ACTIVATED PROTEIN C RESISTANCE UNIVERSITY | Clinical Aspects

Figure 8. Reported prevalences of APC resistance and the factor V:Q506 mutation in the global population.

The origin of the FV:Q⁵⁰⁶ mutation

Several investigators have suggested that the high prevalence of the FV:Q⁵⁰⁶ mutation could be due to the evolutionary advantage it would confer, which has helped to maintain and spread the mutation.^71,77,81 It is possible that the selective disadvantage of a life-long hypercoagulable state could be balanced by, for example, the protection against excessive blood loss during delivery and menstruation. The selective risk of the FV:Q⁵⁰⁶ mutation would also be of less historical importance as people in ancient times were not exposed to modern risk factors for thrombosis (e.g. oral contraceptives, surgery, sedentary lifestyle etc.). The high allelic frequency of FV:Q⁵⁰⁶ in Caucasian populations and its linkage to different polymorphisms in the factor V gene, supports the hypothesis that the mutation occurred as a single event in the ancient European population.^13,74-75,94 The time of this event would be approximately 30,000 years ago, i.e. after the diversion of Africans from non-Africans (140,000 years ago) and after the diversion of Caucasians from Mongolic populations (70,000 years ago), but before the diversion of Caucasian subpopulations. However, since the FV:Q⁵⁰⁶ mutation involves a CpG dinucleotide, which is an established hotspot for mutation, the possibility of recurrent mutations in other races should not be ruled out altogether.⁸⁸

Clinical manifestations of APC resistance

The clinical manifestations of inherited, heterozygous protein defects in familial thrombophilia involving antithrombin, protein C, protein S and factor V (APC resistance) are fairly similar. Mutations affecting the qualitative or quantitative function of these proteins often result in venous thromboembolism at a young age (before the age of 45 years) and are followed by a tendency towards recurrent thrombotic episodes.⁵⁰ The most common manifestation of APC resistance is deep venous thrombosis (DVT) of the lower limbs, with or without pulmonary embolism, which accounts for about 90% of all thrombotic episodes.^50,53,96 Other, less frequent, manifestations include superficial thrombophlebitis⁹⁶ and unusual sites for thrombosis such as the mesenteric,⁹⁶ central retinal,^97-101 portal,^102,103 internal jugular,¹⁰⁴ and cerebral veins.^105,106

Thrombotic risk

The relative risk of DVT for carriers of the FV:Q⁵⁰⁶ mutation compared to non-carriers has been estimated to increase eight-fold for heterozygotes (single defect) and 80-fold for homozygotes (double defect).⁵⁴ Since aging itself is a risk factor for thrombosis, the absolute risk increases with age.⁶⁹ A similarly increased risk of pulmonary embolism has been observed by some investigators,²¹⁶ although this could not be confirmed by others.^217,218 The risk of recurrent venous thromboembolism in carriers of the FV:Q⁵⁰⁶ mutation has been reported to be three to fivefold higher compared to patients without the mutation,^223,286 but again there is no consensus on this matter.²²⁴

FV:Q⁵⁰⁶ is a mild risk factor by itself

The penetration of clinical manifestations among APC resistant individuals is variable, and a majority of heterozygous carriers of FV:Q⁵⁰⁶ actually never experience any symptoms. In fact, not even homozygous carriers will necessarily be affected by thrombosis during their lifetime.⁴³ These facts illustrate that FV:Q⁵⁰⁶ is a mild risk factor per se and that the probability of APC-resistant individuals developing thrombosis is dependent on the coexistence of other risk factors. About 60% of APC-resistant patients have their first thrombotic event in combination with pregnancy, oral contraceptives, trauma or surgery.^16,53,96 Furthermore, because of the high prevalence of APC resistance in the general population, its combination with other genetic defects is not unusual. A wide range of disorders have been reported in connection with APC resistance, implicating its part in the development of thrombotic complications. These include the Budd-Chiari syndrome,^107-109 nephrotic syndrome,¹¹⁰ leg ulcers,^111,112 heparin-induced thrombocytopenia,¹¹³ priapism,¹¹⁴polycythemia vera,¹¹⁵ essential thrombocythemia,¹¹⁵ child-thrombosis,^116-119 cutaneous skin necrosis,^120,121 neonatal purpura fulminans,¹⁶⁵ acute lymphoblastic leukemia,¹⁶⁶ systemic sclerosis,¹⁷⁰ and preeclampsia.²⁸²

Table 5. Prevalence of APC resistance phenotype in patients with venous thrombosis.

