Prothrombotic states may be heritable, acquired or mixed – the result of the environment (e.g. oestrogen use, obesity or other lifestyle factors) interacting with genetic background. To date, a limited number of genetic variants are proven to be independent risk factors for venous thromboembolism. These include mutations in the genes encoding the natural anticoagulants antithrombin, protein C and protein S, and the clotting factors fibrinogen, prothrombin and factor V.
Antithrombin deficiency Antithrombin (previously called antithrombin III) is synthesized by the liver. Its inhibitory effect is not confined to thrombin. It also inhibits the activated clotting factors IXa, Xa, XIa, XIIa and tissue factor-bound factor VIIa. Heparins markedly accelerate the rate of complex formation between antithrombin and the serine proteases.
Within the last decade, our understanding of the basis of familial antithrombin deficiency has been greatly facilitated by advances made in the molecular biology and functional characterization of this inhibitory glycoprotein. Of particular significance has been the recognition that the antithrombin molecule possesses two important functional regions – a heparin-binding domain and a thrombin-binding domain. Two major types of heritable antithrombin deficiency are recognized. Type I is characterized by a quantitative reduction of qualitatively (functionally) normal antithrombin protein. Type II deficiency is due to the production of a qualitatively abnormal protein. In both types of antithrombin deficiency, antithrombin activity is reduced to a variable extent. Type II deficiency is subclassified according to the site of the molecular defect:
(a) Reactive site (RS) – abnormalities residing in the reactive (thrombin binding) site.
(b) Heparin binding site (HBS) – abnormalities residing in the heparin binding site.
(c) Pleiotropic effect (PE) – abnormalities residing in both reactive and heparin binding sites.
Antithrombin assays Only functional assays of heparin cofactor activity will detect both type I and type II antithrombin deficiencies. For routine clinical purposes it is recommended that a heparin cofactor activity assay be used in the initial screen.
The distinction between the subtypes of antithrombin deficiency is of clinical relevance as the incidence of thrombosis is higher in association with type I deficiency and type II deficiency in which the mutation affects the reactive site than in type II deficiency in which the mutation affects the heparin binding site (Finazzi et al, 1987). An initial classification into type I or type II can be made by comparing the result of an immunological assay with the result of the heparin cofactor assay.
Type II heparin-binding variants are associated with a lower risk of thrombosis than type II reactive site defects. However, a heparin-binding variant may increase the attributable risk of an additional thrombophilic defect, such as the factor V Leiden mutation. A short incubation, of 30 s or less, with a low concentration of heparin is required for detection. As most currently used antithrombin activity assays utilize a long incubation, heparin-binding defects are not detected. The distinction between type II defects is therefore only an issue for those centres using assays specifically to detect heparin-binding defects. Crossed immunoelectrophoresis with heparin is a simple tool that may be used to detect type II heparin-binding site variants in those centres that need to identify these defects. Although many mutations associated with antithrombin deficiency have been described, identification of the mutation is not usually necessary for clinical purposes.
Normal ranges and variations Age- and sex-related variations in antithrombin activity (Tait et al, 1993a) and antigen levels are minor, so the reference ranges in healthy populations are narrow. Antithrombin levels are slightly lower in premenopausal women than in men of similar age or post-menopausal women and are slightly lower in women using combined oral contraceptive pills than in non-pill-using women (Tait et al, 1993a). More significant decreases in antithrombin activity are observed in patients on heparin treatment (Marciniak & Gockerman, 1977) and in those with current thrombosis. Profound decreases in plasma antithrombin are seen in disseminated intravascular coagulation, liver disease and the nephrotic syndrome.
Prevalence of antithrombin deficiency and risk of thrombosis The prevalence of type I antithrombin gene mutations in the general population is of the order of 0·02% (Tait et al, 1994). Family studies suggest that antithrombin deficiency is a more severe disorder than deficiencies of protein C or protein S with the majority of patients suffering thrombosis before the age of 25 years (Thaler & Lechner, 1981; Hirsh et al, 1989; Demers et al, 1992). In studies of unselected patients with thrombosis, antithrombin deficiency was reported in 1% (Heijboer et al, 1990) and 0·5% (Mateo et al, 1997). The relative risk of venous thromboembolism is around 25–50-fold for individuals with type I antithrombin deficiency (Rosendaal, 1999).
Protein C deficiency Protein C is a vitamin K-dependent glycoprotein that is synthesized in the liver. Before activation by the thrombin–thrombomodulin complex on the endothelial cell surface, it circulates as a two-chain zymogen. By degrading activated clotting factors Va and VIIIa, activated protein C (APC) functions as one of the major inhibitors of the coagulation system. Activated protein C also reduces platelet prothrombinase activity by degrading platelet-bound factor Va at the receptor for factor Xa. The inhibitory effects of activated protein C are facilitated through the cofactor activity of protein S.
