CHAPTER 127 HEREDITARY THROMBOPHILIA
CHAPTER 127 HEREDITARY THROMBOPHILIA
SCOTT H. GOODNIGHT
JOHN H. GRIFFIN
Major Hereditary Defects
Hereditary Resistance to Activated Protein C
Prothrombin G20210A Gene Polymorphism
Protein C Deficiency
Protein S Deficiency
High Levels of Factor VIII and Other Coagulation Factors
Hereditary Thrombotic Dysfibrinogenemia
Other Potential Thrombophilic Disorders
Diagnosis of Thrombophilia
Therapy of Thrombophilia
Thrombophilia and Pregnancy, Oral Contraceptives, and Hormone Replacement Therapy
Management of Pregnancy in Thrombophilic Women
Pregnancy Loss due to Thrombophilia
Estrogens: Oral Contraceptives and Hormone Replacement Therapy
Hereditary thrombophilia is defined as a genetically determined increased likelihood of thrombosis. An emerging paradigm suggests that thromboembolism is a multicausal disease involving one or more genetic defects in conjunction with acquired risk factors such as inactivity, trauma, malignancy, inflammation, pregnancy, oral contraceptive use, or autoimmune disease. The three most common hereditary defects (found in a substantial proportion of patients presenting with venous thrombosis) include activated protein C resistance caused by replacement of Arg506 by Gln in the factor V gene (factor V Leiden), a prothrombin polymorphism (G20210A) that causes elevated plasma prothrombin levels, and hyperhomocysteinemia. Additional genetic abnormalities include deficiencies of the anticoagulant factors protein C, protein S, or antithrombin. The majority of these thrombophilic defects either enhance procoagulant reactions or hamper anticoagulant mechanisms and thus cause a prothrombotic state due to hypercoagulability of the blood. Venous thrombosis is the most common manifestation of hereditary thrombophilia, although a minority of patients, particularly those with other vascular risk factors, may develop arterial thrombi as well. Less usual presentations of venous thromboembolic disease include abdominal and cerebral vein thrombosis, along with pregnancy loss or other complications due to placental vascular insufficiency and thrombosis. Laboratory assays are now widely available to identify the great majority of patients with thrombophilia. Knowledge of these disorders affects patient management, including the duration of anticoagulant treatment, the use of clotting factor replacement therapy, the need for prophylactic antithrombotic agents, and counseling involving the relative risks of pregnancy and use of oral contraceptives or hormone replacement.
Acronyms and abbreviations that appear in this chapter include: APC, activated protein C; C4BP, C4b-binding protein; DVT, deep vein thrombosis; MTHFR, methylenetetrahydrofolate reductase; PAI-1, plasminogen activator inhibitor 1; PCR, polymerase chain reaction.
Hereditary thrombophilia is defined as a genetically determined increased risk of thrombosis. According to Virchow’s classic (and still useful) triad, risk factors for thrombosis may involve abnormalities in the vessel wall (see Chap. 114), rheology, and/or blood components. Identification of defects in specific blood components, especially plasma factors, has provided molecular insights into the pathogenesis of thrombophilia. The major defects associated with familial thrombophilia are listed in Table 127-1. The first description of hereditary thrombophilia caused by a deficiency of an anticoagulant protein was by Egeberg in 1965.1 Members of the family described in the report suffered from recurrent venous thrombosis, and the disorder was inherited in an autosomal dominant pattern. The plasma of affected family members had reduced amounts of an inhibitor to thrombin, antithrombin III. In 1976, Stenflo and coworkers2 purified and characterized an anticoagulant factor, protein C, from bovine plasma, and subsequently the first patients with hereditary protein C deficiency and thrombosis were described by Griffin and colleagues.3 Three years later protein S deficiency was reported in several families with thrombosis by Schwarz and coworkers4 and Comp and coworkers.5,6 In 1993 Dahlback and coworkers reported three families with venous thrombosis associated with hereditary resistance to activated protein C (APC),7,8 and in 1994 the underlying genetic defect was simultaneously reported by th ree laboratories to involve the factor V mutation of Arg506 to Gln, a defect now often referred to as factor V Leiden.9,10 and 11 At about the same time, mild to moderate hyperhomocysteinemia was also recognized as a risk factor for venous thrombosis,12 although a predisposition to arterial vascular disease due to elevated levels of homocysteine had been known for some time.13 In 1996, a mutation in the 3′-untranslated region of prothrombin was identified and linked to familial venous thromboembolism by Poort and colleagues.14 Many important observations have come from the Leiden Thrombophilia Study of 300 to 500 Dutch consecutive patients presenting with a first episode of venous thrombosis.15,16 With the imminent characterization of about 100,000 human genes, one can anticipate a steady stream of advances in the identification of more genetic defects responsible for hereditary thrombophilia.
TABLE 127-1 MAJOR HEREDITARY DEFECTS ASSOCIATED WITH INCREASED RISK OF VENOUS THROMBOSIS
Specific hereditary thrombophilias can now be identified in 30 to 50 percent of patients presenting with a first episode of venous thromboembolism, with even higher percentages found in subjects with recurrent thrombosis. Patients with hereditary thrombophilia may have more than one hereditary thrombophilia and associated acquired abnormalities such as antiphospholipid antibodies, malignancy, myeloproliferative diseases, or inflammatory disorders (Fig. 127-1). Hereditary prothrombotic states are usually associated with venous rather than arterial thrombosis; however, in association with other risk factors such as smoking or diabetes, recent data suggest that up to 10 percent of arterial thromboses are associated with hereditary thrombophilia.17,18
FIGURE 127-1 Paradigm for genetic contribution to venous thrombosis. Clinically significant venous thrombosis most often follows from the simultaneous presence of an acquired risk factor for thrombosis and one or more genetic factors that convey thrombotic risk. The presence of two genetic factors (i.e., gene-gene interaction) greatly increases the likelihood of thrombosis. Mild genetic risk factors include APC resistance with or without factor V Leiden; the prothrombin G20210A polymorphism causing elevated plasma prothrombin levels; and heterozygous deficiency of protein C, protein S, or antithrombin. Hyperhomocysteinemia is a mild risk factor. Elevated levels (more than 150 percent of normal) of various coagulation factors, including factors VIII, XI, and IX and fibrinogen also appear to be mild risk factors for venous thrombosis. Venous thrombosis patients frequently have two or more genetic risk factors. See Schafer,351 Bertina,15 Rosendaal,16,78 and van Boven.279 Taken from Schafer with permission.351
Venous thrombosis and its complications are important and common medical problems, estimated to occur at a rate of 1.2 events/1000 population per year.19 Thus, for the population of the United States, there will be approximately 201,000 new cases of venous thromboembolism per year, of which 107,000 will be deep venous thrombosis and 94,000 will be pulmonary embolism.
In this chapter, we will discuss the pathogenesis and unique clinical features of each of the more common hereditary thrombophilias (see Table 127-1). Thereafter an approach to the diagnosis and subsequent treatment of these thrombophilic patients will be presented.
MAJOR HEREDITARY DEFECTS
HEREDITARY RESISTANCE TO ACTIVATED PROTEIN C
DEFINITION AND HISTORY
The term activated protein C (APC) resistance is defined as an abnormally reduced anticoagulant response of a subject’s plasma to APC on the basis of in vitro testing. A “normal” range for response to APC is established for the various coagulation or other related assay conditions used to assess response to APC.7,20,21 In 1989, an abnormally poor response to APC was described for several individual patients with venous thrombosis when it was shown that partially purified antibody fractions interfered with expression of APC activity.22,23 and 24 In 1991, familial APC resistance was first reported in one kindred and was ascribed to an APC-resistant factor VIII defect.25 Apparently this mechanism could not be confirmed, and in 1993, the description of three unrelated families presenting with venous thrombosis associated with APC resistance without any identifiable defect stimulated an intensive search for genetic and molecular mechanistic explanations.7 Three laboratories independently reported in May of 1994 that a single genetic defect was associated with APC resistance, involving replacement of G by A at nucleotide 1691 in exon 10 of the factor V gene which causes the amino acid replacement of Arg506 by Gln.9,10 and 11. Further analysis indicated genetic linkage between APC resistance and the factor V gene.26,27 In the published literature and in this chapter, this defect is variously termed Gln506-factor V, Q506-factor V, or factor V Leiden.
