The phenomenon known as acquired activated protein C resistance

British Journal of Haematology, 2001, 115, 767±773

Review

THE PHENOMENON KNOWN AS ACQUIRED ACTIVATED PROTEIN C RESISTANCE

Resistance to the effect of activated protein C (APC), addedto plasma from patients with a history of deep-veinthrombosis, was reported by Amer et al (1990), and thesignificance of inherited resistance was recognized byDahlback et al (1993). In the majority of subjects, inheritedresistance results from the Leiden mutation (factor V Leiden,FVL) in the gene coding for coagulation factor V (Bertinaet al, 1994), although a number of other genetic variationshave been described in the factor V gene associated with anAPC resistance phenotype. Inherited resistance has beenidentified as a significant risk factor for venous thrombosis(Griffin et al, 1993; Koster et al, 1993), late fetal loss(Martinelli et al, 2000) and pre-eclampsia (Dizon-Townsonet al, 1996). In recent years, the potential significance ofresistance occurring in the absence of known mutations ofthe factor V gene has been recognized. A number of authorshave coined the term acquired APC resistance for thisphenomenon.

The investigation of APC resistance by Dahlback et al(1993) was based on the effect of APC on an activatedpartial thromboplastin time (aPTT). Since then, it hasbeen recognized that the originally described aPTT-basedtest is not specific for the Leiden mutation. Indeed, in theintervening years, many strategies have been devised torender coagulation-based APC resistance assessmentsmore specific for FVL (de Ronde & Bertina, 1994;Jorquera et al, 1994; Varadi et al, 1995; Nicolaes et al,1996). Despite this drive to improve specificity, studiesthat used the original aPTT-based test or techniques thatremain sensitive to a variety of causes of APC resistance(Gable et al, 1997; Nicolaes et al, 1997) have shown thatthe degree of resistance (in FVL-negative subjects) relatesto:

X thrombin generation (Clark et al, 1999; Lowe et al,1999);

X venous thrombosis risk (de Visser et al, 1999);X cerebrovascular disease risk (van der Bom et al, 1996;

Kiechl et al, 1999);X pre-eclampsia risk (Clark et al, 2001a);X the presence of antiphospholipid activity (Hampton

et al, 1994);X combined oral contraceptive use (Osterud et al, 1994;

Henkens et al, 1995; Olivieri et al, 1995), in particular third-generation oral contraceptive use (Rosing et al, 1997).

A substantial number of questions are posed by theseobservations. These are typified by the debate resulting from

the observation that use of the third-generation combinedoral contraceptive pill (COCP) is associated with more APCresistance than second-generation COCPs (Rosing et al,1997). The authors used an APC resistance test based uponthe thrombin generation resulting from tissue factor-activated plasma. However, a comparable differential ofresistance has not been seen with aPTT-APC resistance tests(Henkens et al, 1995; Olivieri et al, 1995; Schramm &Heinemann, 1997), a finding that has subsequently beenconfirmed by Rosing's own group (Curvers et al, 1999).Although this variance has highlighted the difficulty ofdiagnosing acquired APC resistance, it is likely to afford anopportunity to understand the mechanism of the additionalrisk of venous thrombosis (venous thromboembolim, VTE)associated with third-generation COCPs (Vandenbroucke &Rosendaal, 1997).

From this debate, it is clear that the detection of acquiredAPC resistance requires consideration of the method oftesting and the population under study. The APC resistancephenotype does, however, correlate with markers of in vivothrombin generation (Freyburger et al, 1997; Clark et al,1999; Lowe et al, 1999) and may indicate an overallprothrombotic phenotype. What information, then, doesAPC resistance testing give us and, given the myriad ofinfluences upon it, can APC resistance testing (in non-FVLsubjects) be tamed to provide a useful tool in the assessmentof thrombotic risk?

