Antiplatelet Factor 4—Heparin Antibodies in Patients with Antiphospholipid Antibodies

Thrombosis Research 95 (1999) 271–279

REGULAR ARTICLE

Antiplatelet Factor 4—Heparin Antibodies in Patients with Antiphospholipid Antibodies Marta E. Martinuzzo, Ricardo R. Forastiero, Yolanda Adamczuk, Gonzalo Pombo and Luis O. Carreras Institute of Cardiology and Cardiovascular Surgery, Division of Haematology, and the Favaloro University, School of Medicine, Favaloro Foundation, Buenos Aires, Argentina. (Received 21 September 1998 by Editor J. Aznar; revised/accepted 15 February 1999)

Abstract Antibodies directed against platelet factor 4-heparin are present in patients with heparin-induced thrombocytopenia (HIT). Additionally, it has been suggested that heparin can be an antigenic target of antiphospholipid antibodies (aPL). We investigated the presence of heparin-platelet factor 4-induced antibodies (HPIA) in 33 patients with aPL. There were 30 patients with lupus anticoagulant, 25 with anticardiolipin antibodies, 21 with anti-b2 glycoprotein I, and 18 with antiprothrombin antibodies. 20 patients had a history of thrombosis and 19 had received heparin during the last 60 months. We found 7 (21.2%) who had HPIA; 5 of them also had anti-b2 glycoprotein I antibodies. Four patients had severe thrombocytopenia and suspicion of HIT. Among them, two presented high positive HPIA results, one of them with positive platelet aggregation test. The third patient showed grey zone HPIA and borderline aggregation test and the fourth one had negative results. Among patients without a history of HIT, 2 who had never Abbreviations: APS, antiphospholipid syndrome; aPL, antiphospholipid antibodies; aCL, anticardiolipin antibodies; ab2, anti-b2; GPI, glycoprotein I; anti-II, antiprothrombin antibodies; LA, lupus anticoagulant; HIT, heparin induced thrombocytopenia; IgG, immunoglobulin G; PF4, platelet factor 4; ELISA, enzyme linked immunosorbent assay; IgM, immunoglobulin M; CL, cardiolipin; OD, optical density; PBS, phosphate buffer saline; HPIA, heparin-platelet factor 4 induced antibodies; IgA, immunoglobulin A; SLE, systemic lupus erythematosus. Corresponding author: Professor Luis O. Carreras, Hematologı´a, Universidad Favaloro, Solı´s 453, (1078) Buenos Aires, Argentina. Tel: (5411) 4378-1146/1145; Fax: (5411) 4381-0323; E-mail: ,[email protected]..

received heparin presented high positive, one a moderate positive, and one a grey zone HPIA result; all of them with negative aggregation tests. Five positive sera samples were incubated with cardiolipin liposomes in the presence of b2 glycoprotein I, and whereas an inhibition greater than 50% was achieved in anticardiolipin and anti-b2 glycoprotein I activities, HPIA results did not change. We demonstrate that HPIA could be frequently found in patients with aPL. They are responsible for HIT in some cases but can also be found in patients who have not received heparin. Whether they predispose patients with aPL to HIT is not known; nevertheless, a close follow-up of heparin treatment in these patients seems to be mandatory.  1999 Elsevier Science Ltd. All rights reserved. Key Words: Antiphospholipid antibodies; Heparin-induced thrombocytopenia; Antiplatelet factor 4/heparin complexes antibodies; Antiphospholipid syndrome; Thrombosis; Thrombocytopenia

T

he antiphospholipid syndrome (APS) is defined by the presence of antiphospholipid antibodies (aPL) associated with thrombosis, recurrent abortion, and/or thrombocytopenia [1,2]. These antibodies are heterogeneous and react against phospholipid-binding proteins or their complexes with anionic phospholipids [3,4]. They can be detected by solid-phase assays: anticardiolipin (aCL), anti-b2 (ab2) glycoprotein I (GPI), and antiprothrombin (anti-II) antibodies; or by means of coagulation tests [5] as lupus anticoagulant (LA). Heparin-induced thrombocytopenia (HIT) is a

0049-3848/99 $–see front matter  1999 Elsevier Science Ltd. All rights reserved. PII S0049-3848(99)00057-2

