Activated protein C resistance phenotype in patients with antiphospholipid antibodies

Activated protein C resistance phenotype in patients with antiphospholipid antibodies JUSTO AZNAR, PIEDAD VILLA, FRANCISCO ESPANA, AMPARO ESTELLES, SALVADOR GRANCHA, and CRISTINA FALC6 VALENCIA, SPAIN

The effect of antiphospholipid antibodies (aPL) on the action of activated protein C (APC) was examined in 32 patients: 19 with lupus anticoagulant (LA), 6 with anticardiolipin antibodies (aCL), and 7 with LA and aCL. Eighteen patients had a ratio of activated partial thromboplastin time (APTT) with APC to APTTwithout APC (APTTratio) <2.06 (cut-off level) and no factor V Leiden mutation; these patients showed APCresistance (APC-R) phenotype. The mean prolongation of APTTafter addition of APC in a control group was 45.3 secondS, with a lower limit of 31.4 seconds. Only 3 of the 18 patients with low API"r ratio had a prolongation of <31.4 seconds; they were classified as true APC-R phenotype, whereas the other 15 patients were classified as spurious APC-R. Of the 3 patients with true APC-R, 2 had deep venous thrombosis, I with pulmonary embolism, and the third had recurrent abortion. Of the other 15 patients, 2 had had ischemic stroke, I had recurrent abortion, and 12 were asymptomatic. Circulating APC level was measured in 14 of the 18 aPL patients with a low APTTratio; it was lower than the normal lower limit in 4 patients and within the lower limit in 2. Three of the 4 patients with reduced APC levels had a history of thrombosis. We conclude that patients with aPL who show APC-R phenotype due to a low APTTratio without the factor V Leiden mutation can be classified into two groups: true and spurious APC-R phenotype. Since those with true APC-R phenotype could have greater thrombotic risk, adequate classification of these patients is important. Moreover, aPL can sometimes interfere with the activation of protein C, thus reducing the circulating levels of APC, and this could constitute another thrombotic risk factor. (J Lab Clin Med 1997;130:202-8)

Abbreviations: aCL = anticardiolipin antibodies; APC = activated protein C; APC-R = resistance to the anticoagulant effect of activated protein C; aPL = antiphospholipid antibodies; APYr = activated partial thromboplastin time; API-r ratio = ratio of AP~ with APC to APT[ without APC; ELISA = enzyme-linked immunosorbent assay; IgG = immunoglobulin G; IgM = immunoglobulin M; LA = lupus anticoagulant; PPACK= D-phenylalanyl-L-prolyI-L-arginine chloromethyl ketone

From the Department of Clinical Pathology and the Research Center, "La Fe" University Hospital. Supported in part by Grants 96/1129 and 96/1256 from Fondo de Investigaciones Sanitarias, Madrid, and by a grant from CAM, Alicante, Spain. Submitted for publication Nov. 12, 1996;revision submitted Feb. 27, 1997; accepted Feb. 28, 1997. Reprint requests: Justo Aznar MD, PhD, Departamento de Biopatologfa, Av. Campanar, 21, 46009 Valencia, Spain. Copyright © 1997 by Mosby-YearBook, Inc. 0022-2143/97 $5.00 + 0 5/1/81992

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esistance to the anticoagulant effect of A P C (APC-R) has recently been described as an important cause of venous thrombosis. 1 This abnormality is associated with the presence of a guanidine-to-adenine substitution at nucleotide 169 of the factor V gene (factor V Leiden).2 High prevalence of this mutation has been found in patients with venous thrombotic events, 3-7 whereas in the general European population prevalence of the mutation is about 3% to 5%. 2,3 Biologic evaluation of A P C - R is based on the determination of two APTTs, one in presence of A P C and the other in its

