow] T) is associated with increased plasma homocysteine levels, especially in conjunction with folate deficiency, and has been described in 5% to 15% of the Western white and Japanese populations. (82) The mutation is uncommon in Indian Asians. (83) This mutation has been associated with premature cardiovascular disease and pregnancy-related vascular disorders. (82,84) An increased risk for venous thrombosis has been described in persons homozygous for the mutation, both alone (85) and in conjunction with FVL, (86) but an overall risk for venous thromboembolism was not confirmed in a meta-analysis. (87) The elevated homocysteine levels observed in persons with the 677C [right arrow] T mutation can be normalized by administration of vitamin [B.sub.12] or folate. (88) Pregnancy-related neural tube defects have also been described in persons homozygous for the 677C [right arrow] T mutation. (89) In screening individuals with thrombophilia, the plasma homocysteine level would be the appropriate initial screening test, followed by the MTHFR 677C [right arrow] T polymorphism if other nutritional causes of hyperhomocystinemia can be excluded.
Platelets play a crucial role in the hemostatic process. After vascular injury, platelet surface glycoprotein receptors rapidly bind to exposed subendothelial extracellular matrix proteins. (90) After adhesion of platelets to the vascular wall, glycoprotein IIb/IIIa ([[alpha].sub.IIb]/[[beta].sub.3] integrin) is involved in platelet-platelet aggregation by binding fibrinogen between adjacent platelets. Deficiencies of surface glycoproteins due to genetic mutations are an uncommon, but well-described cause of bleeding. For example, Glanzmann thrombasthenia is a deficiency of the IIb and/or IIIa subunits of the GP IIb/IIIa receptor. (91)
Glycoprotein IIIa Polymorphisms
In the glycoprotein IIIa gene, a common mutation seen in approximately 15% of the white population leads to a substitution of proline for leucine at amino acid 33 (Leu33Pro); the wild-type allele is known as P[L.sup.A1] (HPA-1a), and the 33 proline substitution is known as P[L.sup.A2] (HPA-1b). (92) The homozygous A2/A2 mutation is well known to be associated with posttransfusion purpura and neonatal alloimmune thrombocytopenia, in which alloantibodies are formed against the A1 allele. (93) In 1996, a report was published that associated the P[L.sup.A2] allele with a risk of acute coronary thrombosis. (94) Many subsequent studies evaluated the significance of the P[L.sup.A2] allele, of which some confirmed the association with coronary artery disease, but more did not. (95) Studies that have examined coronary artery disease in general, compared to acute thrombotic coronary events, suggest that the P[L.sup.A2] polymorphism may be associated with a risk for coronary thrombosis, but not atherosclerosis. (95,96) In an array-based multiplex analysis of 12 candidate polymorphisms, the P[L.sup.A2] polymorphism, along with a 4G polymorphism of the plasminogen activator inhibitor 1 gene, was associated with increased myocardial infarction. (97) There appears to be little association of the P[L.sup.A2] polymorphism with venous thrombosis, and an association with cerebrovascular disease is tenuous. (95) Possible mechanistic associations between the P[L.sup.A2] polymorphism and thrombosis include increased sensitivity to platelet aggregation by various agonists (98) and altered sensitivity to aspirin. (99)
Other Platelet Glycoproteins
In addition to the prominent hemostatic role of GP IIb/ IIIa, other platelet surface glycoproteins, such as the von Willebrand receptor (GP Ib/IX/V) and the collagen receptor (Ia/IIa) are likely candidates for an association with thrombotic disease. Emerging candidate genetic variations include a length polymorphism of GP Iba with a variable number of tandem repeats of 39 bp and a polymorphism at 3550C [right arrow] T leading to a Thr145Met substitution. (95) A silent exonic dimorphism at position 807C [right arrow] T in the gene for the [alpha]2 peptide of the GP Ia/IIa protein leading to increased receptor density may also be associated with myocardial infarction. (100,101) A polymorphism at nucleotide 1648A [right arrow] G in the [alpha]2 peptide of GP Ia/IIIa is another recent candidate for association with coronary artery disease. (102) Few associations between polymorphisms of platelet surface glycoproteins and venous thrombosis have been described, but a recently described polymorphism of a platelet thrombin receptor (protease-activated receptor 1 [PAR1]) has a tentative protective effect on venous thrombosis. (103)
In addition to the plethora of polymorphisms described herein, other polymorphisms have been described recently that have a tentative association with venous thrombosis or increased cardiovascular risk. These polymorphisms include mutations of factor XII, tissue plasminogen activator, plasminogen activator inhibitor 1, thrombospondin, angiotensin-converting enzyme, and insulin receptor substrate 1. (52) It seems as if new polymorphisms are described almost daily, claiming tentative associations with either venous thrombosis or arterial vascular disease. This activity has resulted in a bewildering array of potential risk factors for thrombotic disease, as summarized in Table 2.
