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Activated protein C resistance, the factor V Leiden mutation, and a laboratory testing algorithm. (Original Articles).

ACTIVATED PROTEIN C RESISTANCE AND FACTOR V LEIDEN

The Protein C/Protein S Anticoagulation Pathway

The pathway to inhibit fibrin formation by degrading selected coagulation factors is as complex as the cascade leading to the conversion of fibrinogen to fibrin (Figure 1). (1) Thrombin (factor IIa), the same factor that converts fibrinogen to fibrin and activates platelets, is also responsible for initiating the pathway to inhibit fibrin formation. Thrombin binds to thrombomodulin on the blood vessel wall. When thrombin is bound to thrombomodulin on the surface of an endothelial cell, it becomes activated and can convert protein C to its activated form (activated protein C). Activated protein C is only effective when it is bound to its cofactor, protein S. Protein S is available as a cofactor for protein C only when it is not bound to C4b binding protein. In the basal state, approximately 40% of protein S is free (unbound) and thereby available to serve as a cofactor for activated protein C. The activated protein C/ protein S complex degrades factors Va and VIIIa, and their loss is associated with a decrease in fibrin formation and, therefore, a reduction in the ability to form a fibrin clot. Although not shown in Figure 1, factor V itself acts as a cofactor for activated protein C/protein S in the degradation of factor VIIIa. (2) In addition, a receptor on the endothelial cell, called the endothelial cell protein C receptor, enhances protein C activation by the thrombin-thrombo-modulin complex. (3)

[FIGURE 1 OMITTED]

Activated Protein C Resistance

Activated protein C normally degrades activated factors Va and VIIIa by proteolytic cleavage at specific arginine residues. Individuals with activated protein C resistance have a mutated factor V, such that it is resistant to degradation by activated protein C. (4-7) More than 95% of cases are due to a point mutation, known as the factor V Leiden mutation, at 1 of the 3 arginine cleavage sites in the factor V gene. (4) Two additional, very rare factor V mutations at other arginine cleavage sites have been identified recently, factor V Hong Kong (8) and factor V Cambridge. (9) Factor V Cambridge can cause activated protein C resistance, but factor V Hong Kong has not caused activated protein C resistance in the few cases reported to date. (8,10) Other factor V mutations are also under investigation. (11) Mutations in the factor VIII gene causing resistance to activated protein C are theoretically possible but have not yet been described. Very rarely, activated protein C resistance with thrombosis has developed as the result of an autoantibody (inhibitor) against activated protein C, without an underlying genetic mutation in factor V. (12)

The Clinical Significance of Activated Protein C Resistance

The factor V Leiden mutation is present in 3% to 5% of the general white population in heterozygous form. (13-17) It is less common or rare in other races and ethnic groups, especially those of African or Asian ancestry. The presence of the factor V Leiden mutation confers a genetic risk for thrombosis, which is primarily venous. (18,19) The risk for venous thrombosis is approximately 3- to 10-fold in individuals who are heterozygous for the factor V Leiden mutation. Because the mutation is present in as many as 1 of 20 in the white population, homozygotes are not rare. Homozygous individuals have been reported in different studies to have an approximately 80-fold risk over baseline for thrombosis. (19) In the population of all patients with a venous thrombosis, which includes deep vein thrombosis and pulmonary embolism, approximately 20% of cases are positive for the factor V Leiden mutation. Among the population of individuals who have a family history of thrombophilia, approximately 50% have the factor V Leiden mutation. (6,7,20) Thus, this particular mutation accounts for a significant percentage of people with a thrombotic event or a family history of thrombosis.

Individuals who are positive for the factor V Leiden mutation in heterozygous or homozygous form often need the presence of a second risk factor, which can be genetic or acquired, to produce a thrombotic event. (21) Many of the acquired causes are well known, and include malignancy, trauma, surgery, the use of oral contraceptives or estrogen replacement therapy, and the presence of an antiphospholipid antibody, either as a lupus anticoagulant, an anticardiolipin antibody, or both. Patients who are double heterozygotes for the factor V Leiden and prothrombin G20210A, another high-incidence mutation in the white population, have a further increased risk for thrombosis. (22) The presence of elevated homocysteine, occurring as a result of a genetic cause or an acquired cause (such as low levels of vitamins [B.sub.6], [B.sub.12], or folate), can further increase the risk of thrombosis with the factor V Leiden mutation. (23) The thrombosis risk with the factor V Leiden mutation also increases with age. (24) The increased risk associated with oral contraceptive use combined with the factor V Leiden mutation is synergistic rather than additive. (25) The risk of arterial thrombosis with the factor V Leiden mutation is uncertain, but it appears that factor V Leiden may be more prevalent among myocardial infarction patients who do not have atherosclerosis and/or young patients with certain other risk factors (smoking, hypertension, obesity, high cholesterol, or diabetes) when compared with control groups. (26-28) The incidence and severity of thrombosis with heparin-induced thrombocytopenia did not appear to be affected by the presence or absence of factor V Leiden. (29)