ETIOLOGY AND PATHOGENESIS
APC resistance can be caused by heterogeneous molecular defects, although replacement of Arg506 by Gln in factor V is responsible for APC resistance in the great majority of patients. APC resistance is a laboratory phenotype, whereas Gln506-factor V is a genotype, and the term APC resistance should not be used as a synonym for factor V Leiden. APC resistance caused by defects other than factor V Leiden is associated with increased risk of venous thrombosis28,29 or ischemic stroke.30,31
Theoretically, any genetic abnormality of a protein C pathway component that interferes with the expression of APC activity can cause APC resistance as could acquired abnormalities such as antibodies against protein C pathway components.32,33 Although the causes of many cases of acquired APC resistance are unknown, the majority (more than 90 percent) of hereditary APC resistant subjects have the same genetic abnormality, factor V at G1691A (Arg506Gln), which arose in a single Caucasian founder some 21,000 to 34,000 years ago.34 The molecular mechanism for APC resistance in such probands involves resistance of Gln506-factor Va to proteolytic inactivation by APC,9,35,36 with kinetic studies showing that the Gln506-variant is inactivated 10 times slower than normal Arg506-factor Va.36,37,38 and 39 Gln506-factor Va, whether activated by thrombin or factor Xa, is partially but not entirely resistant to APC, implying that inactivation of Gln506-factor Va by APC can occur in vivo, albeit at a reduced rate. Explanation for only a partial resistance to APC derives from the fact that cleavage of factor Va by APC at Arg306 also occurs, causing complete loss of factor Va activity, although this cleavage is slower than that at the Arg506 site.36,37,38,39 and 40 This finding helps explain why APC resistance due to Gln506-factor V is a rather mild risk factor for venous thrombosis and why a combination of genetic risk factors or a combination of a genetic risk factor plus acquired risk factors for venous thrombosis is found in a significant fraction of symptomatic patients (see below). A nother possibility to help explain the mild risk of venous thrombosis associated with Gln506-factor V is that factor Va may be inactivated in vivo by proteases other than APC that cleave at sites other than residue 506.
There are additional potential molecular defects that might contribute to thrombosis in hereditary APC resistance. In purified clotting factor reaction mixtures, factor V enhances inactivation of factor VIIIa by APC in the presence of protein S,41 and APC resistant subjects carrying Gln506-Factor V are reportedly defective in this APC cofactor activity.42,43,44 and 45 APC resistance caused by rare factor V mutations that replace Arg306 by Thr46 or Gly47 have been reported, although the relationship to relative risks of thrombosis have not been established.48 A factor V haplotype, designated R2, has also been associated with mild APC resistance.49 The molecular mechanisms and thrombotic risks associated with the R2 factor V haplotype which contains normal Arg506 remain to be defined, although it appears that the R2 haplotype is only a risk factor when present along with the Gln506-factor V allele.50
APC is a normal component of circulating blood that contributes to antithrombotic surveillance mechanisms and prevents thrombosis (see Chap 113).51 Normal subjects have a mean APC concentration of 2.3 ng/ml (38 pM) in the circulation,52 and the in vivo half-life of APC in normal adult human subjects as well as in freshly drawn whole blood is approximately 22 min.53,54 Thus, there is continuous activation of the protein C pathway in vivo. In normal subjects, there is an inverse relationship between levels of circulating APC and of thrombin.55 APC levels are increased when thrombin is acutely generated such as during DIC, ischemia, or surgical procedures.
Because circulating APC has such a long half-life, it provides systemic anticoagulation to down-regulate thrombin generation and to limit extension of hemostatic plugs. Hence, genetic or acquired defects that impair the response to APC are understandably prothrombotic. Elevated plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin complexes are found in many subjects heterozygous or homozygous for Gln506-factor V,56,57,58 and 59 presumably reflecting the impairment of the expression of APC’s anticoagulant activity.
APC resistance has been associated with less intrapartum blood loss, suggesting an evolutionary advantage.60 However, pregnancy loss is increased in some women due to thrombosis in the placental vasculature (see below). Hemophilia A patients who also inherit Gln506-factor V have been reported to have less severe hemorrhagic symptoms.61
The factor V Leiden mutation is present in 3 to 12 percent of Caucasians and is rare in other ethnic groups.62,63 and 64 Deep and superficial venous thromboses are the most common manifestations of this disorder, whereas pulmonary embolism and thromboses in unusual locations appear to be relatively less frequent than in subjects with deficiencies in antithrombin, protein C, or protein S.65,66,67,68 and 69 In patients with venous insufficiency leading to leg ulcers, approximately 25 percent were found to have APC resistance70 or factor V Leiden.71 Cerebral, hepatic, and other thromboses have been reported in patients with factor V Leiden.72,73 and 74 About half the patients will have idiopathic (unprovoked) venous thromboembolism, with 20 percent occurring after surgery, and 30 percent in women who are pregnant or taking birth control pills.75 Pregnancy loss and other obstetric complications occur at an increased rate in women with factor V Leiden (see below).
The risk of thrombosis in subjects with factor V Leiden appears to be somewhat lower than in patients from families with deficiencies of antithrombin or protein C76,77; nonetheless, since factor V Leiden is so common, it accounts for the largest proportion of patients presenting with a first thromboembolic event (20 to 25 percent).78 The relative risk of venous thrombosis in patients heterozygous for factor V Leiden is increased by four to eightfold in studies from Europe and North America.20,67,79,80 and 81 The risk of idiopathic venous thromboembolism for men increases with age, from a relative risk of 1.23 at age 40 to 50 years to 5.97 for those aged 70 years and older.79 First-degree relatives of symptomatic carriers of the factor V mutation develop thromboses at a rate of 0.45 percent per year (0.25 percent per year in the 15- to 30-year age group, and 1.1 percent per year in those over 60).75,77 In several studies, recurrent thrombotic events are rather frequent, occurring at a rate of 5 to 10 percent per year following a first thrombosis.82,83 and 84 However, other investigators found no increased rates of recurrence when compared to thrombosis patients without factor V Leiden.85,86 and 87 Homozygous carriers of factor V Leiden have an odds ratio for venous thrombosis of 50 to 100, and it is estimated that approximately half of such individuals will experience a clinically significant episode during their lives.88 Alth ough thromboses in homozygotes are substantially more common than in heterozygotes, the disorder is far less severe than in subjects with homozygous deficiency of protein C, or protein S.56,88,89 Despite the increased thrombotic risk, the presence of factor V Leiden does not increase overall mortality.90,91,92 and 93
Coronary artery thrombosis has been notably associated with the factor V Leiden mutation in young women17 and men94 also displaying other vascular risk factors. The relative risk of myocardial infarction in carriers of V Leiden from the Netherlands is 1.4, which increased to three- to sixfold if other risk factors such as obesity, smoking, hypertension, or diabetes were present.95 Similar findings have been reported for young women from Washington State, with odds ratios of up to 32 for myocardial infarction in V Leiden carriers who were also smokers.17 However, other studies have failed to find a relationship between APC resistance and myocardial infarction or stroke in older individuals.80,96,97 and 98 Factor V Leiden seems to be relatively common in children who develop cerebral infarction or venous thrombosis.99,100 and 101
PREDISPOSING FACTORS FOR THROMBOSIS IN SUBJECTS WITH FACTOR V LEIDEN
Although isolated factor V Leiden is associated with a relatively mild hypercoagulable state, the risk of thrombosis is greatly magnified when other prothrombotic disorders are also present (Fig. 127-1). These additional risk factors may be hereditary (e.g., protein C deficiency or the prothrombin gene mutation), acquired (antiphospholipid antibodies, hyperhomocysteinemia), physical (inactivity or surgery), due to other diseases (malignancy or inflammation), or hormonal (oral contraceptives or pregnancy).78
Multiple hereditary thrombophilic defects (i.e., gene-gene interaction) are quite common, and are found in up to 15 percent of patients presenting with venous thromboembolism.102 Factor V Leiden has been reported in combination with protein C deficiency,9,103,104 and 105 protein S deficiency,106,107 and 108 antithrombin deficiency,109 the prothrombin gene mutation,110,111 and hyperhomocysteinemia.112,113 and 114 In families with combined defects, thromboses occurred more frequently and at an earlier age in the subjects with two separate defects.