* abstract paper

Table 6. Prevalence of the FV:Q⁵⁰⁶ mutation in patients with venous thrombosis according to gene analysis.

* abstract paper

Table 7. Prevalence of the FV:Q⁵⁰⁶ mutation in patients with arterial thrombosis compared with controls according to factor V gene analysis.

Arterial thromboembolism

Although there is a clear link between APC resistance due to the FV:Q⁵⁰⁶ mutation and venous thrombosis, the same link to arterial thrombosis is enigmatic. Several studies have reported the presence of APC resistance in young stroke patients, suggesting that it contributes to the pathophysiology.^{55,122-126,163} Halbmayer et al for example, found that 20% (6 out of 30) of young Austrian stroke patients were APC-resistant according to the classic APC resistance test.⁵⁵ In contrast, other studies of stroke patients showed no increased prevalence of either the APC resistance phenotype or FV:Q⁵⁰⁶ mutation compared to healthy controls (Table 7).^{67,69,93,133-135,169}

The possible correlation between the FV:Q⁵⁰⁶ mutation and the risk of ischemic heart disease, particularly myocardial infarction, has also been investigated. With the exception of two papers,^78,132 the general conclusion is that the FV:Q⁵⁰⁶ mutation is not an important risk factor for arterial thrombosis in heterozygotes,^{67,69,72,75,93,127-131,164} but may have a role in homozygotes.^126,137 Some papers have appeared recently which may help to clarify the situation. These suggest that acquired (or inherited) APC resistance, independent of the FV:Q⁵⁰⁶ mutation, may indeed be an important risk factor for arterial thrombosis.^139-141 These observations are most interesting and they call for an evaluation in different patient groups, using the classic APC resistance test, to explore whether the APC ratio may be predictive for both venous and arterial thrombotic events.

APC ratios in thrombophilic families

Zöller et al investigated 50 thrombosis-prone Swedish families with APC resistance (Figure 9).¹⁶ In three of these families the FV:Q⁵⁰⁶ mutation was not present, suggesting another, as yet unidentified, cause of APC resistance. In total, 308 family members were investigated; 146 normal, 144 heterozygotes and 18 homozygotes. APC ratios were low in all the homozygous and most of the heterozygous cases. APC ratios in the APC-resistant individuals who lacked the mutation ranged from 1.3 to 2.0. Heterozygotes with a history of thrombosis had significantly lower APC ratios than those without thrombosis and none of the heterozygotes with APC ratios >2.0 had experienced thrombosis. Moreover, relatives without the mutation but with thrombotic histories had on average lower APC ratios than those without thrombosis. Significant differences in thrombosis-free survival curves and APC ratios were observed between the groups (Figure 10), thus confirming that APC resistance is an important risk factor for thrombosis. By the age of 33, 8% of the normals, 20% of the heterozygotes and 40% of homozygotes had experienced manifestations of venous thrombosis. The average age for the first thrombotic event was 25 (range 10 to 40 years) for homozygotes and 36 (range 18 to 71 years) for heterozygotes. In the thrombosis-prone families the observed incidence of thrombosis was higher than expected, suggesting that these families have been affected by additional genetic defects.