A few studies suggest that among individuals with factor V Leiden, those with type O blood may have less risk for thrombosis than individuals with type A, B, or AB blood. (30,31) One study found that although factor V Leiden was associated with an increased incidence of deep vein thrombosis overall, the incidence of iliofemoral deep vein thrombosis was actually significantly decreased. (32) The thrombi with factor V Leiden were predominantly distal to the iliofemoral veins. Since pulmonary emboli arise more often from thrombi in the iliofemoral veins than from more distal thrombi, this may help explain why the prevalence of factor V Leiden/activated protein C resistance is lower among patients with isolated pulmonary embolism (8.9%) than in patients with isolated deep vein thrombosis (18.8%). (33)

Treatment of Thrombosis With Factor V Leiden

Individuals with factor V Leiden who develop venous thrombosis are typically treated with heparin followed by oral anticoagulation medications, as are most individuals with venous thrombosis. The recommended duration of anticoagulation therapy does not appear to have reached consensus, and the duration of anticoagulation treatment should be individualized for each patient. (34) A first idiopathic venous thrombosis is usually treated with at least 6 months of anticoagulation therapy. A first event with a reversible risk factor, such as estrogen use, injury, surgery, or immobilization, is typically treated with at least 3 months of anticoagulation therapy (with or without factor V Leiden). For recurrent events, 12 months or indefinite anticoagulation treatment is recommended. (34) Homozygous factor V Leiden is a stronger risk factor than heterozygous factor V Leiden, which should be taken into consideration when deciding the appropriate duration of anticoagulation.

CLINICAL TESTING FOR ACTIVATED PROTEIN C RESISTANCE AND FACTOR V LEIDEN

The Clot-Based Tests for Activated Protein C Resistance

The original test for activated protein C resistance that was offered for use in clinical laboratories was a partial thromboplastin time (PTT) performed in the presence and absence of exogenously supplied activated protein C. (4,20) In normal patients, the activated protein C degrades the patient's factor Va and VIIIa, and on that basis, prolongs the PTT. In patients with a factor V Leiden mutation, the degradation of factor Va does not occur to the same extent and, therefore, the PTT does not become as prolonged. The ratio of the PTT with activated protein C versus the PTT without activated protein C is calculated. In this original test for activated protein C resistance, normal individuals typically had a ratio of 2.0 or greater, and individuals with factor V Leiden typically had a ratio less than 2.0. (It has been recommended that each laboratory determine its own cutoff value.) However, there was considerable overlap between normal subjects and heterozygotes in the 2.0 to 3.0 range of values. (35) The separation of those with the Leiden mutation from those without the Leiden mutation was poorly optimized, leading to sensitivities for this assay of 50% to 86% and specificities of 75% to 98%. (35-37) In addition, the original assay was plagued by inaccuracies in patients who had an abnormal baseline PTT. Any cause of an elevated PTT, such as a deficiency of factors VIII, IX, XI, or XII, or a lupus anticoagulant, had an interfering effect on this original activated protein C resistance assay. A shortening (as opposed to a prolongation) of the PTT from a factor VIII elevation also produced an interference in this original assay. Platelet contamination of plasma specimens can alter the result if specimens are subsequently frozen then thawed. (38,39)