As with antithrombin deficiency, familial protein C deficiency can be classified into two types on the basis of phenotypic analysis using functional and immunological assays. Type I is characterized by parallel reductions of functional and immunoreactive protein C. In type II the functional level is substantially lower than that of the antigen. In contrast to antithrombin deficiency, in which type II deficiency is more common than type I, type I protein C deficiency is more common than type II. The anticipation that the underlying genetic variant and associated phenotype might be predictive of the degree of thrombotic risk has not been realized and phenotypic classification of protein C deficiency therefore serves no useful clinical purpose.
Protein C assays Most functional assays of protein C use the specific activator Protac which is derived from snake venom. The activated protein C formed can be quantified by clotting or chromogenic methods. Both are available in kit form from commercial manufacturers. A standard calibrated against the current International Standard for protein C must be used. Chromogenic assays are simple to perform and will detect all type I defects and the vast majority of type II defects. In the presence of factor V Leiden, misleadingly low protein C activity levels may be obtained with the clotting method (Faioni et al, 1996). Clotting methods also underestimate protein C activity in patients with elevated plasma factor VIII levels (De Moerloose et al, 1988) and in the presence of hyperlipidaemia. Measured protein C activity by clotting assay may be unreliable in the presence of a lupus inhibitor (Simioni et al, 1991).
Protein C antigen assays are available widely and will help distinguish between type I and type II deficiency. As there appears to be no clear relationship between the type of protein C defect and the risk of thrombosis, there is no clinical justification for this extra investigation or for molecular studies to identify the specific mutation.
Reference ranges and variations There is a wide overlap in protein C activity between heterozygous carriers and their unaffected relatives in families with protein C deficiency (Allaart et al, 1993). Protein C activity levels appear to be related to age and sex (Tait et al, 1993b), but this is explained by blood lipid levels (Rodeghiero & Tosetto, 1997). Reduced protein C activity is observed in patients with disseminated intravascular disease (DIC) and in liver disease. Protein C activity is reduced markedly by coumarins.
Prevalence of protein C deficiency and risk of thrombosis The prevalence of heritable protein C deficiency in the general population is approximately 0·2–0·3% (Miletich et al, 1987; Tait et al, 1995) and in unselected patients with venous thromboembolism is around 3% (Heijboer et al, 1990; Koster et al, 1995a; Mateo et al, 1997). The relative risk of venous thromboembolism is between 10- and 15-fold for individuals with protein C deficiency (Rosendaal, 1999).
Protein S deficiency Protein S, another vitamin K-dependent protein, is a cofactor for activated protein C. Approximately 65% of the total plasma protein S is complexed with C4b-binding protein (C4bBP) and has no cofactor activity. The remaining 35%, designated free protein S, remains uncomplexed and is the active moiety. The bioavailability of protein S is closely linked to the concentration of C4bBP, which acts as an important regulatory protein in the activated protein C:protein S inhibitory pathway.
Three types of protein S deficiency are described. In accordance with the classification of antithrombin and protein C deficiencies, type I protein S deficiency is a quantitative defect caused by genetic abnormalities which result in the reduced production of structurally normal protein. Both total and free protein S antigen levels are reduced. Type II protein S deficiency has been characterized as a qualitative (functional) defect, but it has become evident that some individuals with inherited or acquired APC resistance have been incorrectly diagnosed as having type II protein S deficiency (Faioni et al, 1993). In type III deficiency, although free protein S antigen is reduced, the total protein S antigen level is normal. It has been suggested that type I and type III protein S deficiencies may be phenotypic variants of the same genetic disorder (Zoller et al, 1994a; Simmonds et al, 1997).
Protein S assays Three main types of assay are available: for functional protein S, and for total immunoreactive protein S and free immunoreactive protein S.
Functional protein S assays are based on the cofactor activity. Ideally these assays should reflect only free protein S activity, but this is not always the case as separation of free protein S from C4bBP-complexed protein S is not performed in many of the available methods. Functional assays detect all types of protein S deficiency, but some functional assays of protein S are non-specific and have been shown to be sensitive to the inherited APC resistance associated with factor V Leiden and the acquired APC resistance observed in some patients with antiphospholipid antibodies (Faioni et al, 1993).
Several techniques are available for the determination of total immunoreactive protein S. In addition to Laurell assays, these include enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays. There are a number of methodological problems associated with the Laurell technique and it is therefore not recommended. An increasing number of commercial kits and reagents are available for the measurement of total protein S, the majority of which are ELISAs. It is important to establish that the chosen method for measuring total immunoreactive protein S is not influenced by the concentration of C4bBP.