Pregnancy and estrogen-containing oral contraceptives substantially enhance the risk of thrombosis in women with factor V Leiden. Of women who develop venous thromboembolism during pregnancy, 28 to 46 percent will carry the factor V mutation.115,116 and 117 The relative risk of developing thrombosis for heterozygotes during or after pregnancy is increased over threefold, with a higher probability of recurrence (relative risk of 3.86).117 Several studies have examined the risks of thromboembolism in women with factor V Leiden using third-generation oral contraceptives. There is a highly significant increase of 30- to 80-fold in the odds ratio for thrombosis, with an absolute increase in risk from 0.8 to 28.5 per10,000 women per year.118,119 Even higher risks are seen in women who are homozygous for the mutation.120 Data are not yet available on the risk of thrombosis in women with factor V Leiden (with or without a history of thrombosis) who receive hormone replacement therapy. A major side effect of selective estrogen receptor modulators such as tamoxifen or raloxifene may also exert an increased risk of venous thrombosis. Three cases of tamoxifen-associated venous thrombosis were associated with factor V Leiden.121
Coagulation assays and DNA-based assays are available for the identification of patients with APC resistance. Plasma-based coagulation tests depend on the relative prolongation of the activated partial thromboplastin time (aPTT) or other coagulation screening tests caused by the addition of purified APC. Individuals with resistance to APC have less prolongation of the aPTT than normal. Although an aPTT assay was originally used, current assays employ factor V deficient plasma,35 which makes the test informative in most patients with lupus inhibitors, in pregnant patients, in patients with inflammatory states, and in patients on anticoagulants. The test is sensitive and specific when compared with the genetic test for factor V Leiden.122,123,124,125 and 126 Studies using the first-generation assay (see above) have suggested that an abnormally low APC ratio is associated with venous thrombosis, in both the presence and absence of the factor V Leiden mutation28,29 and with ischemic stroke.30,31 Thus, there is clinically relevant information in the classic aPTT-based APC resistance test that is not obtained using factor V deficient substrate plasma. Tissue-factor-based APC resistance assays can provide additional information about plasma components that differentially modulate the protein C pathway,127,128 and 129 such as “anticoagulant” high-density lipoprotein or as yet unidentified factors that are altered by oral contraceptive usage. The presence of platelets or platelet microparticles in plasma tested for APC resistance using aPTT assays,130,131 and 132 as well as autoantibodies against APC,33 can reduce the anticoagulant response to APC, indicating the need to carefully prepare plasma prior to testing.
Many DNA-based assays for the factor V Leiden polymorphism are available. Genomic DNA is isolated, amplified by polymerase chain reaction (PCR), subjected to restriction fragment length polymorphism analysis, and analyzed for G or A at nucleotide 1691.9 Plasma coagulation tests are often used for screening patients, followed by confirmation of positive results with the DNA assay. Only DNA tests clearly distinguish factor V Leiden heterozygosity from homozygosity. “Pseudo-homozygotes” heterozygous for factor V Ledien and for a dysfunctional factor V allele will have very low APC-resistance ratios in the plasma test but will be heterozygous by the DNA assay for factor V Leiden.133,134 and 135
PROTHROMBIN G20210A GENE POLYMORPHISM
DEFINITION AND HISTORY
In 1996, Poort and colleagues reported that a polymorphism in the 3′-untranslated region of the prothrombin gene, namely nt G20210A, was associated with increased risk of venous thrombosis and with elevated levels of plasma prothrombin.14 This polymorphism likely arose as a single mutation in a Caucasian founder,136 and the polymorphism is currently found in 1 to 5 percent of Caucasians.137
ETIOLOGY AND PATHOGENESIS
Replacement of G by A at nt 20210 in the 3′-untranslated region of the prothrombin gene does not alter transcription of the gene but may increase translation, thus resulting in elevated synthesis and secretion of prothrombin by the liver. The elevated level of plasma prothrombin likely contributes directly to increased thrombotic risk by causing increased thrombin generation.
The prothrombin gene mutation is found largely in Caucasian populations.136 In contrast to factor V Leiden, the frequency of the mutation seems to increase from northern Europe to southern Europe, i.e., only 1.7 percent of the population in northern Europe had the abnormality compared with 3 to 5 percent in the south of Europe and the Middle East.137,138 The prothrombin gene mutation is associated with venous thrombosis in all age groups.139 When sequential patients presenting with a first venous thromboembolism are analyzed, 4 to 8 percent of them will have the mutation, and the odds ratio for thrombosis in subjects with prothrombin 20210A is increased approximately 2- to 5.5-fold.14,102,140,141,142,143,144 and 145 In patients with recurrent thromboembolism or a family history of thrombosis, as many as 15 to 18 percent will have the defect compared with 1 to 3 percent of controls in various populations.14,146 As mentioned earlier, the prothrombin variant is associated with elevated plasma levels of prothrombin (e.g., a mean of 132 percent).14 Increased prothrombin activity or antigen is also associated with an increased risk of thrombosis even in the absence of the mutation.147
As in other forms of hereditary thrombophilia, the prothrombin gene mutation has been found in patients with thrombosis in unusual sites, particularly cerebral sinus vein thrombosis.148,149,150,151,152 and 153 For example, in a study of 40 patients with cerebral vein thrombosis, 20 percent had the gene defect (OR 10.2). Many of these thromboses were in young women taking oral contraceptives, which raises the likelihood of thrombosis even higher (i.e., an OR of 150).150
Individuals who are homozygous for the prothrombin gene mutation appear more likely than heterozygotes to develop thrombosis.154,155 and 156 The mutation also occurs in concert with other hereditary thrombophilic states (8 percent in one study).111,140,142,146,157 When the prothrombin variant was associated with factor V Leiden in young symptomatic patients, overall thrombosis rates were increased as well as spontaneous events and thromboses in unusual locations.111
The prothrombin gene mutation appears generally not to be overrepresented in unselected patients with cerebral vascular or coronary artery disease.146,158,159,160 and 161 However, certain selected groups of patients with arterial thrombosis have an increased likelihood of carrying the mutation.95,141,155,162,163 and 164 In young (younger than 50 years) patients with documented ischemic stroke but without other risk factors such as diabetes, hypertension, or hyperlipidemia, 15 percent had the prothrombin gene mutation (giving an odds ratio for ischemic stroke of 5.1).155 The mutation also appears to be associated with an increased risk of myocardial infarction, especially in those with other major risk factors for coronary heart disease such as smoking.95,162 Finally, a large proportion of a group of young women with acute unexplained spinal cord infarction were found to have the mutation.165 All were taking oral contraceptives and most were smokers.
Identification of the mutation in the 3′-untranslated region of the prothrombin gene requires DNA analysis following PCR amplification of the pertinent region.14 Although prothrombin levels are elevated, assay of prothrombin activity or prothrombin antigen is usually not sufficiently sensitive or specific to screen for the presence of the mutation or as a more effective predictor of thrombosis.146,147,157
DEFINITION AND HISTORY
A plasma homocysteine level above the normal range defines hyperhomocysteinemia. Severe hyperhomocysteinemia, also identifiable as homocystinuria, is rare and is an autosomal recessive trait associated with severe defects in cystathionine b-synthase, 5,10-methylenetetrahydrofolate reductase (MTHFR), or possibly other enzymes that affect homocysteine metabolism.166,167 and 168 Such severe abnormalities are associated with neurologic abnormalities, premature cardiovascular disease, stroke, and vascular thrombosis. Mild to moderate hyperhomocysteinemia is an independent risk factor for arteriosclerosis and arterial thrombosis.13,167,169 A meta-analysis of 10 case-control studies concluded that mild hyperhomocysteinemia conveys a significant, though mild, increased risk of venous thrombosis.170
ETIOLOGY AND PATHOGENESIS
Homocysteine is an intermediate in the metabolism of the sulfur-containing amino acids, methionine and cysteine, and homocysteine participates in several metabolic pathways. Remethylation of homocysteine to generate methionine requires the vitamin B12-dependent enzyme, methionine synthase, and 5-methyltetrahydrofolate, which are part of a metabolic pathway that recycles tetrahydrofolate, and 5-methyltetrahydrofolate and involves the enzyme methylenetetrahydrofolate reductase. For the synthesis of cysteine from homocysteine, a transulfuration pathway first involves condensation of homocysteine with serine to generate cystathionine by the vitamin B6-dependent enzyme, cystathionine b-synthase; then deamination and cleavage of cystathionine to yield cysteine and a-ketobutyrate is accomplished by the vitamin B6-dependent enzyme, cystathioninase. The most common known genetic cause of mild hyperhomocysteinemia involves an MTHFR gene polymorphism, nt C677T, that causes a conservative replacement of Ala222 by Val which results in a variant enzyme with reduced specific activity and increased thermolability.113 Homozygosity for this so-called thermolabile form of MTHFR, i.e., homozygosity for TT at nt677, is associated with mild hyperhomocysteinemia.