Hemostasis involves the integration of many procoagulant proteins, natural anticoagulants, fibrinolytic proteins, and platelet surface glycoproteins. It does not take extensive logic to anticipate that thrombosis, with dysregulation of the hemostatic balance, could be affected by many different gene products. Sorting out which abnormalities are associated clinically with venous or arterial thrombosis has led to some generalizations. It appears that venous thrombosis has the strongest association with genetic polymorphisms affecting the natural anticoagulants, due to the prominent role of stasis in venous thrombus formation. The mechanism of atherosclerosis involves lipid dysregulation and cellular proliferation in addition to thrombosis, so the genetic influences are probably even more multifactorial than venous thrombosis. However, since atherosclerosis involves high-flow arteries, it is logical that prothrombotic protein and platelet glycoprotein polymorphisms should play a greater role.
This review of polymorphisms associated with thrombotic disease has highlighted the considerable variability of clinical associations with the various polymorphisms. For every study that finds a positive association with a particular polymorphism, there are several that seemingly find the opposite. This disparity may be due to differences in polymorphism prevalences between ethnic and geographic study populations, but is probably also due to variable sample sizes, other risk factors (eg, smoking, hypertension, diabetes, and lipid levels), variable definitions of study endpoints (eg, myocardial infarction, unstable angina, coronary artery disease, stroke, deep vein thrombosis, and pulmonary embolism), and patient populations. Further large, case-controlled clinical studies evaluating many different polymorphisms simultaneously will be needed to assess whether these abnormalities are truly risk factors for thrombotic disease.
As the list of genetic mutations associated with thrombosis increases, the laboratory faces the challenge of determining which genetic assays to include in screening for thrombotic risk. Cost effectiveness must be taken into consideration, because performing a large panel of tests may be very expensive. However, no single screening assay for thrombotic risk is available, and the presence of 2 or more risk factors may give an additive increased risk of thrombosis, so measurement of more than 1 analyte is necessary. Most investigators would agree that laboratory testing for hypercoagulable risk factors is useful in patients with a history of documented thrombosis, their family members, or patients with high-risk pregnancy. However, the use of 1 or more genetic tests to screen patients before starting oral contraceptives or hormone replacement therapy is controversial, due to the low mutation prevalence in the general population and the small absolute increase in thrombotic risk with these therapies.
When utilizing laboratory screening to determine risk for venous thrombosis, 2 genetic screening tests would be recommended in addition to assays for antithrombin, protein C, and protein S: (1) analysis of APC resistance followed by assay for FVL if APC resistance is detected and (2) the prothrombin 20210G [right arrow] A mutation assay. Evaluation of the MTHFR mutation would be recommended only in patients with elevated homocysteine levels. Until a genetic abnormality is established, evaluation of factor VIII and possibly factor XI coagulant levels may be useful in patients in whom no other abnormalities are detected. For arterial thrombosis, the tentative genetic associations outweigh the established associations, so laboratory testing of prothrombotic markers is not generally recommended, except in individuals with arterial thrombosis at a young age or in an unusual location. Measurement of fibrinogen and homocysteine levels may be useful in some individuals, but routine genetic screening of fibrinogen, procoagulant, fibrinolytic, or platelet polymorphisms must await further clinical confirmation.
Table 1. Acquired Risk Factors for Thrombosis * Myeloproliferative disorders Heparin-induced thrombocytopenia Coumadin skin necrosis Lupus anticoagulant Antiphospholipid syndrome Inflammatory bowel disease Oral contraceptives Nephrotic syndrome Buerger disease Hyperactive platelets Age Obesity Malignancy TTP, DIC, PNH Pregnancy Sepsis Paralysis Trauma Behcet syndrome Surgery * TTP indicates thrombotic thrombocytopenic purpura; DIC, disseminated intravascular coagulation; and PNH, paroxysmal nocturnal hemoglobinuria. Table 2. Clinical Associations of Genetic Polymorphisms * Polymorphism Phenotype Protein C Anticoagulant Pathway Factor V Leiden: 1691G [right arrow] A APC resistance (Arg506Gln) Factor V Cambridge: Arg306Thr APC resistance Factor V Hong Kong: Arg306Gly APC resistance Factor