Free protein S is the active moiety of the total protein S. Consequently, free protein S assays are frequently performed tests in the investigation of heritable thrombophilia. In the most widely used method, separation of free protein S from the C4bBP-bound protein S is achieved by precipitation with polyethylene glycol followed by centrifugation. Results are expressed relative to a pooled plasma calibrated against the current International Standard, either as a proportion of the total protein S or, preferably, against the free protein S content. Assays using monoclonal antibodies for distinct epitopes of free protein S allow direct measurement of free protein S in citrated plasma without the need for a precipitation stage (Aillaud et al, 1996).
It is recommended that in cases in which a functional protein S assay is used as an initial screening test for protein S deficiency, low results should be further investigated with an immunoreactive assay of free protein S.
Reference ranges and variations Protein S levels are slightly higher in men than in women. Protein S levels fall progressively during pregnancy (Clark et al, 1998) and are reduced to a lesser extent in women using oestrogen-containing oral contraceptives or hormone replacement therapy (Comp et al, 1986; Malm et al, 1988; Lowe et al, 1999). Overdiagnosis of protein S deficiency is therefore a risk. Acquired protein S deficiency is also seen in patients on coumarins, in those with antiphospholipid antibodies and in disseminated intravascular coagulation and liver disease.
Prevalence of protein S deficiency and risk of thrombosis The prevalence of protein S deficiency in the general population remains unknown. It has been suggested that the best way to determine protein S deficiency is by measurement of free protein S antigen (Faioni et al, 1997). In the Leiden Thrombophilia Study (Koster et al, 1995a) and in a subsequent population-based case–control study reported from Italy (Faioni et al, 1997), low levels of free protein S antigen were found in around 3% of patients with venous thromboembolism and in 2·1% and 1·3% of the controls, respectively, suggesting that low free protein S levels have a mild effect on the risk of venous thrombosis – increasing the risk by only around twofold. However, the conclusion that protein S deficiency is a mild risk factor may be flawed as it is possible that the prevalence of protein S deficiency in the general population is much lower than 1–2%. The currently available evidence indicates a substantial difference in risk associated with protein S deficiency in thrombophilic families and in unselected consecutive patients, suggesting that the effect in families is the result of interaction with other defects (Koeleman et al, 1995; Zoller et al, 1995).
Activated protein C (APC) resistance and factor V Leiden APC resistance is defined as an impaired plasma anticoagulant response to APC added in vitro. The phenomenon of APC resistance first attracted widespread attention when it was reported that APC resistance co-segregated with thrombosis in families with familial venous thromboembolism (Dahlback et al, 1993). Shortly thereafter it was demonstrated that the majority of patients with familial APC resistance have the same point mutation in the gene for clotting factor V (1691G-A), the ‘factor V Leiden’ mutation (Bertina et al, 1994). Although factor V Leiden is the most common cause of inherited APC resistance, other changes in haemostasis cause acquired APC resistance, e.g. increased plasma levels of factor VIII or the presence of antiphospholipid antibodies. Activated protein C resistance increases with age and in women who use oestrogen-containing contraceptive pills (Olivieri et al, 1995) or hormone replacement therapy (Lowe et al, 1999). The effect is more marked with third-generation than second-generation oral contraceptives. Activated protein C resistance also increases during pregnancy (Cumming et al, 1995; Mathonnet et al, 1996; Clark et al, 1998).
APC resistance tests The most commonly used test system is the activated partial thromboplastin time (APTT). Samples are tested with and without added APC and the resultant clotting times are expressed as a ratio (Dahlback et al, 1993; De Ronde & Bertina, 1994) – the so-called APC sensitivity ratio (APC:SR). When testing for APC resistance it is important to avoid platelet contamination and activation. Reagents, coagulometers and concentrations of APC affect the results obtained using the APTT method. It has been suggested that the methodological variability associated with APTT-based tests for the detection of APC resistance can be reduced by ‘normalizing’ the results by dividing the patient's APC:SR by the APC:SR of pooled normal plasma. If this system is adopted it is important to establish that the normal plasma pool does not include a contribution from an individual who carries the factor V Leiden mutation, as even a single affected donation is sufficient to affect the APC:SR of the pool (Tripodi et al, 1998).