Oral contraceptives

The use of oral contraceptives (OCs) is a much-debated risk factor for thrombosis. Numerous studies have shown that oral contraceptives increase the risk of thrombosis about two to nine-fold depending on the active substance used, compared to non-users.^185-186 However, it is still largely unclear why oral contraceptives cause thrombosis in some women. Similar to pregnancy, increased levels of procoagulant factors, as well as reduced APC ratios and reduced levels of antithrombin and free protein S have been reported in women using OCs.^179,187-190 For the majority these procoagulant changes are negligible, although for a small number of women with a genetic or acquired predisposition for thrombosis, the added prothrombotic influence of OCs may be sufficient to trigger a thrombotic event.¹⁹¹

The risk of venous thrombosis in OC users with APC resistance has been investigated by Vandenbroucke et al.¹⁸⁵ The risk of thrombosis among OC users in this study was shown to increase 4-fold when compared to women not using OCs (baseline risk about 0.01% annually). Women heterozygous for the FV:Q⁵⁰⁶ mutation who did not use OCs showed an 8-fold increase in thrombotic risk, whereas a 35-fold increase in risk was shown in OC users heterozygous for the mutation. Thus, the joint effect of the two risk factors appears to be multiplicative. Through a similar effect, the increase in risk for homozygotes using OCs is several 100-fold (Table 9).^54,185 Clinical studies also demonstrate that OCs leads to an unacceptably high risk of venous thrombosis in females with homozygous FV:Q⁵⁰⁶.¹⁹²

Hellgren et al investigated 28 women with a history of thrombosis in connection with OCs and they found that nine (32%) of the women had APC ratios <2.0, indicating APC resistance.172 None of the women investigated were using OCs at the time the blood samples were taken and in only one of nine patients with APC resistance was an additional risk factor for thrombosis identified.

Considering the high prevalence of APC resistance in the general population, a substantial number of women are placed at a higher risk by using OCs. In North America alone there are about 10 million OC users.¹⁹³ Assuming the allele frequency for FV:Q⁵⁰⁶ is 0.025, then roughly 500,000 would be heterozygous and 6,000 homozygous for the mutation. Since the annual thrombotic risk is about 0.3% for heterozygotes and 3% for homozygotes using OCs, it can be calculated that around 2,000 thrombosis cases per year in North America are caused by the combination of APC resistance and OCs. This figure should be compared to the number of thrombosis cases in OC users without the mutation, which is about 4,000 cases per year. Thus, in the presence of both risk factors (OCs + mutation), venous thrombosis appears to develop in a substantial number of women who would never have had thrombosis in the presence of either risk factor alone. It can be estimated that 1-2% of all cases of venous thrombosis caused by OCs will have a fatal outcome due to pulmonary embolism.

Physicians who prescribe OCs generally interview the woman about her family history to learn whether there are any cases of thrombosis, indicating a genetic predisposition. However, since many homozygous APC resistance patients have asymptomatic parents that are heterozygous, it would not be suspected from interviewdata alone. This fact raises the question as to whether all women should be screened for the FV:Q⁵⁰⁶ mutation prior to prescribing OCs.^190,194-197 The potential reduction in mortality and morbidity cases due to thromboembolism caused by the pill must, however, be weighed against the risk of thrombosis in connection with unwanted pregnancy.

Surgery

Patients undergoing major trauma or surgery, in particular orthopedic or abdominal surgery, generally run a high risk of experiencing thromboembolic events due to a sustained activation of coagulation (Table 10). The precise risk for an individual is determined not only by the type, extent and duration of the surgical trauma, but also by the accumulation of predisposing risk factors such as advanced age, morbidity and thrombophilia.¹⁹⁹ The general and standardized use of heparin for post-operative thromboprophylaxis has reduced thrombotic complications to a great extent during recent decades. However, it still occurs in a substantial number of patients. Thus, it may be beneficial to pre-operatively identify these individuals at higher risk in order to adopt a more individualized and adequate prophylaxis.²⁸³ The latter approach is supported by recent studies, showing that prolonged prophylaxis using LMW heparin in connection with hip replacement resulted in a 50% reduction in venographically verified deep venous thrombosis.^201-202 More studies are needed in this area to evaluate whether individualized prophylactic treatment might be beneficial in patients with genetic risk factors for thrombosis.

At present, the underlying role of APC resistance in postoperative thrombosis is under intense research. In a Danish study it was found that 30% of patients who developed deep venous thrombosis after knee arthroplasty were APC resistant.²⁰⁰ APC resistance was also shown to be a risk factor for arterial reocclusion of vascular grafts in peripheral vascular surgery.^136,138 These studies are highly interesting and suggest that it would be cost-benefical to screen for APC resistance pre-operatively in order to reduce post-operative thrombosis.