FACTOR V LEIDEN-SPECIFIC AND NON-SPECIFICAPC RESISTANCE TESTING

In the assessment of APC resistance, distinction has to bedrawn between factor V mutation-specific and non-specificassessments. In the main, factor V-specific assessmentsinvolve predilution of subject plasma. This may be achievedwith factor V-depleted plasma (Jorquera et al, 1994), whichallows the cofactors of the protein C/protein S system to besupplied by the added normal plasma. The dilution of factorV renders the test highly specific to the presence of factor Vmutations. Similar strategies include predilution of thepatient plasma before assessment of the effect of APC onfactor VIIIa/factor Xa generation (Varadi et al, 1995), theeffect of APC on factor Va generation (assessed in aprothrombinase system) (Nicolaes et al, 1996) or the effectof APC on a modified factor V assay (Le et al, 1995). Thesestrategies exclude inhibitors and anticoagulants, but alsorender the test insensitive to other prothrombotic influ-ences.

In addition to the original aPTT test, other methods of

q 2001 Blackwell Science Ltd 767

Correspondence: Dr Peter Clark, Department of TransfusionMedicine, Ninewells Hospital and Medical School, Dundee DD1

9SY, UK. E-mail: [email protected]

APC resistance assessment have been described that remainsensitive to a variety of influences (Kraus et al, 1995; Datiet al, 1997; Nicolaes et al, 1997). In particular, theassessment (described above) by Nicolaes et al (1997)examines the effect of APC on the generation of thrombin(measured by the formation of thrombin±a2-macroglobulincomplexes with time) from tissue factor-activated plasma. Inthe aPTT-APC resistance test described by Dahlback et al(1993), a ratio of the aPTT obtained in the presence ofadded APC/the aPTT obtained without added APC isderived. With this APC sensitivity ratio (APC:SR), a lowratio indicates high resistance.

COAGULATION FACTORS AND ACQUIRED APCRESISTANCE

A number of coagulation factors influence both the aPTT(Dahlback et al, 1993) and tissue factor (Nicolaes et al,1997) assessments. aPTT-APC resistance correlates posi-tively with factor VIIIc levels during pregnancy (Clark et al,1998), in association with markers of an inflammatorystate (Laffan & Manning, 1996; Mathonet et al, 1996), andin the population as a whole (Lowe et al, 1999). This isconsistent with the association of both factor VIIIc (Kosteret al, 1995; Kraaijenhagen et al, 2000) and blood group(Talbot et al, 1972; Meade et al, 1994) with thrombosis.aPTT-based resistance also correlates positively with factorIXc (Lowe et al, 1999), Xc and XIc levels (Kluft et al, 1999).This may, in part, be confounded by the relationship of thesevariables to each other and the aPTT (Lowe et al, 1997; vanHylckama Vlieg et al, 2000), although factor IXc has beenrecognized as an independent risk factor for venousthrombosis (van Hylckama Vlieg et al, 2000). In pregnancy,aPTT-APC resistance also correlates positively with factorVc (Clark et al, 1998), but this influence is only observed atextreme factor Vc levels in non-pregnant subjects (Colucciet al, 1994; Freyburger et al, 1996). An inverse relationshipto protein S is seen in pregnancy (Clark et al, 1998), andhigher resistance is observed in protein S-deficient subjects(de Ronde & Bertina, 1994). However, no influence onresistance is apparent at `normal' protein S levels (de Ronde& Bertina, 1994; Freyburger et al, 1997; Lowe et al, 1999).No relationship with `normal' levels of protein C has beenreported (Freyburger et al, 1997; Lowe et al, 1999), butincreasing resistance is associated with increasing antith-rombin levels (Freyburger et al, 1997; Lowe et al, 1999).This may indicate a compensatory response to a prothrom-botic state.

aPTT-APC resistance is also associated with lupusinhibitors (Bokarewa et al, 1994). The observation of higherresistance associated with anti-B2 glycoprotein-1 antibodiesand anticardiolipin antibodies (Martinuzzo et al, 1996)suggests that this effect is not solely in vitro interference withthe aPTT. Indeed, anticardiolipin antibodies can inhibit theaction of APC on factor Va (Oosting et al, 1993), and adose±response relationship between antiphospholipid-related resistance and thrombosis has been suggested(Aznar et al, 1997).