R

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absence. A P C - R is defined as a poor anticoagulant response of the patient's plasma to APC, expressed as A P T T r a t i o ) Determination of factor V Leiden mutation includes amplification of the nucleotide region of the mutation, either from genomic D N A or from RNA, followed by a mutation detection step. 2'4'8'9 Comparison of the results obtained from the A P C - R clotting assay with those from the polymerase chain reaction assay for factor V Leiden mutation showed that some patients with a positive A P C - R clotting test did not have the gene mutation. 2 This suggests the existence of either an acquired A P C - R phenotype 1°-14 or another molecular abnormality not yet known. The most frequent cause of A P C - R phenotype is the presence of aPL. 1°'12'15-17 Ehrenforth et al n found A P C - R in 17 out of 48 patients with LA, and Bokarewa et a118 observed the coexistence of L A and A P C - R in 17 out of 78 thrombophilic patients. Other authors, n'13,ls however, found no association between the presence of aCL and APC-R, which suggests that there is no relation between them. ~3 These discrepancies as to the effect of aPL on A P C activity may be a result of the enormous heterogeneity of these antibodies) 9-zl There are different mechanisms by which aPL, and especially LA, could induce a low A P T T ratio. A P L can block A P C function by selectively interfering in the expression of phospholipid-dependent A P C anticoagulant activity a5 and thus give rise to a true A P C - R phenotype. Also, the prolongation of A P T T induced by aPL may yield a falsely low A P T T ratio, 14 resulting in a spurious A P C - R phenotype. Phospholipids play an essential role in the intrinsic pathway of coagulation 22 and in the activation 23 and function of protein C. 15'24-28 Therefore, aPL may prolong the A P T T and even cause bleeding, z9'3° or aPL may interfere in protein C activation or in the A P C function, inducing a thrombophilic state. Because aPL often acts at several levels, it is difficult to predict the clinical expression of aPL activity. The present study examines the question of how to distinguish between true and spurious A P C - R phenotype, characterized by a low A P T T ratio, in patients with aPL. METHODS

Thirty-two patients with aPL (mean age _+ SD, 37 _+ 15 years; range, 5 to 78 years; 28 women, 4 men) were evaluated for APC-R. LA activity was detected in 19 patients, aCL in 6, and both LA and aCL in 7 patients. All 13 aCL patients had IgG-type antibodies, and 1 of them also had IgM-type antibodies. Nine patients had systemic lupus erythematosus. Of the other 23 patients with aPL, 7

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were catalogued as having primary antiphospholipid syndrome because they had thrombotic disease or recurrent abortions: 1 had deep vein thrombosis and another had deep vein thrombosis and pulmonary embolism, 2 patients presented ischemic stroke, and 3 patients had recurrent abortions. None of the patients was under oral anticoagulation at the time of sampling. An interval of more than 3 months separated the thrombotic episode from the time of blood sampling. To evaluate the response of native plasma to APC, a control group of 95 healthy subjects (mean age, 34 _+ 12 years; range, 9 to 63 years; 85 women, 10 men) was studied. To evaluate the response to APC of normal plasma diluted 1 to 5 with factor V-deficient plasma, a control group of 46 healthy individuals (mean age, 36 -+ 12 years; range, 10 to 63 years; 39 women, 7 men) was examined. Informed consent was obtained from all patients and controls before sample extraction. Blood samples were obtained by venipuncture in the cubital vein, anticoagulated with 1/10 volume of 0.13 mmol/L trisodium citrate, and immediately centrifuged at 1500 g for 30 minutes at 4° C. Plasma was collected and stored in aliquots at -70 ° C until used. For DNA studies, venous blood was collected in ethylenediamine tetraacetic acid and kept at 40 C. Leukocytes were isolated within 48 hours and stored frozen until DNA extraction was performed. To measure circulating APC, venous blood was collected into two vacutainer tubes (Becton Dickinson, Meylan, France) containing 1/10 volume of 0.13 mmol/L trisodium citrate. Immediately after sampling (within 30 seconds), 1/100 volume of 0.5 mol/L benzamidine and 0.5 mmol/L PPACK was added to one blood tube and 1/100 volume of 1000 U/ml heparin was added to the other. After incubation at 37°C for 30 minutes, plasma was obtained by centrifugation at 1500 g for 30 minutes at 20° C and stored in aliquots at -80 ° C. A diagnosis of LA was made with the use of screening and confirmatory procedures. Screening tests comprised measurement of APTT, using either lyophilized cephalin and micronized silica (IL test TM APTT reagent, Instrumentation Laboratory, Milano, Italy) or an LA-sensitive APTT reagent (Diagnostica Stago, Asnieres, France) on an ACL 300 R coagulometer (Instrumentation Laboratory), and the diluted Russell viper venom time, using viper venom (Sigma Chemical Co., St. Louis, MO). The screening tests were performed with the platelet-poor plasma of the patients and with 1:1 and 4:1 mixtures of the patient's plasma with a pool of 20 normal plasmas. The confirmatory test (platelet neutralization assay) was a diluted Russell viper venom time in which one volume of plasma sample was first mixed with one volume of a commercial platelet preparation (Platelet extract reagent, Biodata Corporation, Horsham, PA). A plasma sample was diagnosed as containing LA if at least two of the screening tests and the confirmatory test were positive, according to the criteria defined in the Guidelines on LA testing by the Lupus Anticoagulant Working Party of the British Society