Antiphospholipid antibodies

Lupus anticoagulant and anticardiolipin antibodies are acquired antiphosholipid antibodies (APAs) which were initially described in patients with systemic lupus erythematosus (SLE). Recently, the interest in these types of antibodies, which are more correctly described as antibodies directed against phospholipid-protein complexes, has focused mainly on their strong correlation with thrombotic disease, thrombocytopenia and recurrent fetal loss. The association of one of these complications and the presence of APAs has been termed the antiphospholipid syndrome.²⁰³ The mechanism underlying the syndrome is at present unknown, although various interferences in the protein C pathway have been suggested as being a possible pathogenic mechanism for thrombosis.²⁰⁴ One such interference may be the selective blocking of APC on phospholipid surfaces by specific types of APAs, leading to an acquired APC resistance.^41,205-207 Several reports have also demonstrated acquired APC resistance to be a relatively common feature in patients with the antiphospholipid syndrome,^206,208-211 even when the uncertainties of lowered APC ratios caused by APA-induced prolongation of the basal APTT were considered.²⁰⁶ At present, there is no clear clinical evidence to support a higher thrombotic risk among patients with the antiphospholipid syndrome and acquired APC resistance.^210,214,215 Furthermore, the FV:Q506 mutation does not seem to be over-represented in patients with the syndrome compared with healthy controls.^210-212

When to test for APC resistance phenotype and FV:Q⁵⁰⁶genotype

Up to now, the classic APC resistance test has been used mainly as a simple screening assay for the FV:Q⁵⁰⁶ mutation. However, several limitations have been reported for this application. Firstly, this test has a sensitivity and specificity for the mutation which is usually in the range 75-90%.^219-220 Secondly, the test is only reliable if the basal APTT-reaction is within the normal range,²²¹ which therefore disqualifies many APC-resistant patients on anticoagulant therapy from testing.²²²As the classic APC resistance test stands today, it is not recommended for FV:Q⁵⁰⁶ mutation screening. The classic test provides phenotypic information about APC resistance and should primarily be used as a complimentary test to the modified APC resistance test in a thrombosis investigation. A preliminary guideline for how to use the classic and modified APC resistance test is given in Table 11.

Table 11. Preliminary guideline for APC resistance and/or FV:Q⁵⁰⁶ testing.²⁷⁶

APC resistance phenotyping

Interestingly, the APC ratio is not a simple, one-variable reflection of the APC response in vivo. Instead, it seems to reflect an anticoagulant system response that may decrease under a hypercoagulable state.¹³⁹ A poor anticoagulant response to APC, independent of the FV:Q⁵⁰⁶ mutation, could therefore be a thrombotic risk factor or risk marker in a wide range of conditions and circumstances including venous thromboembolism,^16,139 antiphospholipid protein syndrome,²⁰⁹ second trimester miscarriage,¹⁷⁷ systemic sclerosis,¹⁷⁰ ischemic stroke,^140-141 occlusion after vascular surgery,¹³⁸ pregnancy¹⁷⁴ and the use of oral contraceptives.^188-190 Future studies will clarify whether the phenotypic APC ratio obtained from the classic test may serve as a predictor of venous and arterial thrombotic events.

 Figure 12. Relationship between APC-V ratios and the factor V:Q⁵⁰⁶ allele in various sample categories as determined using the Coatest® APC Resistance V method. Complete discrimination was obtained between the normal and mutated factor V genotypes. Instrument: ACL. OAC= oral anticoagulant therapy (from ref. 278 with extension of data for pregnant women).