Like the aPTT test, the tissue factor-APC resistance test is

affected by lupus inhibitors and is unreliable in the presenceof anticoagulants (Nicolaes et al, 1997). Protein S deficiencyis also associated with higher tissue factor-APC resistance(Nicolaes et al, 1997). In pregnancy, a gestation-indepen-dent positive relationship of resistance with cholesterol,antithrombin and protein C activity and an inverserelationship with total protein S has also been reported(Clark et al, 2001b), although there is little other publishedinformation on the influence of coagulation factors on thistest.

OESTROGEN AND ACQUIRED APC RESISTANCE

Heightened APC resistance, as measured by an aPTT-basedmethod, was reported in association with COCPs containing# 37´5 mg of ethinyl oestradiol (Henkens et al, 1995). Inthis study, no distinction between second-generation (levo-norgestrel or norgestimate) and third-generation progesto-gens (desogestrel or gestodene) was made. The bulk ofsubsequent studies has shown that third-generation pro-gestogens are associated with a 1´4- to fourfold increasedrisk of VTE compared with second-generation COCPs(reviewed by Vandenbroucke et al, 2001). As noted above,use of the third-generation COCP is associated with moreAPC resistance than second-generation COCPs (Rosing et al,1997), when estimated by the thrombin generationresulting from tissue factor-activated plasma. Althoughnot seen with aPTT-based APCR assessments, this phenom-enon has been offered as an explanation for the additionalrisk of VTE associated with third-generation COCPs(Vandenbroucke & Rosendaal, 1997). Consistent with

Table I. Demographic influences on non-factor V Leiden APC

resistance.

Parameter Correlation with aPTT-APC resistance

Blood group Oa,b #Smokinga,b,c,d #Cholesterola,b,c,d "Triglyceridea,d "Female gendera,d,e "Agea,c,d "Menopausef #Acute-phase reactantsa,f "Blood pressureb,d "BMIa,d "FH APC resistance*g "Platelet activation²h "Ceruloplasminf,i "

A positive correlation of the parameter with APC resistance is

indicated by " and an inverse relationship by #.*In families with no known mutation of factor V.

²APC resistance observed in platelet-rich plasma.

Letters indicate the source publication: (Tosetto et al, 1997)a;

(Clark et al, 2001a)b; (van der Bom et al, 1996)c; (Lowe et al,1999)d; (Henkens et al, 1995)e; (Kiechl et al, 1999)f; (Tosetto et al,

2000)g; (Taube et al, 1999)h; (Ripoll et al, 1998)i. FH, family history.

768 Review

q 2001 Blackwell Science Ltd, British Journal of Haematology 115: 767±773

observations of COCPs, recent epidemiological studies havesuggested that the use of hormone replacement therapy(HRT) is associated with a threefold relative risk of deep-vein thrombosis (DVT) (Chae et al, 1997; Meade, 1997).A comparable increase in aPTT-APC resistance to thatobserved with oral contraceptive use has been observed inHRT users (Lowe et al, 1999), a relationship that may be

independent of factor VIIIc and IXc (Nebulsi et al, 1993;Woodward et al, 1997).

DEMOGRAPHIC FACTORS AND ACQUIRED APCRESISTANCE

A number of demographic variables also influence the

Fig 1. Potential mechanisms of acquired APC resistance. The potential link between demographic factors and APC resistance (APCR) via

alterations in coagulation factor levels is shown. FVc, factor Vc; PS, protein S; FVIIIc, factor VIIIc; HRT, combined hormone replacement

therapy. A potential linkage from these factors via APCR and thrombin generation to pre-eclampsia (PET), venous thrombosis (VTE) and stroke