The most common cause of rare, severe hyperhomocysteinemia is defective cystathionine b-synthase. Suboptimal levels of folate or vitamins B6 or B12 can also contribute to acquired mild to moderate hyperhomocysteinemia by providing inadequate cofactor levels to support the enzymes that regulate homocysteine metabolism. Conversely, administration of folate with vitamins B6 and B12 can reduce homocysteine levels.171,172 To date, no controlled studies of vitamin therapy to reduce homocysteine levels in venous thrombosis patients have been reported nor has it been proved that this vitamin strategy reduces arterial or venous thrombotic risk.
The exact mechanisms by which hyperhomocysteinemia causes increased risk of thrombosis have not been defined, although there is strong evidence that elevated homocysteine levels cause deleterious prothrombotic alterations in a number of normal vascular functions based on animal model studies, tissue culture experiments, and clinical research (see Chap. 114).173,174 and 175
Hyperhomocysteinemia is commonly associated with venous thromboembolism as well as arterial disease. From 10 to 25 percent of patients with primary or recurrent venous thrombosis have plasma homocysteine concentrations that are greater than the 95th percentile of the distribution in normal individuals (i.e., greater than 17 to 22 µmol/liter).167,169,176,177,178,179 and 180 Meta-analysis of multiple studies suggest that the odds ratio for venous thrombosis is 2.5 to 3.0 if homocysteine concentrations are elevated.170 Coagulation activation markers such as F1.2 or plasma levels of activated protein C are increased in patients with hyperhomocysteinemia and thrombosis, suggesting the presence of hypercoagulability.181,182
The association of hyperhomocysteinema and venous thrombosis is stronger among women (e.g., OR 7), and it also increases with age, rising to an odds ratio of 5.5 for individuals who are older than 50 years.176 In most but not all studies,183 the combination of hyperhomocysteinemia in concert with other hypercoagulable states substantially increases the risk of thromboembolism.112,114,169 For example, in the Physicians Health Study, the odds ratio for idiopathic venous thromboembolism in subjects with hyperhomocysteinemia was 3.4, for those with factor V Leiden it was 3.6, but for subjects with both disorders, the odds ratio was greatly increased to over 20.114 Hyperhomocysteinemia is also a strong predictor of recurrent thrombosis (OR 2–3), with reported recurrence rates of up to 10 percentper year for the first 2 years after cessation of oral anticoagulants.177,180
The thermolabile form of MTHFR has been associated with hyperhomocysteinemia, particularly during periods of folate deficiency.184,185 Although controversial, it appears that homozygosity alone for this enzyme defect (which occurs in 10 to 20 percent of normal individuals) is associated with either an absent or a mild increased risk of venous thromboembolism in the absence of associated thrombotic risk factors.102,113,186,187,188 and 189 However, subjects who are homozygous for the MTHFR variant who also have factor V Leiden or the prothrombin gene mutation may be at a mildly to moderately increased risk of thrombosis.102,113,186,188
Plasma homocysteine concentrations can be measured by HPLC or an immunoassay or by performing a methionine loading test. Both fasting levels and levels after methionine loading have been used to assess hyperhomocysteinemia.190,191,192 and 193 Although the methionine loading test may detect additional subjects with hyperhomocysteinemia, it is not clear that the predictive value for thrombosis is sufficiently increased to warrant the additional effort and cost of this procedure.169,177,193,194 Blood samples for homocysteine levels should be obtained in the fasting state, kept cold, and centrifuged immediately.190,192 Individual measurements reflect average homocysteine concentrations over time (e.g., 4 weeks) reasonably well.190 Serum homocysteine levels are higher than plasma levels, and male values are higher than female values.195 The thermolabile nt 677T variant of MTHFR and mutations in the cystathionine beta-synthase gene can be assessed using DNA-based molecular techniques.196
PROTEIN C DEFICIENCY
DEFINITION AND HISTORY
The first cases of familial heterozygous protein C deficiency (about 50 percent of normal plasma level) associated with venous thrombosis in young adulthood3 and of severe protein C deficiency (less than 1 percent protein C activity) associated with neonatal purpura fulminans197 were reported in 1981. Most typically, hereditary deficiency of protein C results from an autosomal trait in which affected individuals have approximately 50 percent of the normal level of functional plasma protein C. Over 150 different mutations in the protein C gene which are associated with thrombosis have now been reported.198 Heterozygous protein C deficiency conveys a mildly increased risk of venous thrombosis. Fewer than two dozen cases of severe protein C deficiency due to homozygosity or compound heterozygosity have been reported in neonates with purpura fulminans or massive thrombosis. Type I protein C deficiency is defined as a disorder with parallel reductions in both plasma antigen and anticoagulant activity levels, whereas type II deficiency, associated with circulating dysfunctional molecules, involves normal plasma levels of antigen but low levels of anticoagulant activity.
ETIOLOGY AND PATHOGENESIS
Protein C is synthesized in the liver and circulates in plasma as a serine protease zymogen; it is activated by limited proteolysis by thrombin bound to thrombomodulin, possibly with additional acceleration by an endothelial protein C receptor (see Chap. 113). APC is a potent anticoagulant enzyme that down-regulates the blood coagulation pathways by proteolytic and irreversible inactivation of factors Va and VIIIa. Thus, decreased levels of protein C zymogen may impair the inhibition of thrombin generation and contribute to hypercoagulability.
Protein C deficiency occurs in 0.2 to 0.4 percent of normal individuals199,200 and is found in approximately 4 to 5 percent of consecutive outpatients with objectively confirmed deep venous thrombosis.201 Deficiency of protein C is linked to thrombosis (OR 6.5–8),76,201 and many families with hereditary protein C deficiency and thromboembolism have been reported.3,202,203 and 204 The mean age of first thrombosis has been reported to be similar (approximately 45 years) in patients with factor V Leiden and protein C deficiency suggesting similar thrombotic tendencies in the two types of thrombophilia.205 When mortality rates are compared, individuals with protein C deficiency have a normal life-span.206
Variability in clinical expression is a hallmark of the disorder. Subjects identified by screening large numbers of normal individuals (e.g., blood donors) in most instances have neither a personal nor a family history of thromboembolism.199,200 The discrepancy in thrombosis rates between these surveys and studies of families who have striking thrombotic symptoms can be explained in part by the coinheritance of factor V Leiden or another thrombophilic state (Fig. 127-1).9,103,104 and 105,207,208 Polymorphisms in the promotor region of the protein C gene resulting in lower levels of protein C in some of the families could also be involved.209 Recurrent thrombosis in affected families with protein C deficiency is quite common and is unprovoked in about 60 percent of instances.210,211
Deep and superficial venous thrombosis is the most common clinical presentation of protein C deficiency.210,211,212 and 213 By the age of 45, up to 50 percent of heterozygous subjects in clinically affected families will have venous thromboembolism, and half of the episodes will be spontaneous.203 Protein C deficiency has been linked to unusual sites of venous thrombosis including the cerebral and mesenteric veins.210,214 Arterial thrombosis seems to be uncommon, although ischemic stroke and other arterial occlusive events have been reported.76,215
Homozygous protein C deficiency with protein C levels of less than 1 percent produces a fulminating thrombotic diathesis including the dramatic syndrome of neonatal purpura fulminans in affected infants.197,216,217,218 and 219 In a similar scenario, “warfarin skin necrosis,” large areas of thrombotic skin necrosis, appear over central areas of the body (breast, abdomen, genitalia) in subjects with heterozygous protein C deficiency given warfarin.220 In this syndrome in protein C deficient patients, the vitamin K antagonist induces a fall in protein C activity from approximately 50 percent to very low levels because of the short half-life of protein C in vivo (4 to 8 h).221 Because the half-lives of prothrombin, factor IX, and factor X are much longer, a transient hypercoagulable state may arise at the outset of vitamin K-antagonist therapy. Heparin or low-molecular-weight heparin should be used when initiating warfarin treatment in subjects known to be protein C deficient.202
Most laboratories screen for protein C deficiency with a protein C activity assay that employs a highly specific snake venom protease to activate protein C.222,223 Protein C activity is best assessed with an assay that employs a coagulation rather than a chromogenic end point to identify the greatest number of patients with protein C deficiency.224 Immunoassays are used to distinguish type I defects (reduced antigen and activity) from type II disorders (normal antigen, reduced activity).225 Normal ranges for protein C increase with age (4 percent per decade) so that results need to be interpreted against these age-specific norms.222 Protein C gene promotor polymorphisms also influence plasma concentrations of the protein which can vary from 94 to 106 percent,209,226 and liver disease or oral contraceptives can lower or raise protein C levels respectively.227 Consequently, protein C levels of less than 55 percent (in the absence of oral anticoagulants or overt liver disease) suggest protein C deficiency, but levels from 55 to 70 percent must be considered borderline, and repeated testing or family studies should be undertaken.224 The use of DNA-based assays to identify patients with hereditary protein C deficiency is not practical because more than 150 different mutations have been described.198
The diagnosis of hereditary protein C deficiency in patients who are receiving warfarin is particularly difficult. Protein C antigen levels can be compared with antigen levels for other vitamin K-dependent clotting factors such as factor VII or X, but only if careful control ranges are established for the ratios of protein C to two other vitamin K-dependent factors are established.3,202 In most situations, it is necessary to wait until at least 2 weeks after the end of anticoagulant therapy for a reliable diagnosis. Warfarin should not be restarted before laboratory results have been returned to reduce the possibility of warfarin-induced skin necrosis in patients who are later found to have protein C deficiency.