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citrate plus APC inhibitors (see above). In the heparin tube, APC reacts completely and irreversibly with its major plasma inhibitors, protein C inhibitor and cxl-antitrypsin, and the complexes formed are measured by specific ELISAs. The amount of circulating APC is calculated from the difference between the total amount of complexed APC (sample in citrate plus heparin) and the amount of APC complexed in vivo (sample in citrate plus inhibitor). The detection limit of APC is 0.1 ng/ml. Because APC levels are proportional to protein C zymogen levels, values are expressed as the ratio of APC to protein C; for 20 healthy individuals, the ratio was 1.2 _+ 0.3 (mean -+ SD).

4

I

o

3

Oo

< 2

~°o~ tool

RESULTS QI

V

LA(+) aOL(-)

LA(-) LA(+) aCL(+) aCL(+)

Fig. 1. Activated protein C-resistance in three groups of patients with antiphospholipid antibodies. An APTT ratio below 2.06 (cut-off) indicates APC-R.

for Haematology (normal ratios, 0.90 to 1.09; normal platelet neutralization assay result, <10%). The aCL titer and IgG and IgM isotypes were analyzed by an ELISA method (Cheshire Diagnostic Ltd., Chester, England). A sample was considered positive for aCL if it had >15 IgG or IgM phospholipid antibody units. Classic APC-R was assayed by the Coatest APC-R test (Chromogenix AB, M61ndal, Sweden) according to the manufacturer's instructions, on an ACL 300 (Instrumentation Laboratory). The modified APC-R test was performed as reported elsewhere, 31 with some modifications. The patients' plasma was diluted 1:5 with factor V-deficient plasma from Baxter Diagnostica Inc. (Deerfield, IL). APC-R was evaluated by the Coatest APC-R test (Chromogenix AB), and the results were expressed as an APTT ratio instead of a normalized APTT ratio. The APC-R test in the presence of an excess of platelet phospholipids was done as previously reported. 32 Briefly, equal volumes of test plasma and a suspension of washed platelet reagent (Platelet extract reagent, Biodata Corporation) were mixed at 37°C for 5 minutes, and the APTT was then measured using a commercial kit (Coatest APC-R test). Factor V Leiden mutation was detected by polymerase chain reaction amplification and restriction analysis of a fragment of factor V DNA, following the method described by Gandrille et al. 33 Circulating APC levels were measured as reported previously. 3 4 Briefly, the assay requires collection of duplicate blood samples, one in citrate plus heparin and the other in