The limitations in the originally devised assay for activated protein C resistance led to the development of a modified assay. The modified assay has shown sensitivity and specificity values for detection of factor V Leiden that approach 100%. (40) The basis for the modification is that the patient plasma is first diluted 1:5 with factor V--deficient plasma. The presence of the factor V--deficient plasma provides all factors except factor V to offset any PTT-related factor deficiencies and to minimize the effect of an elevation of factor VIII, which would shorten the PTT. The modified assay also contains polybrene, which neutralizes unfractionated heparin (up to 1 IU/mL) or low-molecular-weight heparin in the specimen. Protein S (Figure 1) is a required cofactor for activated protein C. In the original assay for activated protein C resistance, a protein S level less than 20% could produce a false-positive test. (41) In the modified assay, it might be expected that the factor V--deficient plasma into which the patient plasma is diluted would supply sufficient exogenous protein S to counteract the effect of any protein S deficiency in the patient. However, some reports show that protein S can become inactivated during the commercial preparation of factor V--deficient plasma. Therefore, low protein S levels could influence the result if the factor V-deficient plasma reagent used in the assay is deficient in protein S. (42)

One potential major interference in the modified assay is the lupus anticoagulant. Because the modified screening assay is PTT-based, the lupus anticoagulant is capable of producing an interference in the assay. Patients with a lupus anticoagulant should be evaluated directly with a genetic assay to determine if the factor V Leiden mutation is present. Alternatively, the screening assay results are improved if there is a higher dilution of the specimen into factor V-deficient plasma. In one study, the screening assay correlated with the genetic assay when a 1:40 dilution was used instead of 1:5, due to dilution of the lupus anticoagulant. (43) Other researchers have found that adding phospholipid can neutralize the lupus anticoagulant interference in the screening assay. (44)

Other variations of the activated protein C resistance assay have been developed. One reported variation uses a prothrombin-based factor V assay instead of a PTT-based assay, along with factor V--deficient plasma. (45) The major advantage of this assay is that lupus anticoagulants typically do not produce an interference. Another activated protein C resistance assay is a modified Russell viper venom time test. (38,46) In this assay, there is a high concentration of phospholipid that renders the assay largely insensitive to lupus anticoagulants. A factor Xa--based assay has also been developed, which includes factor V--deficient plasma, to assay for activated protein C resistance. (47)

For the first 6 months of life, the reference range for the original assay for activated protein C resistance was found to be higher than in individuals older than 6 months. (48) A modification of the assay for these infants was developed. It involved a 1:11 dilution of the patient plasma in factor V--deficient plasma, (49) in contrast to the 1:5 ratio for adults. The higher dilution in newborns correlated better with the results from DNA analysis than did the 1:5 dilution. (49)

A theoretical disadvantage to the use of factor V--deficient plasma in the modified assay is that if indeed a patient appears with a genetic mutation in the factor VIII gene that impairs its degradation by activated protein C, this abnormality will not be detected in the modified assay. Factor V--deficient plasma contains normal factor VIII molecules and, therefore, its use will mask any mutation in the patient's factor VIII that allows it to resist degradation by activated protein C. However, as noted, at the current time, this is only a theoretical disadvantage because no such factor VIII mutations have been identified.

One method of reporting a result for activated protein C resistance involves the use of a normalized ratio. This ratio is produced by dividing the result of the activated protein C resistance assay for the patient by the result of the activated protein C resistance assay for normal pooled plasma. Although this may reduce intralaboratory or interlaboratory variability in the assay, it does not appear to significantly improve the diagnostic efficiency for activated protein C resistance (50-52); therefore, the extra calculation does not appear to be necessary.

The original assay, despite its limitations, is undergoing evaluation to determine whether the numerical test result for activated protein C resistance provides information regarding hypercoagulability in the absence of factor V Leiden by a DNA-based method. In the absence of the factor V Leiden mutation, a result in the low end of the reference range correlated with a 2.5-fold increased risk for venous thrombosis, after adjusting for factor VIII levels. (53) However, at present, it would be difficult to interpret or apply this knowledge in the care of an individual patient.

DNA Analysis for the Factor V Leiden Mutation

The gold standard assay for the factor V Leiden mutation involves the use of polymerase chain reaction (PCR) methodology. (52,54) The factor V Leiden mutation is a point mutation at nucleotide position 1691, which results from replacement of a guanine residue with an adenine residue. This alteration in the gene leads to a substitution of arginine with glutamine at amino acid residue number 506. The change in the nucleotide at position 1691 eliminates an MnlI restriction site. The sample used for this assay must be whole blood, and not plasma, because cellular material with DNA is required for the assay. A common mistake in test ordering is to submit a plasma specimen (which has essentially no cells) and request a genetic test.