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drug-induced immunoglobulin-mediated thrombocytopenia that is frequently complicated by lifethreatening thrombotic complications. They are usually of immunoglobulin G (IgG) isotype and the main antigen is the complex of heparin (or other high-sulphated polysaccharides) and platelet factor 4 (PF4) [6]. It has been suggested that heparin is one of the antigenic targets of some aPL. On the other hand, patients with APS frequently receive heparin as treatment for thrombotic events. A high prevalence of HIT was found in patients with LA and chronic thromboembolic pulmonary hypertension [7]. Furthermore, Lasne et al. [8] found heparin-related antibodies in some patients with primary APS. In this study we evaluated the presence of antiheparin-PF4 antibodies by enzymelinked immunosorbent assay (ELISA) in 33 patients with aPL (with and without APS) and investigated the possible cross-reactivity between these antibodies and those directed against phospholipid-protein cofactors.

1. Materials and Methods 1.1. Patients We studied 33 patients with aPL. Patients characteristics and clinical manifestations are shown in Table 1. Venous thrombotic events have been documented by venography and/or Doppler ultrasound for deep vein thrombosis and ventilation-perfusion lung scan and/or pulmonary angiography for pulmonary embolism. Chronic thromboembolic pulmonary hypertension was diagnosed when mean resting pulmonary artery pressure was above 25 mmHg during right heart catheterization with a mean pulmonary wedge pressure below 12 mmHg, associated with at least one segmental or larger perfusion lung scan defect with ventilation perfusion “mismatch.” Computed tomography scan or nuclear magnetic resonance and arteriography have been used for assessing stroke and arterial thrombosis, respectively. The study was approved by the Ethics Committee of the Institute and was carried out in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from each patient.

1.2. Blood Collection Blood was obtained by clean venipuncture from the antecubital vein and collected into plastic tubes containing 0.11 mol/L sodium citrate in a ratio of nine parts of blood to one of anticoagulant. After double centrifugation at 2,500 g for 15 minutes, platelet-poor plasma was immediately assayed. For sera preparation, blood was collected into glass tubes and allowed to clot at 378C, and then centrifuged at 1,500 g for 10 minutes. All sera were stored at 2808C until use. For platelet aggregation studies 20 mL of blood from three different normal donors were collected in plastic tubes containing 0.11 mol/L sodium citrate in the same ratio mentioned above. Blood was centrifuged at 800 g for 15 minutes and platelet-rich plasma was collected and corrected at 4003109/L.

1.3. Detection of Lupus Anticoagulant Plasma samples were evaluated for the presence of LA activity by using screening tests: partial thromboplastin time performed with PTT-LA (Diagnostica Stago, Asnie`res, France); dilute Russell viper venom time using rabbit brain cephalin and viper venom from Sigma (Sigma Chemical Co., St. Louis, MO, USA). For inhibitor identification, these clotting tests were carried out on 1:1 and 4:1 mixtures of patient and normal plasmas. As confirmatory tests, platelet neutralisation procedure (in both the activated partial thromboplastin time and dilute Russell viper venom time systems) and tissue thromboplastin inhibition assay by using undiluted and 1:500 dilution of the recombinant thromboplastin (Innovin, Dade International Ink, Miami, FL, USA) were performed. In some cases the Textarin/Ecarin assay was also used. LA was diagnosed when at least one of the screening and one of the confirmatory procedures were positive, according to previously defined criteria [9].

1.4. Measurement of Anticardiolipin Antibodies Anticardiolipin antibodies [IgG and immunoglobulin M (IgM) isotypes] were measured by using a standardised ELISA [10]. Polystyrene plates were from Linbro Titertek (ICN-Biomedicals, Horsham, PA, USA). Cardiolipin (CL), adult bovine serum, alkaline phosphatase-conjugated goat antihuman IgG or IgM and p-nitrophenyl phosphate were

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Table 1. Patients’ data including clinical manifestations and heparin treatment during the last 60 months Patient

Age

Sex

Received heparin

Clinical Manifestations

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

23 37 58 41 47 48 39 39 46 32 22 22 45 56 38 62 36 65 62 35 47 30 31 43 75 35 69 37 25

F M F F F F F F F F F F M M M M M F M M M F M M F F M M M

Yes Yes Yes Yes Yes No No Yes Yes No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No