The originally described APC resistance test is abnormal in subjects with acquired APC resistance and in those who have a prolonged baseline APTT due to, for example, clotting factor deficiencies or anticoagulant therapy. It is therefore not diagnostic of factor V Leiden. Predilution of the test plasma in factor V-deficient plasma increases the sensitivity and specificity of the APTT-based APC: SR as a screen for factor V Leiden (Jorquera et al, 1994; Trossaert et al, 1994). This modification makes the test close to 100% specific and sensitive to factor V Leiden in both healthy controls and patients with suspected VTE (Svensson et al, 1997; Tripodi et al, 1997) and may be reliably used if DNA analysis is not available. However, there is evidence that the APC:SR determined with the original unmodified test correlates with venous thrombosis risk, irrespective of whether or not factor V Leiden is present (Zoller et al, 1994b; Bertina et al, 1995; De Visser et al, 1999; Rodeghiero & Tosetto, 1999). The specificity of the modified APC:SR test means that individuals who have increased APC resistance for reasons other than the possession of the factor V Leiden mutation will be overlooked if the original APC:SR test is omitted from the screening procedure. The clinical value of detecting APC resistance in the absence of factor V Leiden is unknown.
Detection of the factor V Leiden mutation Detection of the factor V Leiden mutation relies on amplification of the nucleotide region close to the exon–intron boundary in exon 5 of the factor V gene from either genomic DNA or from mRNA followed by a mutation detection step.
Prevalence of the factor V Leiden mutation and risk of thrombosis In Caucasian populations, factor V Leiden is much more common than any of the other heritable thrombophilias having a reported prevalence of between 2% and 15% (Rees et al, 1995) and is more prevalent in individuals of Northern European extraction than in those from Southern Europe (Rosendaal et al, 1995; Ridker et al, 1997). Depending on patient selection, factor V Leiden is found in 20–50% of patients presenting with a first episode of venous thromboembolism (VTE) (Koster et al, 1993; Rosendaal et al, 1995) and in more than 50% of probands from selected families with familial thrombophilia (Griffen et al, 1993). Heterozygous carriers have a three- to eightfold increased risk of venous thrombosis (Koster et al, 1993; Ridker et al, 1995a; Rosendaal et al, 1995) and homozygotes have an 80-fold increased risk (Rosendaal et al, 1995).
Familial APC resistance in the absence of the factor V Leiden mutation Occasionally familial APC resistance occurs in the absence of factor V Leiden (Zoller et al, 1994b; Bertina et al, 1995). One identified cause is a mutation in the 306 APC cleavage site (factor V Cambridge) (Williamson et al, 1998). A specific factor V gene haplotype (HR2) has been shown to occur more frequently in individuals with APC resistance ratios beneath the 15th percentile than in those with higher ratios or in normal controls (Bernardi et al, 1997). Co-inheritance of this HR2 haplotype with factor V Leiden may increase the risk of VTE above that associated with factor V Leiden alone (Faioni et al, 1999).
Prothrombin G20210A mutation The G→A transition at nucleotide 20210 in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increased risk of venous thrombosis (Poort et al, 1996). In the absence of a specific phenotypic test for the presence of the variant 20210A allele, DNA-based procedures are required. The 20210A transition is not associated with the introduction or loss of a specific restriction enzyme recognition site and detection methods that do not require the use of restriction enzyme digestion of the amplified polymerase chain reaction (PCR) product have been devised.
Prevalence of the prothrombin G20210A mutation The prevalence in Northern Europe is around 2% in the healthy population and 6% in unselected patients with a first thrombosis (Poort et al, 1996). Higher prevalences have been reported in Southern Europe in which prothrombin G20210A is the most prevalent heritable thrombophilic defect (Souto et al, 1998). The risk of venous thrombosis in heterozygous carriers of the 20210A allele is estimated to be around three times that in non-carriers.
Dysfibrinogenaemia Over 250 cases of heritable dysfibrinogenaemia have been reported. Most cases are asymptomatic and found coincidentally, but in about 20% there is an increased tendency to arteriovenous thromboembolism and in 25% a bleeding tendency (Haverkate & Samama, 1995). In some there is a low fibrinogen functional level. The prevalence of fibrinogen abnormalities in patients with venous thrombosis is low (0·8%), but a high incidence of post-partum thrombosis and an increased risk of pregnancy loss have been reported in women with thrombophilic fibrinogen variants (Haverkate & Samama, 1995). Several mechanisms to explain the thrombotic tendency have been proposed, including defective lysis of the abnormal fibrin or defective binding of thrombin to the abnormal fibrin with consequent elevated plasma thrombin levels.
Other defects It is probable that there are other as yet unidentified heritable abnormalities of haemostasis associated with an increased risk of thrombosis. Some additional candidates such as dysplasminogenaemia and heparin cofactor II deficiency have been studied. However, none has been demonstrated conclusively to contribute to heritable thrombophilia.