Thrombosis Research 135 (2015) 368–374

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Regular Article

Endothelial and platelet microparticles in patients with antiphospholipid antibodies K.A. Breen a,⁎, K. Sanchez b, N. Kirkman b, P.T. Seed c, K. Parmar a, G.W. Moore b, B.J. Hunt a,c a b c

Guys and St.Thomas’ NHS Foundation Trust, London, United Kingdom Viapath, Guys and St.Thomas’ NHS Foundation Trust, London, United Kingdom King’s College, London, United Kingdom

a r t i c l e

i n f o

Article history: Received 9 June 2014 Received in revised form 3 November 2014 Accepted 30 November 2014 Available online 4 December 2014 Keywords: Antiphospholipid antibodies Antiphospholipid syndrome Microparticles

a b s t r a c t Background: The antiphospholipid syndrome (APS) is the association of thrombosis and recurrent pregnancy loss and/or pregnancy morbidity with persistent antiphospholipid antibodies (aPL). Previous studies of microparticles in patients with APS/aPL have mainly been small and findings, contradictory. Objectives: To quantify endothelial and platelet microparticle levels in patients with isolated antiphospholipid antibodies or primary antiphospholipid syndrome (PAPS). Patients/Methods: We measured endothelial and platelet microparticle levels by flow cytometry in 66 aPL/PAPS patients and 18 healthy controls. Results: Levels of circulating platelet (CD41 and CD61) and endothelial microparticles (CD51 and CD105) were significantly increased in patients with PAPS and aPL compared to healthy controls. There were correlations between platelet and endothelial microparticles levels in all patients with aPL. Conclusions: Platelet and endothelial microparticles are increased in all patient groups within this cohort of patients aPL. Whether they may have a role in the pathogenesis of APS merits further study. © 2014 Elsevier Ltd. All rights reserved.

Introduction Microparticles (MP), first described in 1967 [1], are small membrane fragments of 0.1-1 μm in diameter released from cell surfaces of endothelial cells, platelets, leucocytes and trophoblast cells with platelet microparticles constituting the majority of circulating microparticles [2]. MP express antigens specific to their cell origin providing a method for detection. MP play a role in inflammation, thrombosis and angiogenesis through cell-cell interaction and signalling and although present in healthy individuals are elevated in many disease states including inflammatory disorders, prothrombotic disorders, malignancy and infection. Cell activation and apoptosis by varied stimuli such as cytokines, thrombin or hypoxia, leads to microparticle release from the cell surface. Microparticles express antigens according to the cell from which they are derived and their size and composition can differ according to stimulus for release. Expression of negatively charged phospholipids by microparticles provides a procoagulant surface, which allows binding of coagulation factors and promotes formation of prothrombinase complexes [3]. Numerous adhesion molecules, receptors, enzymes and ⁎ Corresponding author at: Department of Haemophilia and Thrombosis; 1st floor, North Wing, St.Thomas’ Hospital, Westminster Bridge Rd., London SE1 7EH, United Kingdom. Tel./fax: +44 2071887263. E-mail address: [email protected] (K.A. Breen).

http://dx.doi.org/10.1016/j.thromres.2014.11.027 0049-3848/© 2014 Elsevier Ltd. All rights reserved.

major histocompatibility complexes (MHC) are expressed on leucocyte, endothelial and platelet microparticles and this, in addition to their size, forms the basis for microparticle detection. Microparticles have been implicated in the pathogenesis of inflammation [4], thrombosis [4] and malignancy [5]; and have been studied in conditions of cardiovascular disease such as coronary artery disease and stroke [6], haemolytic states such as TTP [7] and sickle cell disease, inflammatory conditions including Crohn’s disease in addition to pregnancy, miscarriage [8,9], pre-eclampsia and venous thrombosis [10,11]. Antiphospholipid syndrome (APS) is an immune disorder associated with thrombotic and obstetric complications in association with persistent antiphospholipid antibodies [12]. To date, there have been a small number of studies of microparticles in patients with PAPS or healthy individuals with antiphospholipid antibodies (aPL) and findings have been variable. Studies have usually included only patients with a history of thrombosis or patients with isolated aPL in association with other immune disorders, and only one study to date has included patients with obstetric complications associated with aPL [13]. Three studies of endothelial microparticles levels in patients with isolated aPL and thrombotic complications of aPL demonstrated increased endothelial microparticle levels in patients but study numbers were small [14–16]. Another study including only patients with obstetric complications of aPL (n = 9) showed no difference in EMP compared to healthy controls [13]. Platelet microparticles were found to be elevated in a study of patients with both thrombotic and obstetric complications of aPL [17] and in another