30 31 32 33

50 17 19 26

F M F M

No No No No

PE, CTPH, thrombocytopenia PE, CTPH, thrombocytopenia AT, thrombocytopenia SLE, stroke, fetal loss, thrombocytopenia DVT, PE Stroke SLE DVT, fetal loss SLE, DVT SLE, fetal loss SLE SLE, fetal loss DVT, AT DVT, AT AT AT AT PE, CTPH DVT, PE, CTPH PE, CTPH DVT, PE, CTPH PE, CTPH DVT, PE AT, stroke Fetal loss Fetal loss Mitral valve replacement Migraine Autoimmune hepatitis, severe hypoprotrombinemia with bleeding manifestations Asymptomatic Asymptomatic Asymptomatic Asymptomatic

Abbreviations: F5female, M5male, PE5pulmonary embolism, CTPH5chronic thromboembolic pulmonary hypertension, AT5arterial thrombosis, SLE5systemic lupus erythematosus, DVT5deep vein thrombosis.

from Sigma. Optical density (OD) was read by using a MS2 TitertekPlus Microplate Reader (ICNFlow, ICN Biomedicals, Costa Mesa, CA, USA). International standards (Louisville APL Diagnostics, Louisville, KY, USA) and our own control sera were used for the standard curve calibration. Results were expressed as standard units for either IgG or IgM. Inter- and intraassay variabilities were less than 10% for each isotype. Titres higher than 10 standard units of IgG or IgM were considered positive.

1.5. Measurement of Anti-b2GPI Antibodies We used the homemade ELISA for ab2GPI of IgG and IgM isotypes previously reported [4,11]. Briefly,

microtiter plates (Nunc-Immunoplate, MaxiSorp, Kamstrup, Roskilde, Denmark) were irradiated by electron beam at 100 kGy. Wells were coated with 2 mg of purified human b2GPI (Behringwerke AG, Marburg, Germany). Horseradish peroxidase-conjugated rabbit antihuman IgG F(ab9)2 fragment, antihuman IgM antibodies and o-phenylenediamine were from Sigma . OD at 492 nm was measured in the MS2 Reader and nonspecific binding of each serum sample was determined on phosphate buffer saline (PBS)-coated wells and subtracted. Positive and negative controls were included in each plate. Inter- and intraassay coefficients of variation averaged 10.9 and 3.1%, respectively. Our own negative and positive controls were run in each plate in order to control the day-to-day

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variation. The cutoff values (OD31000555 for IgG and 50 for IgM) were assessed by the method of percentiles (99th) by using 50 normal sera.

1.6. Measurement of Anti-II Antibodies We performed the homemade ELISA as previously described [4]. Polystyrene plates (Nunc-MaxiSorp) were coated with 1 mg/well of purified human prothrombin (Stago). ELISA was performed as described above for ab2GPI. The absorbance of control wells (PBS) was subtracted from the absorbance in the prothrombin-coated wells to account for nonspecific binding. Inter- and intraassay precision yielded coefficient of variance of 10.1 and 4.2%, for IgG and IgM. As described for ab2GPI, our own controls were run in each plate. The cutoff values (OD31000) were 67 for IgG and 53 for IgM.

1.7. Detection of Heparin-PF4-Induced Antibodies Heparin-PF4-induced antibodies (HPIA) [IgG, immunoglobulin A (IgA), and IgM] were detected in patients’ sera by ELISA [12] using polystyrene plates coated with PF4 complexed with heparin in estoechiometric proportions (Asserachrom HPIA, Stago). Cutoff values established by the manufacturer for lot used were OD at 490nm<0.200 for negative results, between 0.200 and 0.400 for the grey zone and .0.400 for positive results. The lower limit was confirmed in our laboratory at OD,0.200 by testing 20 normal control sera samples.

1.8. Platelet Aggregation Test for HIT The platelet aggregation test for HIT was performed by a routine assay as previously described [13], by using patients’ heat inactivated sera and platelet-rich plasma from three different healthy donors.