PROTEIN S DEFICIENCY
DEFINITION AND HISTORY
Familial heterozygous protein S deficiency associated with venous thrombosis was first reported in 1984.4,5 and 6 Since then, many other families with the disorder have been identified.76,228,229,230,231 and 232 Many different mutations in the protein S gene (over 100) associated with thrombosis have been identified.233 Severe deficiency (less than 1 percent of normal protein S levels) due to homozygous or compound heterozygous defects has been reported in only a few infants who presented with neonatal purpura fulminans.234,235 and 236 Protein S enhances the anticoagulant activity of APC, and hence currently available functional assays of protein S measure APC-cofactor activity using protein S–depleted plasma as substrate. Type I protein S deficiency is defined as parallel reductions in both antigen and anticoagulant activity levels in plasma whereas type II deficiency, associated with circulating dysfunctional molecules, involves normal plasma levels of antigen but low levels of anticoagulant activity. Protein S reversibly associates with the plasma complement factor, C4b-binding protein (C4BP), previously known as proline-rich lipoprotein. In normal plasma, approximately 60 percent of protein S is bound to C4BP, and 40 percent is free; importantly, only the free form of protein S functions as a cofactor for APC. This gives rise to another type of protein S deficiency, designated type III deficiency, in which free protein S is low while total protein S antigen is usually in the low normal range.
ETIOLOGY AND PATHOGENESIS
Protein S is principally synthesized in the liver, but other organs may be important sites for its synthesis, including the endothelium, kidney, testes, and brain (see Chap. 113). Because protein S is a cofactor for APC (see Chap. 113), decreased levels of free protein S may impair the down-regulation of thrombin generation and contribute to hypercoagulability. Protein S also exhibits anticoagulant activity that is independent of APC by directly binding to and inhibiting factors Va, VIIIa, and Xa,237,238,239,240,241 and 242 suggesting that deficiency of protein S could also contribute to hypercoagulability by failing to impair factors Va or Xa in the absence of APC. At present, only the APC-cofactor activity of protein S is routinely assayed because there is no generally available standardized assay for the anticoagulant activity of protein S that is independent of APC.
In several studies, approximately 3 percent of unselected outpatients presenting with venous thromboembolism have low levels of protein S201,243,244; higher prevalences are reported for patients under 50 years of age and for patients with a personal or family history of venous thrombosis. The odds ratio for thrombosis in patients with free protein S deficiency has been variably reported to be 1.6,201 2.4,243 8.5,76 and 11.5230 (see below). After an initial venous thrombosis, recurrence rates in protein S–deficient patients average 3.5 percent per year.212,213,245
Deep venous thrombosis and pulmonary embolism are the most common forms of thrombosis associated with protein S deficiency, although superficial vein thrombophlebitis and thrombosis in unusual sites also occur.210,211 and 212 As in other forms of thrombophilia, about 50 percent of thromboses are unprovoked.211 Arterial thrombosis has been reported in a significant number of protein S–deficient patients, particularly in those who smoke or have other thrombotic risk factors.18,106,246,247 Neonatal purpura fulminans has been reported in rare infants with homozygous or compound heterozygous protein S deficiency and very low levels of protein S.234,235 and 236 Warfarin-induced skin necrosis has also been reported in association with protein S deficiency.248
Acquired forms of protein S deficiency are rather common. Oral contraceptive usage decreases plasma protein S levels. Reduced levels of free protein S are regularly found in pregnancy (e.g., as low as 20 to 30 percent of normal),249,250 in patients who are taking oral anticoagulants, and in disseminated intravascular coagulation, liver disease, nephrotic syndrome, inflammatory conditions, and acute thromboembolism.251,252,253 and 254 Protein S deficiency can also occur in concert with the lupus anticoagulant255,256 and as a result of autoantibodies to protein S following varicella or other infections in children.257,258,259,260 and 261
The likelihood of thrombosis varies widely in patients with protein S deficiency. In general, population-based case control studies yield low odds ratios for thrombosis,201 whereas family studies show a high rate of venous thromboembolism in protein S–deficient relatives compared with nonaffected family members.230 Some of the patients identified in the case control studies may have had an acquired deficiency of protein S which was temporary.224 Even more important, several of the families with protein S deficiency have been found to have a second thrombophilic defect, most commonly either factor V Leiden,106,108 or the prothrombin nt 20210A gene mutation.157 Other risk factors, particularly smoking and obesity, also increase the risk of thrombosis in protein S–deficient family members.18,230,231
Laboratory assays of plasma protein S must be chosen and interpreted with care because the protein circulates both free and bound to C4BP. Moreover, normal ranges differ for males compared with females and depend on age. Free protein S antigen or APC-cofactor anticoagulant activity are better than total protein S antigen in screening for hereditary protein S deficiency.201,262 Free protein S antigen can be assayed using monoclonal antibodies specific for free protein S.263,264 Protein S activity assays may be affected by coexisting APC resistance, although the second-generation assays in which factor V–deficient plasma is used as substrate have improved specificity.265,266 and 267 Assessment of total and free protein S plus protein S activity should allow the classification of patients with protein S defects into types I, II, or III. Type I and type III deficiencies may actually be phenotypic variants of the same disease, because within families, different individuals carrying the same DNA mutation in the protein S gene can present with laboratory findings indicating either type I or type III deficiency.262 Type II deficiency, i.e., normal free protein S antigen with reduced protein S activity, is quite uncommon224 so that screening patients with free protein S antigen levels is clinically reasonable. In normal patients, there is an excellent correlation between free protein S antigen and anticoagulant activity. The lower limit of the normal range for free protein S is lower in females than in males (55 percent versus 65 percent)268; protein S is remarkably sensitive to hormonal status in females.
A coagulation assay for protein S anticoagulant activity independent of APC has been described in which the APTT is determined in the absence and presence of anti–protein S neutralizing polyclonal antibodies added to the test plasma. The APTT is shorter in the presence of antibodies, and the ratio of clotting times is indicative of protein S anticoagulant activity.269 At present, the clinical utility of this interesting assay has not been demonstrated, and the assay is not accessible for routine laboratories.
The high frequency of acquired protein S deficiency makes identification of hereditary defects more difficult. Common acquired conditions leading to low protein S levels should be excluded and tests repeated before making a diagnosis of hereditary thrombophilia. Family studies may also be useful. Oral anticoagulant therapy markedly reduces protein S antigen and activity levels. Assays are not often useful during pregnancy, because the low concentrations of protein S normally seen at that time cause diagnostic confusion.249 Diagnosis of hereditary protein S deficiency using DNA techniques is not favored unless the defect has previously been established in the family because there are numerous different mutations in the protein S gene causing protein S deficiency.