V HR2 haplotype Mild APC resistance Thrombomodulin 1418C [right arrow] T Unknown (Ala455Val) Thrombomodulin -- 33G [right arrow] A Unknown EPCR 23 bp insertion in exon 3 Unknown Procoagulant Proteins Prothrombin 20210G [right arrow] A Increased FII Fibrinogen Bcl-1 allele [beta] chain Increased fibrinogen Fibrinogen -- 148C [right arrow] T in No increased fibrinogen B [beta] promoter Fibrinogen 448G [right arrow] A Increased fibrinogen in B [beta] Fibrinogen Thr312Ala in [alpha] chain ? Abnormal FXIII cross-linking Factor VII Arg353Gln Low-normal FVII Factor VII H7H7 Low-normal FVII Factor VII G73A Low-normal FVII Factor XIII A subunit Va134Leu Increased activity Homocysteine Metabolism Cystathionine [beta]-synthase 833T Homocysteinemia [right arrow] C 5,10-methylene tetrahydrofolate reductase Thermolabile enzyme, (MTHFR) 677C [right arrow] T mildly increased Hcy Platelet Surface Glycoproteins GP Illa Leu33Pro (P[L.sup.A2] or HPA-lb) Increased sensitivity to platelet activation; altered sensitivity to aspirin GP lba VNTR Unknown GP lba 3550C [right arrow] T (Thr145Met) Unknown GPla/lla, [alpha] 2 1648A [right arrow] G Altered surface expression of receptor Thrombin receptor PAR-1-5061 Unknown [right arrow] D Association With Venous Polymorphism Thrombosis Protein C Anticoagulant Pathway Factor V Leiden: 1691G [right arrow] A Clear risk factor (Arg506Gln) Factor V Cambridge: Arg306Thr Tentative Factor V Hong Kong: Arg306Gly Tentative Factor V HR2 haplotype Tentative Thrombomodulin 1418C [right arrow] T Unlikely (Ala455Val) Thrombomodulin -- 33G [right arrow] A Possible EPCR 23 bp insertion in exon 3 Suggestive Procoagulant Proteins Prothrombin 20210G [right arrow] A Likely risk factor Fibrinogen Bcl-1 allele [beta] chain Possible Fibrinogen -- 148C [right arrow] T in Not known B [beta] promoter Fibrinogen 448G [right arrow] A Not known in B [beta] Fibrinogen Thr312Ala in [alpha] chain Possible Factor VII Arg353Gln Not known Factor VII H7H7 Not known Factor VII G73A Not known Factor XIII A subunit Va134Leu Possibly protective Homocysteine Metabolism Cystathionine [beta]-synthase 833T Possible [right arrow] C 5,10-methylene tetrahydrofolate reductase Tentative (MTHFR) 677C [right arrow] T Platelet Surface Glycoproteins GP Illa Leu33Pro (P[L.sup.A2] or HPA-lb) Unlikely GP lba VNTR Not known GP lba 3550C [right arrow] T (Thr145Met) Not known GPla/lla, [alpha] 2 1648A [right arrow] G Not known Thrombin receptor PAR-1-5061 Possibly [right arrow] D protective Association With Arterial Polymorphism Disease Protein C Anticoagulant Pathway Factor V Leiden: 1691G [right arrow] A Not likely (Arg506Gln) Factor V Cambridge: Arg306Thr Not studied Factor V Hong Kong: Arg306Gly Not studied Factor V HR2 haplotype Not known Thrombomodulin 1418C [right arrow] T Tentative (Ala455Val) Thrombomodulin -- 33G [right arrow] A Not known EPCR 23 bp insertion in exon 3 Not known Procoagulant Proteins Prothrombin 20210G [right arrow] A Perhaps in selected populations Fibrinogen Bcl-1 allele [beta] chain Tentative Fibrinogen -- 148C [right arrow] T in Tentative B [beta] promoter Fibrinogen 448G [right arrow] A Tentative in B [beta] Fibrinogen Thr312Ala in [alpha] chain Possible Factor VII Arg353Gln Possibly protective Factor VII H7H7 Possibly protective Factor VII G73A Possibly protective Factor XIII A subunit Va134Leu Possibly protective Homocysteine Metabolism Cystathionine [beta]-synthase 833T Not known [right arrow] C 5,10-methylene tetrahydrofolate reductase Tentative (MTHFR) 677C [right arrow] T Platelet Surface Glycoproteins GP Illa Leu33Pro (P[L.sup.A2] or HPA-lb) Tentative, but inconsistent GP lba VNTR Tentative GP lba 3550C [right arrow] T (Thr145Met) Inconsistent GPla/lla, [alpha] 2 1648A [right arrow] G Inconsistent Thrombin receptor PAR-I-5061 Not known [right arrow] D * G indicates guanine; A, adenine; C, cytosine; T, thymidine; Arg, arginine; Gin, glutamine; Thr, threonine; gly, glycine; Ala, alanine; Val, valine; APC, activated protein C; EPCR, endothelial protein C receptor; FII, factor II (prothrombin); FXIII, factor XIII; ?, probably abnormal FXIII cross-linking but mechanism hasn't been verified; FVII, factor VII; Hcy, homocysteine; VNTR, variable nucleotide tandem repeat; PAR-1, protease-activated receptor 1.
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Accepted for publication September 27, 2001.
From the Department of Clinical Pathology, The Cleveland Clinic Foundation, Cleveland, Ohio.
Presented at the 10th Annual William Beaumont Hospital Seminar on Molecular Pathology, DNA Technology in the Clinical Laboratory, Royal Oak, Mich, March 8-10, 2001.
Reprints: Kandice Kottke-Marchant, MD, PhD, Department of Clinical Pathology, The Cleveland Clinic Foundation, L30, 9500 Euclid Ave, Cleveland, OH 44195 (e-mail: email@example.com).
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