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study including patients with thrombotic complications of aPL [16]. However two other studies found no difference in platelet microparticles in patients with thrombotic and obstetric complications of aPL [13,18]. The primary aim of this study was to measure levels of circulating endothelial and platelet microparticles in a large group of patients with thrombotic and obstetric complications of aPL, and isolated aPL and compare to healthy controls. The secondary aim was to assess if there was a correlation between platelet and endothelial levels. Materials and Methods Patients Ethical approval was obtained from London Surrey Borders Research Ethics Committee. Samples were obtained from non-pregnant female patients attending outpatient clinics at our institution who had APS according to International Consensus statement criteria [12], or had persistent aPL without associated complications. Patients with SLE and other inflammatory disorders, acute illness, intercurrent infection or malignancy were excluded in view of potentially increased microparticle levels in these patient groups. The control group was recruited from healthy hospital staff who were not known to have aPL or any of their associated complications. Blood Sample Collection and Processing Blood was drawn by flawless venepuncture into Vacuette® evacuated collection tubes (Greiner Bio-One, Stonehouse, UK) containing citrate 0.105M in a ratio of 9:1 and stored at room temperature for up to 3 hours prior to analysis. Samples for measurement of microparticle procoagulant activity were centrifuged at 2000g for 15 min at 4°C. Plasma was removed and centrifuged again at 2000g for 15 min at 4°C. Following this step, plasma was divided into 500μl aliquots and further centrifuged at high-speed (12,000g) for 2 minutes at 4°C and then stored at −80°C until use. Samples for measurement of platelet and endothelial microparticles were centrifuged at 2000g for 15 min at 4°C, plasma removed and centrifuged again at 2000g for 15 min at 4°C. Plasma was then divided into 500μl aliquots, and stored at −80°C until use. Samples from patients with aPL/APS and controls were processed in the same manner. aPL Determination Patients with PAPS or isolated aPL had demonstrated positive testing for aPL (lupus anticoagulant, IgG/IgM anti-cardiolipin antibodies, or IgG/ IgM anti-β2-glycoprotein I antibodies) on two or more occasions greater than 12 weeks apart [12]. Solid Phase Assays Anticardiolipin antibodies (isotypes IgG and IgM) were quantified by indirect ELISA using AEUSKULISA® Cardiolipin-GM reagents (Grifols UK, Cambridge, UK). Anti-β2-glycoprotein I antibodies (isotypes IgG and IgM) were quantified by indirect ELISA using QUANTA Lite® reagents (INOVA Diagnostics Inc. San Diego, CA, USA). Positive cut-off values for both were determined according to Sydney Criteria (N 40 GPL/MPL or N 99th percentile)[12]. Lupus Anticoagulant Detection Lupus anticoagulant (LA) detection in compliance with published guidelines [19] was determined by dilute Russell’s viper venom time (DRVVT) and dilute APTT (DAPTT), accompanied by appropriate confirmatory tests. DRVVT was performed with Life Diagnostics LA Screen and LA Confirm reagents (Life Therapeutics, Clarkston, GA, USA). DAPTT was

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performed using PTT-LA (Diagnostica Stago, Asniéres, France) in the screen with a platelet neutralisation procedure employing Biodata Platelet Extract Reagent (Alpha Laboratories, Hampshire, UK) in the phospholipid-dependence confirmatory test. Patients on oral anticoagulation additionally received screening with Taipan snake venom time (TSVT)[20] employing Diagen Taipan venom (Diagnostic Reagents, Thames, UK) with an Ecarin time confirmatory test using E. carinatus venom (Diagnostic Reagents)[21,22]. All elevated screens received the confirmatory test plus a screen and confirmatory test on 1:1 mixing studies with normal plasma. Technoclone Lyophilised Platelet Poor Plasma, (Pathway Diagnostics Ltd, Dorking, UK) was used as the normal plasma throughout. LA assays were performed on a Sysmex CS2000i analyser, (Sysmex UK, Milton Keynes, UK). CRYOcheck™ Normal Reference Plasma, (Alpha Laboratories, Hampshire, UK) was used as the normal plasma throughout.