1.9. Inhibition Studies Cardiolipin liposomes were prepared by evaporating CL in ethanol to dryness under a stream of nitrogen and then resuspended in 200 mL of PBS and vortexed for 5 minutes. 100 mL of serum di-

luted with 700 mL of pooled normal plasma (as a source of b2GPI) were mixed with CL liposomes. The final concentrations of b2GPI and CL in the mixture were 200 and 1350 mg/mL, respectively. The mixtures were incubated for 2 hours at 378C. After microcentrifugation at 15,000 g for 15 minutes, the supernatants were collected and stored at 2208C until use. Five sera from patients with aPL and HPIA positive results were processed. One control mixture of each one and PBS instead of liposomes were performed and processed in the same way. The serum of a patient with HIT (HPIA1) without aPL and a normal pooled serum were processed and used as HPIA positive and normal control, respectively. aCL, ab2GPI, and HPIA were measured on the supernatants. Inhibition was calculated according to the formula shown in Eq. (1): [(ODpat PBS2ODNC PBS)2(ODpat Lip2ODNC Lip)] 3100 ODpat PBS2ODNC PBS

(1)

where pat5patient, NC5normal control, PBS5 phosphate buffer saline, Lip5preincubation with cardiolipin liposomes.

2. Results Patients’ characteristics and their clinical features are shown in Table 1. There were 20 patients with primary APS, four with APS secondary to systemic lupus erythematosus (SLE), two SLE without APS, three miscellaneous, and four asymptomatic. Among the 33 patients evaluated, there were four with severe thrombocytopenia (platelet count ,803109/L) and high suspicion of HIT (patients one through four). Table 2 shows that HPIA ELISA gave a high positive result in two (patients one and two), one of them presenting a positive aggregation test. Patient three had HPIA within the grey zone and borderline positive aggregation test whereas patient four had negative results in both tests. Among patients without thrombocytopenia, there were high positive HPIA results in two who had never received heparin (patients six and seven) and in one who had received unfractionated heparin 28 months before because of a pulmonary embolism (patient five). Additionally, patient eight presented a result within the grey zone and

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Table 2. Laboratory data from patients with aPL Patient 11 21 31 42 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

LA

aCL

ab2GPI

anti-II

HPIA (10003OD)

Aggregation Test

1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 BL1 1 1 2 1 1 2 2 2 2 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1

1 1 2 1 1 1 2 1 BL1 1 2 2 1 1 2 2 2 2 1 2 BL1 2 1 1 1 1 2 2 1 1 1 1 1

1 1 2 1 1 2 2 1 1 1 2 2 1 2 2 2 2 2 1 2 BL1 2 2 1 1 2 2 1 1 1 1 1 1

2158 1280 372 187 660 1335 1115 265 50 111 108 125 138 128 155 94 90 151 80 102 114 185 91 87 105 120 99 90 88 50 83 108 85

1 2 BL1 2 2 2 2 2 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

Abbreviations: LA5lupus anticoagulant, aCL5anticardiolipin antibodies, ab2GPI5anti-b2 glycoprotein I antibodies, anti-II5Antiprothrombin antibodies, HPIA5heparin-platelet factor 4 induced antibodies, HIT5heparin induced thrombocytopenia, BL5borderline, ND5not determined. 1 HIT diagnosis positive, 2high suspicion of HIT without confirmation.

had also received heparin 48 months before as treatment for deep vein thrombosis. All these patients presented negative aggregation tests. LA and aCL were positive in all HPIA positive patients (n57). Moreover, five and four of them also had ab2GPI and anti-II, respectively. Sera from patients one, two, five, six, and seven, serum from a patient with HIT without aPL as control, and a normal pooled sera were incubated with CL liposomes or PBS in the presence of normal plasma as a source of b2GPI. After the inhibition experiments, aCL, ab2GPI (IgG isotype for both assays), and HPIA ELISAs were performed on the supernatants.

Anticardiolipin and ab2GPI activities were clearly inhibited: aCL from 45 to 100% inhibition and ab2GPI from 56 to 100% (Figure 1A and B). On the contrary, HPIA titres did not change after incubation with CL liposomes (Figure 1C).

3. Discussion HIT should be diagnosed basically by two criteria: (1) one or more clinical event associated with this syndrome (isolated thrombocytopenia, or associated with a new thrombotic event) and (2) labora-

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Fig. 1. Sera from five HPIA positive patients with aPL and one control patient with heparin-induced thrombocytopenia without aPL were incubated for two hours at 378C in the presence of cardiolipin liposomes with b2GPI or PBS alone. After centrifugation the supernatants were assayed in the aCL (A), ab2GPI (B) and HPIA (C) ELISAs.