Evaluation of APC-R. The classic A P C - R test in 95 healthy individuals produced an A P T T ratio of 2.52 _+ 0.23 (mean _+ SD; normal limits, 2.06 to 2.98). The test was also run in 32 patients with aPL (Fig. 1), and an A P T T ratio <2.06 was observed in 18. Since none of these 18 patients showed factor V Leiden mutation, all were classified as A P C - R phe-

notype. To assess whether the observed A P C - R phenotype was true A P C - R or a technical artifact caused by a prolonged basal APTT, we evaluated A P T T prolongation after the addition of APC. The m e a n prolongation in the control group (n = 95) was 45.27 seconds, with a lower limit of normality of 31.39 seconds (Table I). When this approach was applied to the 18 aPL patients who had a below-normal A P T T ratio, only 3 of them showed an A P T T prolongation <31.39 sec (Table II, group L). The other 15 aPL patients showed a normal A P T T prolongation (see Table II, group H). Therefore, patients in group L were catalogued as true A P C - R phenotype and those in group H as spurious A P C - R phenotype, especially those whose prolongation was much longer than 31.39 seconds. When APC was added to plasma from 46 healthy individuals, previously diluted 1 to 5 with factor V deficient plasma (modified APC-R test), the mean A P T F prolongation was 63.27 seconds (see Table I), with a lower limit of normality of 52.39 seconds. When APC was added to the diluted plasma from 11 aPL patients with low A P T T ratio, all of those who had been classified as spurious APC-R phenotype by the classic APC-R test (undiluted plasma; see Table II) were also classified as spurious APC-R by the modified test. However, only 1 of the 2 patients who showed true APC-R phenotype with the classic test was classifted as having true APC-R phenotype with the modified test, which suggests that the modified test does not adequately detect APC-R phenotype in patients with aPL.

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Table I. APTT in the absence or presence of APC, APC-induced APl-r prolongation, and API-i- ratio in the plasma of healthy individuals

Classic test* (n = 95)

Mean Mean Mean Mean Mean Mean

Modified test t (n = 46)

+ 2SD - 2SD + 2SD - 2SD

APTT (A)

API"r with APC (B)

APC-induced API"r prolongation (B-A)

APTT ratio (B/A)

29.75 33.73 25.77 37.58 43.78 31.38

75.13 91,35 58.91 100.85 114.09 87.61

45.27 59.15 31.39 63.27 74,15 52,39

2.52 2.98 2.06 2.67 2,99 2,35

Data are expressed in seconds. *The assay was performed with undiluted test samples. tThe assay was performed after a 1 to 5 dilutionof the test plasma with factor V-deficient plasma.

Table II. APT[ in the absence or presence of APC, APC-induced API-r prolongation, and APT[ ratio in the plasma of patients wth aPL as determined by the classic APC-resistance test Patient number

Clinical event

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

IS IS None None None None None None None None None RA None None None

16 17 18

DVT+ PE DVT RA

API"r (A)

API"r with APC (B)

APC-induced API"r prolongation (B-A)

APIT ratio (B/A)

58.4 68.8 39.2 77.2 60,5 47,7 48.0 86.7 56.0 52.1 78.6 102.9 121.4 142.2 66.8

103.4 115.6 73.8 129.6 116,8 89.2 89.7 165.4 106.1 86,3 129.3 171.9 190,5 195.5 108,6

45.0 46.8 34.6 52.4 56.3 41.5 41.7 78.7 50,1 34.2 50.7 69.0 69.2 57.3 41,8

1.77 1.68 1.88 1.68 1.93 1.87 1.87 1.91 1.89 1.66 1.64 1.67 1.57 1.40 1.63

34,4 58.7 50.9

57.8 80.0 72.2

23.4 21.3 21.3

1.68 1.36 1.41

Data are expressed in seconds. Patients are grouped according to whether their APC-induced APTT prolongationtime was higher(H) or lower (L) than the normal lower limit (31.39 sec). DVT, deep venous thrombosis; IS, ischemic stroke; PE, pulmonaryembolism; RA, recurrent abortions.