(CVA) is outlined. Higher factor VIIc levels (FVIIc) are associated with higher triglyceride (TRIG) levels. The mechanism of triglyceride-relatedAPCR is unknown. Only the additional coagulation effect of third-generation combined oral contraceptives (third-generation COCPs) over

second-generation COCPs is shown. Third-generation COCPs are associated with higher tissue factor-APC resistance (not aPTT-APCR) and

higher FVIIc levels than second-generation COCPs. The mechanism of higher tissue factor-APC resistance is unknown, but there may be a

direct effect of FVIIc. #A link between factor V Leiden (FVL), factor V Cambridge (FVC), or the HR2 haplotype (HR2) and cerebrovasculardisorders is not proven. Letters indicate the source publication: (Tosetto et al, 1997)a; (Clark et al, 2001a)b; (Clark et al, 1998)c; (Rosing et al,

1997)d; (Lowe et al, 1999)e; (de Visser et al, 1999)f; (Laffan & Manning, 1996)g; (Mathonet et al, 1996)h; (Clark et al, 1999)I; (van der Bom et al,

1996)j; (Kiechl et al, 1999)k; (Bertina et al, 1994)l; (Williamson et al, 1998)m; (Bernardi et al, 1997)n; (van Baal et al, 2000)8; (reviewed by

Winkler, 1998)p.


Review 769

aPTT-APC resistance test (Table I). These reflect the positiveinfluence of oestrogen, factor VIII and acute-phase reactantson APC resistance (Fig 1), and the majority are also inkeeping with the concept of APC resistance as a phenotypicmarker of venous and arterial disease risk. The relationshipbetween smoking and a reduction in APC resistanceappears, however, to be incongruous. There is, indeed,conflicting evidence that smoking is a risk factor for VTE(Marks & Emerson, 1974; Emerson & Marks, 1977; Prescottet al, 1978; Goldhaber et al, 1997). However, the relation-ship of smoking to APC resistance is consistent with themitigating effect of smoking on the risk of pre-eclampsia(van der Velde & Treffers, 1985; Klonoff-Cohen et al, 1993).Serum lipid levels are an accepted risk factor for arterialdisease but are not generally considered to be a risk factorfor venous thrombosis. Small preliminary studies have,however, suggested a link between serum lipids, thrombingeneration (MacCallum et al, 2000) and VTE (Kawasaki et al,1995, 1997).

APC RESISTANCE AS AN INDEPENDENT RISKFACTOR

Given the number of potential confounders (Table I), it isperhaps not surprising that only a few studies haveattempted to determine whether acquired APC resistanceis an independent risk factor for thrombotic disease. Thesestudies have avoided the use of artificial cut-offs tocharacterize subjects as having APC resistance and haveconsidered resistance as a relative phenomenon. In 1996, areport from the Rotterdam Study described a 2´6-fold (95%CI 1´2±5´6) increased risk of cerebrovascular disease whensubjects with the highest quintile of resistance werecompared with those with the lowest (van der Bom et al,1996). No significant difference in the risk was observedwhen the data were corrected for age, sex, cholesterol leveland FVL status. In a population study by Kiechl et al (1999),a dose±response relationship between quintiles of resistanceand the occurrence of femoral artery and carotid arterystenosis was observed. When adjusted for sex, menopausestatus, smoking and factors related to factor VIII levels (age,the aPTT and acute phase-reactants), a strengthening of therelationship was observed. Furthermore, no significantdifference in the three- to fourfold risk of stenoses wasobserved when the data were adjusted for the presence ofthe Leiden mutation. As noted above, smoking is associatedwith lower APC resistance and a reduced risk of pre-eclampsia (PET). In a study of FVL-negative subjects, thedegree of resistance in the first trimester of pregnancyrelated to the risk of development of pre-eclampsia in theindex pregnancy (Clark et al, 2001a). A sevenfold risk ofpre-eclampsia was observed for those in the highest quartilewhen compared with those in the lowest. In this study,regression for those factors that confounded the relationship(smoking and booking blood pressure) revealed a persistingrelationship between APC resistance and the risk ofsubsequent PET. The confounding of aPTT-APC resistanceby factor VIIIc levels was addressed directly by de Visser et al(1999). In FVL-negative, lupus inhibitor-negative subjects,

the highest quartile of resistance was associated with afourfold risk of venous thrombosis when compared withthose subjects in the lowest quartile. Forty-five per cent ofthose subjects in the highest quartile had a factor VIIIc level. 150 iu/dl. Although adjustment for FVIIIc levels, age andsex did not abolish the relationship with thrombosis, therelative risk associated with the highest quartile wasreduced to 2´5 (95% CI 1´5±4´2). Thus, APC resistanceappears to be an independent marker of a prothromboticphenotype.