Microparticle Procoagulant Activity Assay Microparticle procoagulant activity was also measured by a specific ELISA (Zymuphen MP-Activity ELISA kit, Hyphen Biomed, Surrey, UK). Briefly, microparticles present in a plasma sample bind to Annexin A5 coated wells and expose their phospholipid surface. On addition of FXa-FVA, calcium ions and purified prothrombin, thrombin generation occurs and is measured by cleavage of a chromogenic thrombin substrate, which produces absorbance at 405nm, phospholipid concentration being the rate-limiting step. Intra- assay and inter-assay CVs were 3-8% and 5-10% respectively.

Isolation and Quantification of Platelet and Endothelial Microparticles To determine if microparticles were of platelet or endothelial origin, samples were labeled with a CD41 or CD61 fluorescently labeled antibody to detect platelet microparticles and CD51 or CD105 labeled antibodies to detect endothelial microparticles by flow cytometer. Two different antigens were used to confirm microparticle cell origin since microparticles are known to express different antigens according to the stimulant of their release [5].

Method A 500μl aliquot of platelet-free plasma was thawed at room temperature. 10μl of sample was incubated with CD41 PECy5, CD61 PECy7, CD51 FITC, and CD105 PE fluorescently labeled antibodies (Beckman Coulter, California, USA) in each of 2 polypropylene tubes. Samples were incubated in the dark at room temperature for 30 minutes. Nine hundred μl of filtered PBS based enzyme free cell dissociation solution was added, followed by 100 μl of flow count fluorospheres (renumeration beads) of a known concentration to tube 2 only and samples immediately analysed. Data was acquired using a FC500 flow cytometer. Tube 1 was used to gate the negative population. This gate was then used to collect positive events from analysis of tube 2. Regions corresponding to microparticles were defined using forward light scatter versus side angle light scatter intensity dot plot representation. Microparticles were defined as elements positively labeled by CD41, CD61, CD51 or CD105 monoclonal antibodies. Microparticle quantification (absolute counts) was determined using the following formula (previously described [15]):

Absolute Count ðcells=μLÞ ¼ ðTotal Number of Cells Counted = Total Number of Fluorospheres CountedÞ  Flow‐Count Fluorospheres Conc:

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Table 1 Demographic details of patients included in the study. Variables

APS/aPL (n = 66)

Thrombotic APS (n = 37)

Obstetric APS (n = 11)

Isolated aPL (n = 18)

Controls (n = 18)

P value

Age: Median (range) Sex (female: male) Ethnicity (White:Asian:Black) Lupus anticoagulant positive IgG or IgM ACL (% patients) IgG or IgM anti-β2GPI Thrombotic APS (venous:arterial:both) Obstetric APS (early & late) † Warfarin Heparin Aspirin

47 (23-61) 47:0 34:1:2 23 (62%) 11 (30%) 18 (49%) 21:16:5 8 34 0 2

47 (23-61) 47:0 34:1:2 23 (62%) 11 (30%) 18 (49%) 21:16:5 8 34 0 2

41 (32-54) 11:0 9:1:1 10 (56%) 0 (0%) 3 (61%) 0 11 0 0 7

43.5 (19-73) 18:0 14:2:2 15 (83%) 2 (11%) 6 (33%) 0 0 0 0 8

37.5 (20-58) 18:0 12:4:2 0 (0%) 0 (0%) 0 (0%) 0 0 0 0 0

0.05 n/a 0.15

Key: ACL - Anti-cardiolipin antibodies, β2GPI - β2 glycoprotein-I, APS – antiphospholipid syndrome. † Early obstetric complications defined according to International Statement Criteria [12].

4.25) for CD105. No differences were observed between patients with obstetric APS or isolated aPL and healthy controls.

Statistical Analysis Statistical analysis was performed using Stata-11 (Statacorp,Texas). Data were not normally distributed so was logarithmically transformed. Multiple regression analysis was used to determine differences between groups adjusting for age, ethnicity and medications (aspirin and warfarin). Group analysis included comparison of microparticle levels of all patients with PAPS/aPL with healthy controls as well as comparison of aPL subgroups with healthy controls according to aPL related complication (thrombosis, obstetric or none). Correlations between platelet and endothelial microparticles levels were determined, and in addition, correlations between platelet and endothelial microparticles levels and microparticle procoagulant activity were determined.