In the PCR assay, after DNA is isolated from whole blood, the mutation site is amplified by PCR, and the PCR product is digested with MnlI. The presence of the factor V Leiden mutation is indicated by the absence of an MnlI site at position 1691. The testing by PCR allows heterozygotes and homozygotes to be identified specifically. It is not recommended to omit the clot-based screening test (using factor V--deficient plasma) and directly evaluate patients with a genetic test for factor V Leiden, because the genetic tests are much more costly and labor intensive than the clot-based screening assay.

Other DNA-based assays for factor V Leiden have been described. One of the most commonly used commercially available methods, other than PCR, is an invasive signal amplification reaction (Invader assay, Third Wave Technologies, Inc, Madison Wis). (55) DNA is first isolated in the same manner as for PCR. The DNA is subsequently mixed in a microtiter plate with 2 DNA probes. One probe binds only to wild-type (normal) factor V DNA at nucleotide position 1691. The second DNA probe binds only to factor V Leiden DNA at the same nucleotide position 1691. In simplest terms, the detection system generates a fluorescent signal when a probe is bound to the target DNA with no mismatch at position 1691. With wild-type individuals, the wild-type probe binds to nucleotide 1691, but the Leiden probe does not. With patients homozygous for factor V Leiden, the Leiden probe binds to nucleotide 1691, but the wild-type probe does not. With patients heterozygous for factor V Leiden, both probes bind. In one study involving 372 samples, this assay correctly identified 95.8% of 48 heterozygotes and 100% of the 316 wild-type and 7 homozygous individuals (99.5% for the 3 groups overall) as compared with PCR. (56) In a smaller study involving 48 samples, the Invader assay correctly identified all 48 specimens as compared with PCR (30 wild type, 16 heterozygous, 2 homozygous). (57) In a larger study involving 1369 individuals, the Invader assay correctly identified 100% of 102 heterozygous and 3 homozygous individuals, and 99.7% of the 1264 wild-type individuals, compared with PCR. (58) In these 3 studies, DNA was quantified prior to analysis to ensure that sufficient DNA was present.

Diagnostic Algorithm for Assessment of Activated Protein C Resistance and Factor V Leiden

Figure 2 shows an algorithm for the diagnosis of activated protein C resistance and the factor V Leiden mutation. In the initial box, the command is to perform the screening test for activated protein C resistance. The second box asks the question about whether the patient has a lupus anticoagulant. Independent of whether the activated protein C resistance assay is the original one or a modified one, as just discussed, the lupus anticoagulant can interfere with the screening assay for activated protein C resistance (as noted in the third box on the far left side of the algorithm). As noted, this situation mandates the performance of a genetic test for the factor V Leiden mutation to determine if the mutation is absent, present in heterozygous form, or present in homozygous form. The middle boxes of the algorithm ask whether the activated protein C resistance screening assay is the original one or the modified one involving the use of factor V--deficient plasma. If the assay is the original one and does not involve the use of factor V--deficient plasma, there are many interferences as noted earlier, including those which increase the PTT (and possibly the prothrombin time as well), which can produce false-positive or false-negative results in the activated protein C resistance assay. For that reason, if an abnormal result were detected using the original assay, it would be highly recommended to repeat the assay using the modified test. If the modified test is performed and the result is normal, because the sensitivity and specificity approximate 100%, genetic testing for the factor V Leiden mutation is not necessary. If on the other hand, there is an abnormality in the modified activated protein C resistance assay, then a genetic test (PCR is the most commonly used test at this point) for the factor V Leiden mutation is performed.

[FIGURE 2 OMITTED]

SUMMARY

The assessment for activated protein C resistance is initiated by performance of a screening test using factor V-deficient plasma as a diluent for the patient plasma. Dilution into factor V-deficient plasma eliminates most interferences, but lupus anticoagulants can still interfere unless additional assay modifications are performed. Because this assay has sensitivity and specificity values approximating 100%, it can be used to determine if further analysis at the genetic level is necessary. When the screening assay is abnormal, the patient can be evaluated with DNA testing to determine if the factor V Leiden mutation is absent, present in heterozygous form, or present in homozygous form.

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Accepted for publication November 27, 2001.

From the Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston.

Reprints: Michael Laposata, MD, PhD, Room 235 Gray Bldg, 55 Fruit St, Massachusetts General Hospital, Boston, MA 02114 (e-mail: mlaposata@partners.org).
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