DEFINITION AND HISTORY
Antithrombin, also known as antithrombin III, is a plasma protease inhibitor that neutralizes thrombin by irreversibly forming a 1:1 complex. The rate of inhibition of thrombin or other serine trypsinlike proteases by antithrombin is catalyzed by heparin. The first family with hereditary antithrombin deficiency and thrombosis was reported by Egeberg in 1965.1 Since then, many more families have been described.270,271 and 272 A database of over 250 mutations in the antithrombin gene is available273 and can be accessed via the internet (http://www.med.ic.ac.uk/dd/ddhc). Type I antithrombin deficiency is defined by low levels of antigen and activity in the absence or presence of heparin. Type II deficiency involves the presence of dysfunctional molecules in the plasma and is defined by normal levels of antigen with defects that affect either the inhibitor’s active center, which complexes with the target enzyme’s active site, or the inhibitor’s heparin binding site which mediates heparin-dependent acceleration of antithrombin’s action. Severe deficiency of antithrombin (less than 5 percent) is very rare, involves defects in heparin-dependent enhancement of antithrombin, and is associated with severe venous and arterial thrombosis.274,275,276 and 277 Type I antithrombin deficiency is found in 0.023 percent of normal individuals in Scotland, whereas type II defects, mostly in asymptomatic individuals and families, is much more common and found in 0.16 percent of people screened.278
ETIOLOGY AND PATHOGENESIS
Antithrombin is a major protease inhibitor that neutralizes factors Xa, IXa, XIa, and thrombin in reactions accelerated in the presence of heparin or by heparan sulfate on endothelial surfaces (see Chap. 113). Therefore, defects in antithrombin compromise the normal inhibition of the coagulation pathways and cause a hypercoagulable state. Molecular antithrombin defects can involve either the reactive center that combines with the active site of the coagulation proteases or the heparin binding region that mediates heparin-dependent acceleration of antithrombin-protease reactions (see Chap. 113).
Antithrombin deficiency is found in approximately 1 percent of consecutive outpatients under 70 years old with a first objectively documented venous thrombosis (see references76,77 and 78), and the odds ratio for thrombosis in patients with antithrombin deficiency is approximately 10 to 20 and is notably greater than in subjects with factor V Leiden.76,78,201,279 Recurrence rates have been reported to be quite high in the first year after a thrombosis (12 to 17 percent) in selected patients with type I antithrombin deficiency,213,245 but lower rates have been reported in other studies (about 4 percent per year).211,280 There is no evidence that there are differences in clinical severity between patients with heterozygous type I defects and those with type II mutations involving the thrombin binding site. Mortality rates are not increased in these patients.281,282 Patients with type II mutations of the heparin-binding site have few if any thrombotic episodes, although homozygous mutations affecting heparin binding are associated with thromboembolism.280
Venous thrombosis of the lower extremities, which occurs at an early age and peaks in the second decade of life, is the most common symptom in antithrombin deficiency.280 Superficial venous thrombosis appears to be somewhat less common than in protein C or protein S deficiency, or in APC resistance.76,210,211 Thrombosis in unusual sites such as the mesenteric or cerebral veins has been reported.210,211 Arterial thrombosis occurs infrequently (about 1 percent of affected patients).283 As previously indicated for patients with other forms of hereditary thrombophilia, gene-gene and gene-environment interactions markedly increase the risk of thrombosis in subjects with antithrombin deficiency by 5-fold and 20-fold respectively.279 Patients with severe antithrombin deficiency, i.e., activity levels less than 5 percent, are exceedingly rare, most likely because the profound deficiency state causes fetal loss in utero. A few infants with homozygous defects involving the heparin-binding region of the molecule have survived, but most have suffered severe venous and arterial thrombosis.274,275,276 and 277 No patients homozygous for reactive center defects have been identified, leading to the speculation that complete deficiency of antithrombin is incompatible with life.
Resistance to the anticoagulant effects of heparin has been observed in some patients with antithrombin deficiency. However, heparin resistance is quite common in general patients with thrombosis. Up to 40 percent of patients without antithrombin deficiency will require more than 40,000 units of heparin daily to prolong the APTT into the therapeutic range.284 Both acute thrombosis and several days of heparin therapy can decrease antithrombin levels, occasionally to as low as 50 percent of normal, which may lead to an erroneous diagnosis of hereditary antithrombin deficiency.285,286 Acquired conditions leading to lowered levels of antithrombin are common and include liver disease, DIC, nephrotic syndrome, chemotherapy with asparaginase, and preeclampsia.287,288,289,290 and 291
Antithrombin deficiency screening assays should first be performed in the presence of heparin because defects may involve either the reactive center of the inhibitor or the heparin-binding site. If initial results are abnormal, then assays that measure the ability of the inhibitor to neutralize thrombin in the absence of heparin (progressive antithrombin activity) should be done to characterize the abnormality. Antithrombin activity assays that utilize a chromogenic substrate are widely available.292 Most laboratories now use factor Xa or bovine thrombin in their antithrombin assays to avoid the inhibitory effects of heparin cofactor II on human thrombin.293,294 The normal range for antithrombin levels in normal plasma is quite narrow (i.e., 84 to 116 percent).293 Antithrombin antigen measurements are used to help distinguish type I from type II defects. Crossed immunoelectrophoresis using an antithrombin antibody in the presence and absence of heparin can help identify defects in the heparin-binding portion of the molecule.295
In general, patients with type I deficiency and many of those with type II disorders involving the thrombin binding site will have antithrombin activity levels of 40 to 60 percent. Levels of 60 to 84 percent can be due to other type II defects but frequently are a result of acquired antithrombin deficiency such as occurs with mild liver disease, acute thrombosis, or heparin therapy. If these confounding conditions are present, measurement of levels should be repeated and family studies performed if possible.
HIGH LEVELS OF FACTOR VIII AND OTHER COAGULATION FACTORS
DEFINITION AND HISTORY
Based on analysis of the frequency of factor VIII levels that exceed 150 percent of normal values, an elevated factor VIII level has been defined as a significant independent risk factor for venous thrombosis.296,297 Both factor VIII activity and antigen levels are increased.297 The increased risk was similar to that of heterozygosities for factor V Leiden or prothrombin G20210A. Preliminary reports of other epidemiologic studies indicate that elevated levels (higher than 150 percent of normal) of factors XI, IX, X, and V are also risk factors for venous thrombosis.
ETIOLOGY AND PATHOGENESIS
Although factor VIII is an acute phase reactant and elevations can be caused by inflammation, it appears that factor VIII elevations in venous thrombosis patients are not commonly caused by systemic inflammation.297,298 Therefore, elevated factor VIII levels are likely to be directly pathogenic by increasing coagulability of blood via the blood coagulation pathways. Studies of factor VIII levels in different families indicate a significant genetic influence in addition to the known influences from levels of von Willebrand factor, blood group antigens, and the presence of inflammation. Elevations of factor VIII and other coagulation factors of the intrinsic coagulation pathway, e.g., factors XI, IX, X, or V, may contribute to hypercoagulability by increasing thrombin generation.
The clinical presentation of patients with elevated factor VIII levels is not known to differ from that of patients with the other genetic risk factors described in this chapter.
Factor VIII procoagulant activity is measured with routine coagulation assays commonly used to screen for hemophilia (see Chap. 112 and Chap. 123). In venous thrombosis patients, factor VIII antigen levels correlate with activity measurements.297
HEREDITARY THROMBOTIC DYSFIBRINOGENEMIA
DEFINITION AND HISTORY
Dysfibrinogenemia is defined as a qualitative defect in the molecule due to a mutation in the gene for one of fibrinogen’s polypeptide chains. The hereditary dysfibrinogenemias represent a heterogeneous group of abnormalities that can be asymptomatic or cause either thrombosis or bleeding. Initial reports of dysfibrinogenemias associated with thrombophilia appeared in the 1960s from several laboratories. For a detailed treatment of dysfibrinogenemia, see Chap 124.
ETIOLOGY AND PATHOGENESIS
For normal hemostasis, fibrin is formed after release of fibrinopeptides from fibrinogen due to proteolysis by thrombin and subsequent polymerization of fibrin monomers. Fibrin is then stabilized by covalent cross-links introduced by factor XIIIa. Plasmin-dependent proteolysis of fibrin either to limit formation or growth of a thrombus or to clear fibrin in a timely and normal fashion during healing is essential. Defects in fibrinogen that cause abnormal fibrinolysis cause thrombosis, either because fibrin is not cleared in a normal fashion or because the growth of a normal hemostatic plug is not limited. Specific defects causing hypofibrinolysis can involve alterations of plasmin cleavage sites in fibrin or of sites that promote assembly of components of the fibrinolytic system, e.g., binding sites for plasminogen or plasminogen activators (see Chap. 124).