FROM OBSERVATION TO INTERVENTION

The relationship with such a wide variety of prothromboticinfluences (Table I, Fig 1), markers of in vivo thrombingeneration (Clark et al, 1999; Lowe et al, 1999) andthrombotic disease (van der Bom et al, 1996; de Visser et al,2000; Clark et al, 2001a) suggests that APC resistance is acommon mechanism whereby a number of risk factors forarterial and venous thrombosis promote thrombogenesis.

Could this disparate phenomenon usefully be used as ascreening tool to determine risk? A number of questionswould have to be answered before this could be considered:

X What type of test should be used? Clearly, eachresistance assessment gives different demographic andcoagulation information. At present, there is greaterexperience in the use and interpretation of the aPTT-basedtest. It would seem likely that this would become the mostwidely applicable acquired APC resistance screening tool.

X How should testing be standardized? Standardizationhas already been recommended for aPTT-APC resistancescreening for FVL (de Ronde & Bertina, 1994) and, given thevariety of influences upon it, an aPTT test for acquiredresistance would require population-specific referenceranges.

X When is the appropriate time to test? Some resistancerelates to acute-phase reactants, and the link between thisand future thrombotic risk is unknown. Furthermore,testing for acquired resistance can only be achieved onnon-anticoagulated patients.

More importantly, in addition to these technical con-siderations, a number of fundamental principles of screen-ing require to be addressed:

X Will screening alter management? It is not yet clearwhether this question has been answered for heritableresistance (Ridker et al, 1995; Rintelen et al, 1996; Simioniet al, 1997; Eichinger et al, 1999; Kearon et al, 1999).Elevated levels of in vivo markers of thrombin generation areseen in heritable APC resistance (Martinelli et al, 1996;Simioni et al, 1996), and these markers may be reduced byanticoagulation (Takahashi et al, 1993; Koefoed et al,1997). Owing to the attendant risk of bleeding, the use ofanticoagulants requires a subtle balance of risks andbenefits. Unlike FVL, acquired APC resistance does relateto a number of thrombotic risk factors, such as cholesterol,blood pressure and body mass index (BMI) (Table I). It isconceivable that intervention to reduce these thromboticrisk factors could offer additional modalities of therapy toreduce APC resistance and thrombotic risk.

770 Review


X How is an abnormal resistance defined? A number ofstudies have shown that there is a dose±response relation-ship between the degree of resistance and the developmentof disease. That the risks of vascular disease (in those withthe lowest quartile/quintiles of resistance) are comparablewith those observed in heritable resistance is remarkable,given that the degree of resistance is much less. Interventionto reduce disease would clearly require a new definition ofabnormal or unacceptable resistance. The move fromprogressive risk to action limits is, thankfully, not new inthe field of vascular disease.

The discovery of heritable APC resistance has transformedour understanding of VTE. The subsequent discovery ofcorresponding defects in the factor V gene has resulted in aconcentration on refinement of coagulation tests to improvetheir specificity and sensitivity for these mutations. In recentyears, the significance of non-Leiden resistance has beenrealized. Much work is still required to determine whetherthis prothrombotic phenotype usefully can be added toroutine thrombophilia screening.

Department of Transfusion Medicine,Ninewells Hospital and Medical School,Dundee, and Department ofHaematology, Royal Infirmary,Glasgow, UK

Peter ClarkIsobel D. Walker

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Keywords: activated protein C (APC) resistance, contra-ception, pregnancy, thrombosis.


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The phenomenon known as acquired activated protein C resistance