Screening for FV:Q⁵⁰⁶

The modification of the APC resistance test, in which sample plasma is prediluted in an excess of stabilized factor V-deficient plasma, improves the discrimination for the FV:Q⁵⁰⁶ mutation dramatically.^229-236 The modification also allows for the testing of patients on anticoagulant therapy and strongly reduces the influence of preanalytical variables such as storage and plasma handling. Evaluation of this test using different categories of clinical samples showed that the specificity and sensitivity for the presence of the FV:Q⁵⁰⁶ mutation was 100% (Figure 12).²⁷⁸ The robustness and simple format of the modified test, together with its high discrimination between normal and mutated factor V genotypes, makes it an ideal tool for FV:Q⁵⁰⁶ mutation screening. An important point when prediluting the sample plasma in factor V-deficient plasma is that phenotypic expressions related to factors other than factor V are lost. In order to obtain phenotypic and genotypic information for a correct thrombophilia diagnosis, one approach could be to analyze each plasma sample using both the classic and the modified APC resistance test.²³³ This strategy identifies APC-resistant patients, both with and without the FV:Q⁵⁰⁶ mutation, and keeps the need for confirmatory gene analysis to a minimum.

Management of APC resistant patients

A potentially large number of thrombosis-prone individuals with APC resistance will most likely be identified in the near future. This of course raises the question of patient management. At present there are no established guidelines for managing thrombotic patients with APC resistance, although it is generally agreed that they should be treated in the same way as the patients with antithrombin, protein S and protein C deficiency.²⁴⁹ An acute thrombotic episode should be treated conventionally with heparin for 5 to 10 days, followed by an oral anticoagulant (warfarin) within 24 hours to produce an International Normalized Ratio (INR) of 2.0 to 3.0. Patients should be given general advice on how to minimize the thrombotic risk, including dietary advice, cessation of smoking and avoiding long periods of immobility. Thrombophilic women should avoid oral contraceptives and all patients should be notified that they may require special treatment prior to surgical, medical or obstetric procedures that carry an increased thrombotic risk.

Anticoagulant treatments require individual considerations

Because only a proportion of the subjects with a heterozygous defect develop thrombosis, it is unjustifiable to put symptom-free APC resistant individuals on thromboprophylaxis solely on the basis of having a genetic defect. It is, however, essential that asymptomatic individuals in thrombosis-prone families are carefully counseled with respect to their defect and offered short-term prophylaxis in special situations where there is an extra risk of thrombosis. Women with a history of thrombosis and who have a known genetic defect may require anticoagulation throughout pregnancy, preferably by using dose-adjusted subcutaneous heparin.⁶⁸ As a rule, all thrombophilic women should be offered thromboprophylaxis in conjunction with delivery and puerperium. Homozygous and heterozygous patients with a second anticoagulant defect should be given preventive therapy in all risk situations and long-term therapy should be considered if thrombosis is recurrent.

The prescription of oral contraceptives

When a woman has experienced venous thrombosis after oral contraceptive use, it is recommended that she is tested for the possible presence of the FV:Q⁵⁰⁶ mutation. If she is heterozygous for the mutation she should be carefully informed about her thrombotic risk and counseled about the type of contraceptive she should use in the future. The mere fact that she has had thrombosis indicates that the risk in her case is significant. If she is homozygous for the mutation, she should be strongly recommended to discontinue the use of oral contraceptives. As in any investigation of a young patient with venous thrombosis and APC resistance, it is also recommended to search for other causes of inherited thrombophilia.

Summary

The APC resistance phenotype is mainly diagnosed using the classic APTT-based test, which detects both inherited and acquired APC resistance. A modified APTT-based test using predilution of sample plasma with factor V deficiency plasma has been developed with high discrimination for factor V-related APC resistance. The factor V:Q⁵⁰⁶ mutation is highly prevalent among Caucasians (2-13%), it is present among people of Northern India (4%) and Saudi Arabia (2%), but is very rare among, for example Japanese, Eskimoes and Australian aborigines. The race-dependent distribution of the mutation may explain the higher incidence of thrombosis reported for Caucasians. The most common clinical manifestation of APC resistance is deep venous thrombosis, with or without pulmonary embolism. The relative risk of venous thrombosis for carriers of the factor V:Q⁵⁰⁶ mutation has been estimated to increase 8-fold for heterozygotes and 80-fold for homozygotes. The mutation does not seem to be an important risk factor for arterial thromboembolism, although acquired (or inherited) APC resistance not attributable to the mutation may be a risk factor for stroke as well as for venous thrombosis. The current recommendations for treating APC resistant patients are similar to those for other established inherited thrombophilic defects.

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