A P C - R test in the p r e s e n c e of a n e x c e s s of platelet

phospholipids. When patients with aPL were evalu-

ated using a modified APC-R test 32 in which an excess of platelet phospholipids was added, all the patients showed a normal APTT ratio. This indicates that both the true and spurious APC-R phenotypes detected in the 32 aPL patients were caused by the elevated levels of circulating aPL in these patients. In control experiments, addition of the same excess amount of phospholipids to normal plasma did not significantly reduce the APTI" ratio. 32 Evaluation of circulating A P e levels. Since it has been reported that aPL has an inhibitory effect on the

protein C system, we hypothesized that some of the aPL present in patients with aPL could block the activation of protein C, with subsequent reduction in circulating APC levels. To prove this hypothesis, we evaluated the plasma APC levels in 14 aPL patients with APC-R phenotype. Four patients (29%) had APC/protein C ratios lower than the normal lower limit (i.e., <0.6), 2 patients (14%) had ratios just within the normal lower limit (0.6 and 0.7), and the other 8 patients (57%) showed normal APC/protein C ratios. Clinical aspects. Two of the three aPL patients with true APC-R phenotype had venous thrombotic episodes (deep vein thrombosis or deep vein thrombo-

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Table III. APTT in the absence or presence of APC, APC-induced APTT prolongation, and APTT ratio in the plasma of patients with aPL, using the modified APC-resistance test Patient number

H

Clinical event

1 2 4 5 8 10 11 12 14

IS IS None None None None None RA None

16 17

DVT+ PE DV-I-

APTr (A)

AP'rr with APC (B)

APC-induced APTr prolongation (B-A)

APTr ratio (B/A)

55.8 75.3 97.9 59,9 66,8 58.7 115.4 102.9 143.1

116.2 155.2 157.6 125.2 160.4 115.1 222.8 171.9 289.0

60.4 79.9 59.7 65.3 93.6 56.4 92.6 69.0 145.9

2.08 2,06 1.61 2.09 2.40 1.96 1.94 1,67 2.02

68.6 45.5

125.3 83.8

56.9 38.4

1.83 1,84

Data are expressedin seconds. Patientsare grouped as in Table II. Abbreviationsas in Table II.

sis plus pulmonary embolism), and the other had recurrent abortions (see Table II). Two of the 15 patients with spurious APC-R had ischemic stroke, and 1 had recurrent abortions. None of the 14 aPL patients with APTT ratio >2.06 (i.e., no APC-R) had experienced thrombotic events but one had recurrent abortions. Three of the four patients with a reduced APC/ protein C ratio had a history of thrombosis, two involving ischemic stroke and the other venous thrombosis. Of the four patients with a history of thrombosis included in this study, only three could be evaluated for circulating APC. All three of the patients with a history of thrombosis who were evaluated for circulating APC showed reduced APC levels. DISCUSSION

Phospholipids are essential components of the intrinsic pathway of blood coagulation22 and of the protein C activation 23 and anticoagulant function of APC. 1s'24.2s Therefore, inhibition of phospholipids may result in a prolongation of the APTF and/or the prothrombin time, an interference in the anticoagulant function of APC which could induce an acquired APC-R or a reduction in the level of circulating APC. APC-R genotype is induced by a point mutation in the factor V gene that produces substitution of an arginine for a glutamine at position 506 in the molecule of factor V. APC-R phenotype is defined as a poor anticoagulant response to APC in the absence of the mutation in the factor V molecule. Because of its heterogeneity, aPL can interfere with phospholipid functions at several steps. 19-21It may preferably inhibit phospholipids that act on the activation of the coagu-