Endothelial Microparticles Levels of circulating endothelial microparticles (both CD51 and CD105) were significantly increased in aPL/APS patients compared to healthy controls; ratio of geometric means, with CI: 2.27 (1.22 - 4.25) for CD51, 1.95 (1.09 - 3.51) for CD105. When patients were analysed according to subgroup, levels of endothelial microparticles were significantly higher in patients with thrombotic complications compared to healthy controls; ratio of geometric means with CI: 2.25 (1.19 - 4.25). No differences were observed in levels between patients with obstetric APS or isolated aPL and healthy controls.

Results Microparticle Procoagulant Activity 66 patients with aPL and 18 healthy controls were included in the study. Of the patients with aPL; 37 had thrombotic APS (8 of the patients included in this group also had obstetric complications), 11 had obstetric APS and 18 had aPL with no known associated complications. Patient demographics are described in Table 1. Results of microparticle analysis are demonstrated in Table 2.

Microparticle procoagulant activity did not differ significantly between patients with PAPS/aPL and healthy controls. Patients were also analysed according to subgroup, no differences were observed in patients with APS (thrombotic APS, obstetric APS, both thrombotic and obstetric APS) or isolated aPL and healthy controls.

Platelet Microparticles

Correlations Between Markers

Levels of circulating platelet microparticles (both CD41 and CD61) were significantly increased in aPL/APS patients compared to healthy controls; ratio of geometric means, with CI: 2.14 (1.13-4.08) for CD41, 1.89 (1.04-3.45) for CD105. When patients were analysed according to subgroup, levels of platelet microparticles were significantly increased in patients with thrombotic complications compared to healthy controls; ratio of geometric means, with CI: 2.52 (1.27 – 5.01) for CD41, 2.22 (1.16 -

Significant positive correlations were observed (see Fig. 1) between both platelet microparticle markers, CD41 and CD61(r = 0.96). Similarly, significant positive correlations were observed between endothelial microparticle markers, CD51 and CD105(r = 0.98). Platelet and endothelial microparticle markers also had significant positive correlations (CD41:CD51 r = 0.84, CD41:CD105 r = 0.85, CD61:CD51 r = 0.82, CD61:CD105 r = 0.85). Microparticle procoagulant activity did not

Table 2 Microparticle levels by patient group including comparisons with healthy controls. Plasma level

PAPS/aPL (n = 66)

Thrombotic APS subgroup (=37)

Obstetric APS subgroup (n = 11)

Isolated aPL subgroup (n = 18)

Healthy controls (n = 18)

CD41 (MP/ul) Geometric means (95% CI) CD61 (MP/ul) Geometric means (95% CI) CD51 (MP/ul) Geometric means (95% CI) CD105 (MP/ul) Geometric means (95% CI)

1554 (1174-2058) 2.14 (1.13-4.08) 661 (508-860) 1.89 (1.04-3.45) 1564 (1189-2057) 2.27 (1.22-4.25) 801 (620-1037) 1.95 (1.09-3.51)

1842 (1222-2775) 2.52 (1.27-5.01) 789 (536-1161) 2.22 (1.16-4.25) 1868 (1249-2794) 2.68 (1.36-5.26) 941 (644-1377) 2.25 (1.19-4.25)

1788 (825-3872) 2.44 (0.91-6.55) 639 (309-1325) 1.80 (0.71-4.57) 1545 (723-3301) 2.21 (0.84-5.83) 668 (327-1368) 1.60 (0.64-3.99)

994 (566-1743) 1.36 (0.60-3.07) 461 (271-783) 1.30 (0.60-2.80) 1076 (619-1869) 1.54 (0.69-3.43) 632 (375-1064) 1.51 (0.71-3.21)

731 (419-1276)

Key: Microparticle levels by patient group (Geometric means with 95% CI), including comparisons with healthy controls.