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tory evidence for heparin-dependent immunoglobulin (usually IgG) using specific and sensitive tests [6]. Recently, an ELISA for HPIA that uses heparin-PF4 complex as antigen has been developed [13], and high but not complete concordance with functional assays has been found [13–15]. We report here seven positive results of HPIA among 33 patients with aPL, where three of them developed HIT. However, we also found high positive results in two patients who had never received heparin. Recently, Lasne et al. [8] found that three out of 20 patients with primary APS presented borderline or positive HPIA results and one of them gave a weak positive result in the functional serotonin release assay. They found these antibodies in patients without HIT syndrome, like some patients from our study. Moreover, Auger et al. [7] reported that HIT was significantly more frequent in patients with chronic thromboembolic pulmonary hypertension with LA than in those without LA, indicating a close relationship between aPL and HIT. On the other hand, aCL have been found in about one third of patients with heparin-induced thrombocytopenia [16], but a comparable high prevalence of aCL was detected in the control group receiving heparin without developing thrombocytopenia. There was no concordance between functional aggregation test and HPIA-ELISA results in our positive patients. Considering that the HPIAELISA detects antibodies of A, G, and M immunoglobulin isotypes, and that functional assays only detect IgG, the other isotypes could be implicated in HPIA positive results of some patients. In addition, the low prevalence of positive results in the functional aggregation test could be due to the use of platelet-rich plasma instead of washed platelets, that is less sensitive [12,17]. It was reported by several authors that reactivity against heparin or other glycosaminoglycans may be found in sera from patients with aPL [18–22], though others did not confirm this observation [23]. It was shown that heparin added to sera samples in the ELISA for aCL or antiphosphatidylserine antibodies inhibited the specific binding of aPL to different extent. Ermel et al. [21] also found that heparin affinity chromatography absorbed over 80% of the IgG aCL antibodies in sera from women with aPL and recurrent fetal loss. Interestingly, Shibata et al. [19] showed the presence of IgG antiheparin antibodies in the immunoglobulin fractions purified from

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seven patients with APS. They showed that antibody reactivity to heparin was inhibited in fluid phase by incubation with heparin, heparan sulfate, or CL with b2GPI. They also demonstrated that antiheparin antibodies were due to a specific recognition of a disaccharide present in the pentasaccharide that binds antithrombin III, rather than low-affinity electrostatic interactions. Additionally, it has been shown that the charge is not the only important feature for heparin inhibition of aPL reactivity and that heparin inhibited the aPL and not b2GPI binding to phospholipid [20]. Pengo et al. [22] found that aCL from four APS patients bound to heparin after recognising b2GPI, and postulated that “in vivo” binding of these antibodies to heparin-like substances could inhibit the antithrombotic properties of endothelial cells. Furthermore, an inhibition of heparin-accelerated formation of antithrombin III-thrombin complexes by antiheparin antibodies present in APS has been reported by some investigators [18,19]. In our study we demonstrated that binding of the antibodies to heparin-PF4 complexes was not inhibited by preincubation of samples with cardiolipin liposomes in the presence of b2GPI, although aCL and ab2GPI were inhibited to considerable extents. Heparin can form complexes with b2GPI by crossed immunoelectrophoresis as shown by McNally et al. [24]. They also demonstrated that these complexes have increased aPL cofactor activity compared to b2GPI. Our data suggest that antibodies detected by HPIA ELISA in these patients are directed against heparin or heparin-PF4 complexes and that they are different antibodies from aCL or ab2GPI, because they are not removed by adsorption with CL liposomes. This is in accordance with the production of antiheparin antibodies in animal models of autoimmunity [25] and the high titres of antiheparan sulfate antibodies found in patients with SLE [26]. Despite the similarities found between the pathogenic mechanisms involved in APS and HIT [27], the role of HPIA in the development of thrombosis of patients with APS is actually unknown. Furthermore, whether these antibodies are pathogenic or responsible for HIT development in these patients is also not known. Therefore, a close follow-up of aPL patients under heparin treatment seems to be mandatory.

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