Patients with hereditary thrombotic dysfibrinogenemias usually present with venous thrombosis at a young age (e.g., 27 to 32 years old).299 An occasional patient will have both thrombosis and bleeding (usually postpartum hemorrhage).299 An increased rate of spontaneous abortion and stillbirth is also observed.299 Approximately 20 percent of reported cases of hereditary dysfibrinogenemia have been associated with thrombosis, whereas about 30 percent manifested bleeding, and the remainder were clinically silent.299 As summarized in detail in Chap. 124, several dozens of reports of abnormal fibrinogens associated with thrombosis have appeared.300 When a large number of patients presenting with thromboembolism were screened, the prevalence rate of dysfibrinogenemia was found to be 0.8 percent.299
Prolongation of a dilute thrombin time and/or reptilase time due to delayed fibrin polymerization is common with dysfibrinogenemia, as is a disparity between measurement of immunoreactive and clottable fibrinogen. More sophisticated testing often demonstrates abnormal fibrinogen structure or resistance of the fibrin to fibrinolysis. Unfortunately there are no assays readily available that measure the key properties of fibrinogen that are likely to cause thrombosis in patients with dysfibrinogenemia, and therefore this defect may be underdiagnosed.
Therapy relies on anticoagulants. In some instances, the administration of cryoprecipitate as a source of normal fibrinogen should be considered for surgical procedures, both to raise low concentrations of fibrinogen into a hemostatic range and possibly to reduce the risk of thrombosis by dilution of the abnormal prothrombotic fibrinogen.
OTHER POTENTIAL THROMBOPHILIC DISORDERS
Hereditary defects in the fibrinolytic system (see Chap. 116) and in thrombomodulin are potential thrombophilic risk factors. Japanese families with several hereditary dysplasminogenemias301,302 and 303 and hypoplasminogenemias304 have been identified, but these abnormalities were not associated with thrombosis in subjects other than the propositus. Heterozygous plasminogen deficiency is found more often in Asian than in Caucasian populations. One kindred with elevated levels of plasminogen activator inhibitor 1 (PAI-1)and thrombosis was subsequently shown to have protein S deficiency.231 As yet, an association between defects in the fibrinolytic system and thrombosis has not been firmly established.305 Several mutations in the thrombomodulin gene have been discovered in families with thrombosis.306,307 and 308 The genetic defects are scattered throughout the thrombomodulin gene and are associated with variable levels of soluble thrombomodulin in the plasma.307 In aggregate, thrombomodulin defects appear to be relatively common, being found in approximately 5 percent of a group of 200 patients with thromboembolic disease.307 It is not yet clear whether one or more of these mutations constitute a major risk factor, whether they may contribute to the risk of thrombosis in patients with other forms of thrombophilia such as protein S deficiency, or whether they are neutral polymorphisms. At this junc ture, routine screening of thrombosis patients for fibrinolytic or thrombomodulin defects is probably not indicated.
DIAGNOSIS OF THROMBOPHILIA
Plasma or molecular assays are now widely available for each of the common hereditary hypercoagulable states (Table 127-2). Up to 50 percent of patients presenting with a first deep vein thrombosis (DVT) will be found to have an abnormal laboratory test suggesting a thrombophilic defect. Those with recurrent venous thromboembolism or a strong family history of thrombosis are even more likely to have evidence of thrombophilia. Multiple disorders in the same patient are common; e.g., in one series of patients, 16 percent had more than one type of thrombophilia.102
TABLE 127-2 LABORATORY EVALUATION FOR HEREDITARY THROMBOPHILIA
Comprehensive testing for patients with venous thromboembolism should include: an APC resistance test (followed by a factor V Leiden mutation if needed), prothrombin gene mutation analysis, plasma homocysteine concentration, protein C activity (by a clotting assay), protein S activity assay (or free protein S antigen), antithrombin activity, factor VIII activity assay, and fibrinogen concentration (clottable) with a dilute thrombin time (+/– reptilase time) (see Table 127-2). Additional tests for antiphospholipid antibodies should also be considered in patients suspected of having acquired thrombophilia (see Chap. 128). The most appropriate tests for patients with arterial thrombosis are less clear. However, plasma homocysteine, antiphospholipid antibody studies, lipoprotein(a) concentration, and colony assays to search for covert myeloproliferative disorders should be considered. Factor V Leiden, the prothrombin gene mutation, protein S, and other tests for “venous” thrombophilia may prove useful in some patients with premature coronary heart disease or stroke, particularly if other risk factors are present such as smoking, hypertension, diabetes, or obesity. If test results for the more common disorders are normal but the likelihood of a familial hypercoagulable state is high, tests for other causes of thrombosis might be helpful. Experimental tests to consider are assays for elevated levels of factors XI, V, and IX, the 4G4G promoter polymorphism in the PAI-1 gene, a plasminogen activity assay, or perhaps molecular assays for defects in thrombomodulin.
A laboratory evaluation for thrombophilia should be considered if the results of testing could make a difference in the clinical care of the patient or family members. Examples of a potential clinical impact include:
Changes in the duration or intensity of oral anticoagulant therapy
Administration of specific therapy (e.g., antithrombin concentrates, vitamins for homocysteinemia)
More intense prophylaxis for high-risk situations (e.g., surgery, acute illness, immobility)
Better accuracy in estimates of the future risk of thrombosis in clinical settings (e.g., surgery or pregnancy)
Counseling of women as to the risks of oral contraceptives, pregnancy, or hormone replacement therapy
Study of family members at risk of thrombosis
Routine testing of all female family members of a patient found to have factor V Leiden prior to starting oral contraceptives is probably not indicated.75,309 However, if the family has a strong family history of venous thromboembolism in women who were pregnant or taking birth control pills or if the patient has had an episode of venous thrombosis, then test results could help in deciding whether to recommend oral contraceptives or alternative methods of birth control.309
Children with venous or arterial thrombosis (particularly stroke) are also likely to have an underlying thrombophilic disorder. Of these defects, factor V Leiden is the most common, but other disorders including the prothrombin 20210A gene mutation, protein C deficiency, elevations in lipoprotein(a), and antiphospholipid antibodies have been reported.99,100,310,311,312 and 313
Laboratory testing is best performed several weeks after completion of a course of oral anticoagulants in patients with thrombosis, to avoid confounding effects of acute thrombosis or heparin or warfarin therapy on the assay results. However, stopping anticoagulants in some patients with a high risk of recurrent thromboembolism may not be advisable. With the exception of assays for protein C and protein S, all other thrombophilic factors can be assayed in patients taking oral anticoagulants. Options for assessment of protein C or protein S levels in patients requiring warfarin include comparing their relative antigen levels with other “benchmark” vitamin K-dependent clotting factors3,202 or obtaining assays on family members. Alternatively, heparin or low-molecular-weight (LMW) heparin can be substituted for warfarin for a period of time (approximately 2 weeks) prior to drawing blood for analysis.
THERAPY OF THROMBOPHILIA
Thrombophilia patients who develop a DVT or PE are initially given standard venous thromboembolism treatment with heparin or LMW heparin for acute therapy and warfarin for longer-term protection. Warfarin has been shown to effectively prevent recurrent thromboembolism at an INR range of 2 to 3.314,315 Higher intensities of warfarin are unnecessary and will increase the risk of bleeding. The absolute risk of major hemorrhage with oral anticoagulants in patients with venous thromboembolism averages 1 to 3 percent per year, with a fatality rate of 0.2 to 0.4 percent per year.314,316
The optimal duration of anticoagulant treatment in patients with thrombosis and a history of thombophilia is an important clinical question.314,317 Warfarin therapy is usually given for 6 months315 following a thrombotic event (see Chap. 132). However, longer treatment may be indicated in patients with hereditary thrombophilia if the risk of additional thromboemboli substantially outweighs the risk of bleeding due to oral anticoagulants. The lack of reliable data on the absolute risks of recurrent thrombosis in patients with one or more thrombophilic states makes clinical decision making more difficult.314,317 However, several factors make recurrent thrombosis more likely, and, if present, longer-term treatment is more appropriate:
A spontaneous rather than a provoked thrombosis or pulmonary embolism316
A high odds ratio for thrombosis; e.g., antithrombin deficiency (OR = 8) versus factor V Leiden (OR = 2.5)
A strong family history of thrombosis (suggesting the presence of multiple hereditary defects)
A history of recurrent thromboses
Multiple inherited or acquired risk factors (e.g., factor V Leiden + prothrombin G20210A gene mutation; or factor V Leiden + antiphospholipid antibodies)
Permanent rather than temporary major risk factors
Unusual or life-threatening thromboses (e.g., cerebral vein or mesenteric thrombosis; ileofemoral deep venous thrombosis with multiple pulmonary emboli).