354 (210-599) 698 (404-1206) 418 (250-700)

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Fig. 1. Correlations between microparticle platelet (CD41, CD61) and endothelial markers (Cd51, CD105) and microparticle procoagulant activity.

correlate with any of the platelet or endothelial microparticle markers (CD41, CD61, CD51 or CD105). Discussion To date, few studies of microparticles in patients with APS have been performed, patient numbers have mostly been small (excluding a study by Jy et al. [16]), patients included were usually patients who had aPL associated with systemic lupus erythematosus (SLE) and patients with aPL associated complications of pregnancy morbidity were rarely studied. This is one of the largest studies to date to assess MP levels in patients with aPL. We assessed levels of both endothelial and platelet microparticles in a large group of patients with persistent presence of aPL, all associated complications of aPL and without associated autoimmune disease. There have been a small number of studies of endothelial and platelet microparticle levels in patients with aPL and findings have been variable (summarized in Table 3). We found patients with PAPS/aPL had significantly increased levels of both endothelial and platelet microparticles compared to healthy controls. Similar findings of elevated levels of endothelial microparticles have been found in previous studies of patients with APS/aPL [14–16]. In addition, we found patients with thrombotic complications of aPL had significantly elevated levels of endothelial microparticles when patients were compared according to subgroup, but levels were not elevated in patients with obstetric complications of aPL or in patients with isolated aPL. While it is difficult to ascertain whether this is as a result of a previous thrombotic episode or may predispose to an increased risk of future thrombosis, it is noteworthy none of the patients recruited to the study had a known thrombotic event for at least 3 months prior to recruitment. Combes et al. [15] also found elevated levels of endothelial microparticles in patients with thrombotic complications of aPL but only a small number of patients were included in this study. In their later study [14], although they found levels of endothelial microparticles were higher in patients with APS, they found no difference in levels between patients with thrombotic complications of aPL compared to those with isolated aPL. Jy et al. [16] had similar findings in their study. Finally, most patients included in the previously discussed studies included patients with only thrombotic complications or isolated aPL and most patients had aPL secondary to underlying autoimmune disease. Patients recruited

to our study were a clearly defined group of patient who had only primary APS or isolated aPL without underlying autoimmune diseases and had either aPL related thrombotic or obstetric complications. We have also demonstrated significantly increased levels of platelet microparticles in patients with PAPS/aPL compared to healthy controls. In addition, patients with aPL associated thrombotic complications had significantly higher levels of platelet microparticles but those with obstetric complications and isolated aPL did not. Previous studies of platelet microparticles in patients with APS have failed to show any difference in levels of platelet microparticles in patients with APS compared to controls [13,16–18]. However, although this was the case in the study by Jy et al. [16], when they performed subgroup analysis, they showed elevated platelet microparticle levels in patients with thrombotic complications of aPL. There are several potential explanations for variations in findings from studies of microparticles in patients with APL. As discussed previously, most of the studies were small and only included a very small number of patients (mostly b 10) with aPL secondary to other autoimmune disorders, usually SLE [14,15,17,18]. Microparticle antigen expression varies according to stimulant of release [5] and this may somewhat explain the variation in findings since different antigens were targeted in the studies. CD51 (vitronectin) is a glycoprotein which binds serine proteases including plasminogen activator inhibitor (PAI-1) and the terminal complement complex and CD105 (endoglin) is a membrane glycoprotein and acts a receptor for transforming growth factor-β (TGF-β). CD41 (glycoprotein IIb) and CD61 (glycoprotein IIIa) are commonly used for platelet microparticle detection. Previous studies of platelet microparticles in APS have used anti CD146 [18]. anti CD31 [13,16] and CD42 [23] in addition to anti CD41 [18] antibodies, and anti CD31 [16], CD 41 [13], CD42 [23] and CD144 [13] in addition to CD51 [14,15] in studies of endothelial microparticles. The rationale for choice of antigenic markers used in these studies is not explained in depth likely due to difficulty in comparing protocols for flow cytometric detection of specific antigens due to differences between instruments and their settings. Detection and measurement of microparticles is subject to wide variation due to lack of standardization. Scientific standardization committees are working towards standardization of microparticle enumeration techniques. Minor changes in any of the multiple steps required for microparticle analysis (including phlebotomy, time to centrifugation,

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Table 3 Studies of microparticles in APS. Study

Number of patients

APL related complications

APL subtype

Markers studied

Findings

Comments

Galli M et al. [32]

7

5 thrombotic 2 none

LA or IgG ACL

Procoagulant activity assessed by prothrombinase activity EMP (CD51)

Prothrombin-ase activity inhibited by aPL.

Not all subtypes of aPL studied

Increased EMP in those with aPL vs controls. Increased EMP with thrombotic complications of aPL vs those without.

Small patient numbers. Not clear if aPL persistent or of relevance in setting of infection/malignancy. Only patients with LA included

No difference in PMP levels between groups Increased levels with SLE and APS plasma vs. controls Increased procoagulant activity with APS plasma only vs. controls

Not all subtypes of aPL studied

5 PAPS 8 SLE/ APS 6 aPL (AID) 9 aPL (infection) 2 Isolated aPL 30 healthy controls Joseph et al. 20 PAPS [17] 14 SLE/APS16 SLE Dignat-George HUVEC incubated with et al. [14] SLE + APS plasma

LA only Thrombosis Thrombosis None None None None Thrombosis/ LA, IgM or Obstetric/None IgG ACL LA, IgM or IgG ACL

Dignat-George 23 PAPS et al. [14] 14 SLE/APS 28 SLE/aPL 23 SLE (apl neg) 25 thrombosis (no aPL/AID) 25 healthy controls Pereira J et al. 6 SLE/APS [18] 24 SLE 20 healthy controls

Thrombosis Thrombosis None None None

Jy W et al. [16] 60 PAPS 28 APL 39 healthy controls

Thrombosis None

EMP (CD31+/42-) LA, ACL or anti-β2-GPI PMP (CD31+/42+) antibody

Alijotas-Reig et al. [13]

Obstetric

PMP (CD41) LA, ACL or anti-β2-GPI EMP (CD31+/CD144+/CD41-) antibody LMP (CD45)

Combes et al. [15]

9 APS

LA, IgM or IgG ACL

PMP EMP levels following incubation EMP procoagulant activity assessed by clotting time ratios EMP (CD51)

PMP (CD61, CD146)

Not all subtypes of aPL studied

Increased EMP in PAPS, SLE/APS, SLE/aPL vs. controls No difference in aPL + thrombosis vs. isolated aPL No correlation with thrombosis

No patients with aPL related obstetric complications included Not all subtypes of aPL studied

No difference in PMP levels between patients with SLE/APS vs SLE alone. No correlation with thrombin generation EMP increased in APS/aPL vs controls but not in thrombotic complications vs those without. PMP increased in thrombotic complications of aPL vs those without but not in APS/aPL vs. controls No differences in PMP,EMP,LMP compared to aPL negative

Very small patient numbers.

aPL persistence determined by 2 positive tests N6weeks apart No patients with aPL related obstetric complications included High speed spin not used for PMP

Small patient numbers

Key: LA – Lupus anticoagulant, ACL- Anti-cardiolipin antibodies, β2 GPI - β2 glycoprotein-I, APS – antiphospholipid syndrome, SLE- systemic lupus erythematosus, AID – autoimmune disease, aPL – antiphospholipid antibodies, EMP – endothelial microparticles, PMP – platelet microparticles, LMP – leucocyte microparticles

conditions of centrifugation, sample storage and analytical technique) can result in significant changes in microparticle levels [24–26]. In our study, all patients had phlebotomy performed in a similar manner and samples were centrifuged within three hours of collection. Following centrifugation, both patient and control samples were immediately frozen at -80°c and thawed under similar conditions prior to processing. It has been questioned whether freezing may affect microparticle levels but few studies performed comparing microparticle levels in samples which are stored at -80°C to those stored at either 4°C or room temperature. Simak et al. found no significant differences in endothelial cell microparticle levels in samples stored at -80°C compared to samples stored at 4°C [27] while Shet et al. similarly showed no significant differences in samples stored at -80°C compared to samples stored at room temperature [28]. Some groups advocate ‘snap’freezing immediately in liquid nitrogen but if not possible, as long as study and control samples are treated similarly, there should be minimal differences between samples [29]. Microparticles are detected by flow cytometry using methods predetermining their size and antigen expression. Quantification of microparticle levels is through flow cytometric detection of the number of events by forward scattering and the density of events by side scattering. Although flow cytometry is the method of choice for detection of microparticles, recent debates have also suggested limitations of this method. As discussed previously, protocols are difficult to compare due to differences between instruments and their settings, and therefore results are difficult to reproduce. Microparticle size is determined

using beads of a predefined diameter and it is now thought microparticles size may lie outwith this diameter potentially underestimating microparticle levels since flow cytometry has limited sensitivity for microparticle detection, particularly when the diameter is below 500μm [30,31]. In addition, antigen labeling is required for detection of MP from specific cell lines and it is thought ELISA may be more specific in detecting weakly expressed antigens, as well as being a cheaper method for doing so [25]. We found microparticle procoagulant activity in patients with PAPS/ aPL to have no significant difference compared to healthy controls (p = 0.05). Similarly, when patients were analysed according to aPL related complications, no significant differences in procoagulant activity were found. Previous studies assessing microparticle procoagulant activity in patients with aPL have used different methods of assessing procoagulant activity to the method used in this study, and results from these studies have also had contradictory findings. In addition, microparticle procoagulant activity has mostly been assessed in vitro. Galli et al. assessed prothrombinase activity of platelet microvesicles incubated with aPL and β2 glycoprotein-I and found prothrombinase activity to be inhibited by aPL [32]. Dignat-George et al. [14] showed endothelial microparticle released from endothelial cells reduced normalized clotting time ratio in a clotting assay derived to assess endothelial microparticle procoagulant activity and in their study of patients with SLE/APS, Pereira et al. demonstrated measured endogenous thrombin potential of plasma from patients included in the study. They demonstrated increased

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thrombin generation with patient plasma, but failed to show any correlation with aPL [18]. It has been proposed that assays measuring procoagulant activity may be more representative of microparticle function in vivo rather than using assays to quantify microparticle levels [5]. Previous studies of MP in association with aPL have shown similar findings of lack of correlation between functional assays of microparticle procoagulant activity and quantification by flow cytometry [29,33]. We used a functional ELISA assay which aims to measure procoagulant activity by using annexin V to capture microparticles since annexin V binds to negatively charged phospholipid. However, annexin V is not sensitive or specific for microparticles [34] and it has previously been shown that microparticles captured in this way may be underestimated [35,36]. Annexin V forms a protective layer on phospholipid membranes and acts as an anticoagulant by inhibiting binding and activation of coagulation complexes [37]. aPL have been shown to disrupt this annexin V anticoagulant shield [38] so it is possible that aPL present in the plasma may have interfered with the assay by disrupting the annexin V coat on the ELISA plate wells and therefore not capturing all microparticles present in the assay. In addition, many of the patients included in the study were anticoagulated with Vitamin K antagonists (n = 34) but this is not known to interfere with microparticle levels or activity. In vitro studies have shown that aPL target endothelial cells [39,40] and are capable of causing endothelial cell activation [41]. APL induce monocyte adhesion and increased expression of ICAM-1, VCAM-1 [41] and vWF [42] on cultured human umbilical vein endothelial cells incubated with aPL. Anti- β2-GPI antibodies are capable of causing endothelial cell activation when bound to endothelial cells [43,44] as shown by increased monocyte adhesion and expression of ICAM-1, V-CAM-1 and E-selectin [45]. Evidence of in vivo endothelial cell activation has been demonstrated in murine models of APS by increased leucocyte adherence to endothelial cells in association with aPL [46] and it is hypothesised that this may lead to aPL related thrombotic complications since thrombus formation is decreased in ICAM-1, VCAM-1 and P-selectin deficient mice infused with aPL [47]. The C5b-9 complex has been shown to induce vesiculation of the endothelial cell phospholipid membrane [48], we and others have demonstrated increased complement activation in patients with APS/aPL [49–51]. Elevated levels of endothelial microparticles found in patients with thrombotic complications of APL may represent underlying endothelial cell activation. Similarly, aPL have been shown to induce platelet agglutination [52] and aggregation at subnormal concentrations of platelet agonists [53], elevated levels of platelet microparticles in patients with thrombotic complications of aPL potentially representing underlying platelet activation. Shortcomings of the study included number of patients included in the study. Although this has been one of the largest studies to date of microparticles in patients with aPL, it would have been preferable to have larger numbers. Thirty four of the 66 patients with aPL included in this study were receiving oral anticoagulation and 17 were taking aspirin and although neither are known to interfere with assays measuring microparticle procoagulant activity, we would liked to have included more patients with aPL not taking any form of anticoagulation or antiplatelet therapy. In this study, only endothelial and platelet microparticles were measured but since leucocyte microparticles in addition to endothelial and platelet microparticles make up the majority of circulating microparticles, it would have potentially been useful to measure leucocyte microparticles. This study has shown evidence of increased endothelial and platelet microparticles in a large group of patients with thrombotic and obstetric APS and isolated aPL. We suggest the microparticle procoagulant assay is not suitable for use in patients with aPL due to probable interfence of aPL with the assay methodology. How microparticles may lead to the complications associated with APS is worthy of further exploration.

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Authorship BJ Hunt and KA Breen designed, executed and analysed the study. KA Breen and BJ Hunt wrote the first draft of the paper. K Parmar and scientists in Viapath, Guys and St.Thomas’ NHS Foundation Trust performed the lab analyses. GW Moore and KA Breen interpreted the aPL assay findings. P Seed performed statistical analysis of the data. All authors were involved in the first draft of the paper. Contributions None. Disclosure of Conflicts of Interest None. Acknowledgements We would like to acknowledge the Haematology Association of Ireland for providing funding for this study. References [1] Wolf P. The nature and significance of platelet products in human plasma. Br J Haematol 1967;13:269–88. [2] Kaplanski C, Pauley CJ, Griffiths TG, Kawabata TT, Ledwith BJ. Differentiation of rat oval cells after activation of peroxisome proliferator-activated receptor alpha43. Cancer Res 2000;60:580–7. [3] Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ. Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity. J Biol Chem 1989;264:17049–57. [4] Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis. Scand J Immunol 2007;66:159–65. [5] Zahra S, Anderson JA, Stirling D, Ludlam CA. Microparticles, malignancy and thrombosis. Br J Haematol 2011;152:688–700. [6] Simak J, Gelderman MP, Yu H, Wright V, Baird AE. Circulating endothelial microparticles in acute ischemic stroke: a link to severity, lesion volume and outcome. J Thromb Haemost 2006;4:1296–302. [7] Jimenez JJ, Jy W, Mauro LM, Horstman LL, Ahn YS. Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: findings from brain and renal microvascular cell culture and patients with active disease. Br J Haematol 2001;112: 81–90. [8] Laude I, Rongieres-Bertrand C, Boyer-Neumann C, Wolf M, Mairovitz V, Hugel B, et al. Circulating procoagulant microparticles in women with unexplained pregnancy loss: a new insight. Thromb Haemost 2001;85:18–21. [9] Carp H, Dardik R, Lubetsky A, Salomon O, Eskaraev R, Rosenthal E, et al. Prevalence of circulating procoagulant microparticles in women with recurrent miscarriage: a case-controlled study. Hum Reprod 2004;19:191–5. [10] Bucciarelli P, Martinelli I, Artoni A, Passamonti SM, Previtali E, Merati G, et al. Circulating microparticles and risk of venous thromboembolism. Thromb Res 2012 May; 129(5):591–7. [11] Owen BA, Xue A, Heit JA, Owen WG. Procoagulant activity, but not number, of microparticles increases with age and in individuals after a single venous thromboembolism. Thromb Res 2010;127:39–46. [12] Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. [13] Alijotas-Reig J, Palacio-Garcia C, Farran-Codina I, Zarzoso C, Cabero-Roura L, Vilardell-Tarres M. Circulating Cell-Derived Microparticles in Women with Pregnancy Loss. Am J Reprod Immunol 2011 Sep;66(3):199–208. [14] Dignat-George F, Camoin-Jau L, Sabatier F, Arnoux D, Anfosso F, Bardin N, et al. Endothelial microparticles: a potential contribution to the thrombotic complications of the antiphospholipid syndrome. Thromb Haemost 2004;91:667–73. [15] Combes V, Simon AC, Grau GE, Arnoux D, Camoin L, Sabatier F, et al. In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest 1999;104:93–102. [16] Jy W, Tiede M, Bidot CJ, Horstman LL, Jimenez JJ, Chirinos J, et al. Platelet activation rather than endothelial injury identifies risk of thrombosis in subjects positive for antiphospholipid antibodies. Thromb Res 2007;121:319–25. [17] Joseph JE, Harrison P, Mackie IJ, Isenberg DA, Machin SJ. Increased circulating plateletleucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis. Br J Haematol 2001;115:451–9. [18] Pereira J, Alfaro G, Goycoolea M, Quiroga T, Ocqueteau M, Massardo L, et al. Circulating platelet-derived microparticles in systemic lupus erythematosus. Association

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Endothelial and platelet microparticles in patients with antiphospholipid antibodies.

The antiphospholipid syndrome (APS) is the association of thrombosis and recurrent pregnancy loss and/or pregnancy morbidity with persistent antiphosp...
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