lation proenzymes, on the activation of protein C, or on the anticoagulant APC function. 1°'35-38 In the first case, inhibition would result in a prolongation of the APTI', which could give a falsely low APTI" ratio and, therefore, a spurious APC-R phenotype. Our results show that, although 11 (58%) of the 19 patients with LA and 6 (86%) of the 7 patients with LA and aCL showed apparent APC-R phenotype, only 1 of the 6 patients with aCL (17%) had it. These results are in agreement with previous reports 1~'13'18 showing a higher percentage of APC-R in patients with LA than in patients with aCL. However, only 3 of the 18 patients with APC-R phenotype (reduced APTT ratio) showed a reduced prolongation of the APTI" after addition of APC. Therefore, only these 3 patients had a true APC-R phenotype. The other 15 patients with a reduced APTI" ratio but normal APC-induced APTI" prolongation should be catalogued as spurious APC-R. It would therefore seem that use of the APTF ratio to test APC-R in patients with aPL is not reliable, and it should be replaced by use of the APC-induced prolongation of APTT. The false-positive results observed when the APTT ratio is used to detect APC-R are probably caused by the basal prolongation of APTF by circulating aPL even before addition of APC) 4 The modified APC-R test, 31 in which the plasma tested is first diluted with factor V-deficient plasma, is being widely used to rule out many of the falsepositive APC-R phenotypes detected with the classic test. 39'4° Using the modified assay, we re-evaluated the aPL patients shown to have APC-R phenotype on the classic test and found that one patient who was classified as true APC-R with the classic test (patient #16) had a normal APC re-

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sponse on the modified assay (Table III). This result could have been caused by the fact that the dilution of the test plasma with factor V-deficient plasma reduced the level of aPL. If this were the case, patients with low or moderate levels of aPL could have false-negative results on the modified assay. Therefore, the modified A P C - R test does not seem to be the best assay for detecting true A P C - R phenotype in patients with aPL. Further studies with a larger number of patients are needed to clarify this point. Nevertheless, we recommend the use of the classic assay, but the results should be expressed in terms of the APC-induced prolongation of A P T T instead of the A P T F ratio. Of course, when a true A P C - R phenotype is detected in an aPL patient, it is advisable to screen for factor V Leiden to exclude an A P C - R genotype. As a consequence of our study, we propose a new classification of APC-R: (1) inherited A P C - R (genotype and phenotype), including factor V Leiden and other possible mutations, and (2) acquired A P C - R (phenotype), including true A P C - R and spurious APC-R. Our results show that two of the three patients with true A P C - R phenotype had a history of venous thrombosis and the other had recurrent abortions. In contrast, none of the 15 patients with spurious A P C - R phenotype had venous thrombosis, only 1 had recurrent abortions, and 2 had had ischemic stroke. This suggests that true A P C - R phenotype is an independent risk factor for venous thrombosis but spurious resistance is not. Therefore, an adequate identification of A P C - R phenotype seems to be mandatory. Our data also indicate that none of the 18 patients with aPL and A P C - R had factor V Leiden, which suggests that A P C - R genotype is not more frequent in patients with aPL than in the normal population. Since phospholipids are essential for normal protein C activation, 23 interference of aPL with this activation 41-44 could reduce the circulating A P C level. We evaluated the A P C levels in 14 of the 18 aPL patients who showed a low A P T T ratio. Four of the 18 patients had low APC/protein C ratios, 2 had ratios within the lower limit, and the other 8 had normal ratios. This suggests that in some patients the aPL interferes with protein C activation. The finding that 3 of the 4 patients with aPL and low circulating A P C levels had a history of thrombosis but none of the other 10 aPL patients with normal A P C levels did suggests an increased risk of thrombosis in patients with a combination of two or three risk factors (i.e., low circulating levels of A P C and A P C - R phenotype and/or aPL).

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In conclusion, true A P C - R phenotype caused by in vivo interference of aPL with the A P C anticoagulant function may induce thrombotic events. Differential diagnosis of true and spurious A P C - R in aPL patients can be made by measuring the APCinduced prolongation of A P T T instead of the A P T T ratio. Moreover, our results directly show that aPL can interfere with the activation of protein C, thus reducing the level of A P C in circulation, which may constitute an additional risk of thrombosis in aPL patients.

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