Decisions as to long-term anticoagulant therapy are best tailored to individual patients. A thorough assessment should include: (1) an estimate of the future risk of thrombosis; (2) an estimate of the future risk of major or fatal hemorrhage; and (3) patient preferences (e.g., impact of the decision on occupational or social situations).
Prophylactic oral anticoagulation therapy is usually not warranted in subjects who have not yet suffered a thrombotic event but who are discovered to have hereditary thrombophilia because of family testing or some other reason. In this instance, the risks of hemorrhage due to warfarin (about 1 to 3 percent per year) clearly outweigh the risk of thrombosis (e.g., 0.4 percent per year in asymptomatic individuals with APC resistance). In contrast, most authorities would recommend long-term antithrombotic treatment for patients who have suffered recurrent thromboses and who have more than one hereditary or acquired hypercoagulable state. More clinical trial data are needed before persuasive clinical guidelines can be recommended for patients with narrower risk/benefit ratios; e.g., a young patient with a first spontaneous but extensive DVT and a single prothrombotic disorder such as the factor V Leiden mutation.
If oral anticoagulant therapy is not used, an alternative approach includes intensive antithrombotic prophylaxis (e.g., LMW heparin) for events with a high risk of thrombosis such as surgery, infectious (e.g., pneumonia) or inflammatory diseases (e.g., inflammatory bowel disease), or prolonged periods of inactivity. Effective prophylaxis should reduce the risk of thromboembolism by about half, since approximately 50 percent of thromboses in patients with hereditary hypercoagulable states can be attributed to a known provoking factor. Other recommendations include patient education as to the signs and symptoms of acute DVT or PE, facilitation of diagnostic testing should symptoms occur, and continued follow-up in case new laboratory tests or clinical recommendations appear in the future.
Specific therapies are available for some thrombophilic disorders. Antithrombin concentrates are now widely available and can be administered for surgery, major trauma, and at the time of delivery in patients with antithrombin deficiency.318,319 and 320 Protein C and activated protein C concentrates are under development and, when available, may be useful in infants or children with homozygous protein C deficiency, or in heterozygous subjects during surgery or other major stresses.321,322 and 323 Cryoprecipitate is a source of normal fibrinogen, which can be useful for replacement of normal fibrinogen in patients with hereditary dysfibrinogenemia. Finally, although not yet proved to prevent thrombosis in patients with hyperhomocysteinemia, B vitamins (folic acid, pyridoxine, B12) effectively lower homocysteine concentrations into the normal range.171,172
THROMBOPHILIA AND PREGNANCY, ORAL CONTRACEPTIVES, AND HORMONE REPLACEMENT THERAPY
MANAGEMENT OF PREGNANCY IN THROMBOPHILIC WOMEN
Pregnancy substantially increases the risk of thrombosis in women with thrombophilia.324 To date, treatment guidelines supported by clinical trial data for the treatment of these women are not available. Screening all women for a thrombophilic state prior to pregnancy does not seem warranted based on an extremely high cost:benefit ratio.325,326 Similarly, routine heparin prophylaxis for previously asymptomatic individuals known to carry the V Leiden gene mutation or other mild thrombophilic defects is not indicated. Prophylactic heparin (or LMW heparin if proved safe and effective in pregnancy) should be considered for women with a history of venous thromboembolism, particularly if the prior thrombosis was related to pregnancy or oral contraceptives.324,327,328 The potential antithrombotic benefit should offset the risks of heparin-induced osteopenia, bleeding, or heparin-induced thrombocytopenia. Venous thromboembolism that occurs during pregnancy requires therapeutic doses of heparin for the remainder of the pregnancy, followed by postpartum anticoagulants for at least 4 to 6 weeks.324 Antithrombin concentrates (along with low-dose heparin) should be considered for women with hereditary antithrombin deficiency during the peripartum period or during complications of pregnancy.
PREGNANCY LOSS DUE TO THROMBOPHILIA
Thrombophilia is a cause of fetal loss and other complications of pregnancy, most likely due to thrombosis of the placental vasculature.326,329 Fetal loss is often manifested by stillbirth (second or third trimester) rather than first trimester miscarriage.329 In addition, severe preeclampsia, fetal growth retardation, and placental infarction have all been linked to maternal or fetal hypercoagulable states.330,331,332,333 and 334 Most of the hereditary thrombophilic states have been implicated. Factor V Leiden has been linked to fetal loss (OR 2–3),329,335,336,337 and 338 as have protein C deficiency (OR 2.3), protein S deficiency (OR 3.3), and antithrombin deficiency (OR 5.2).329 If combined defects are present, the odds ratio for fetal loss increases to 14.3.329 Hyperhomocysteinemia has been associated with placental abruption, placental infarction, and stillbirth; moreover, homozygosity for the thermolabile MTHFR defect has also been linked to pregnancy complications.330,331,334,339 Although quantitative data are not available, pregnancy loss appears to be increased in women with hereditary thrombotic dysfibrinogenemia.299
When adverse outcomes of pregnancy were combined, including stillbirth, preeclampsia, abruptio placentae, and fetal growth retardation, 52 percent of affected women were found to have thrombophilia compared to a rate of 17 percent in women with normal pregnancies.330 Diagnostic studies for thrombophilia should be considered for women with recurrent midtrimester pregnancy loss or other adverse pregnancy outcomes, particularly if future studies suggest that antithrombotic treatment (e.g., low-dose heparin, LMW heparin, or aspirin) is effective.340
ESTROGENS: ORAL CONTRACEPTIVES AND HORMONE REPLACEMENT THERAPY
Oral contraceptives increase the risk of thrombosis in women with hereditary thrombophilia.118,119,341,342 and 343 For example, the odds ratio for thrombosis in women with factor V Leiden who use third-generation oral contraceptives is increased 30- to 50-fold.118,119 In absolute numbers this represents an increase in risk from 1/12,500 women per year without V Leiden to 1/400 women per year in women with factor V Leiden.119 The thrombotic risk associated with birth control pills is greater in women who are homozygous for factor V Leiden.120 Screening for the factor V mutation prior to the administration of oral contraceptives is probably not cost-effective. By one estimate, it would be necessary to screen 2.25 million women to detect 90,000 women with V Leiden, in order to prevent one death from venous thromboembolism by withholding the oral contraceptives.344 However, if a woman with factor V Leiden has a history of thrombosis or is homozygous for the mutation, then avoidance of oral contraceptives would certainly be prudent.119,120,345 Oral contraceptives probably should not be recommended for women known to be deficient in antithrombin, protein C, and possibly protein S.341
Whether to recommend hormone replacement therapy in women with hereditary thrombophilia is a particularly difficult question. The relative risk of venous thrombosis with replacement estrogens is significantly increased by a factor of 2 to 4 when large groups of women are studied, but the absolute risk of thrombosis is quite low; i.e., 1 excess thrombosis per 5000 women per year.346,347,348,349 and 350 However, studies are not yet available to estimate any potential increase in the relative or absolute risk of thrombosis in women who use hormone replacement therapy and carry the factor V Leiden mutation or other genetic thrombophilic risk factors.
Use of selective estrogen receptor modulators such as tamoxifen or raloxifene increases the risk of venous thrombosis. Three cases of tamoxifen-associated venous thrombosis associated with factor V Leiden have been reported.121 Given the increasing use of selective estrogen receptor modulators for treating or preventing breast cancer and osteoporosis, it will be important to determine whether thrombophilic genetic risk factors (e.g., those listed in Table 127-1) will increase the liklihood of selective estrogen receptor modulator–associated venous thrombosis.
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn