Exploring the plasmatic platelet-activating factor acetylhydrolase activity in patients with anti-phospholipid antibodies

To explore the role of plasmatic platelet-activating factor acetylhydrolase (PAF-AH), a marker of cardiovascular risk, in patients with anti-phospholipid antibodies (aPL). PAF-AH activity was assessed in a series of 167 unselected patients screened for aPL in a context of thrombotic events, risk of thrombosis or obstetric complications and in 77 blood donors. 116/167 patients showed positive results for at least one aPL among IgG/IgM anti-prothrombin/phosphatidylserine (aPS/PT), anti-cardiolipin (aCL), anti-beta2-glycoprotein I (aβ2GPI) or lupus anticoagulant (LAC), while 51/167 patients resulted aPL-negative. LAC+ patients disclosed higher PAF-AH than LAC-negative (22.1 ± 6.4 nmol/min/ml vs. 19.5 ± 4.1 nmol/min/ml; p = 0.0032), and aPL-negative patients (p = 0.03). Patients presenting positive IgG aβ2GPI disclosed higher PAF-AH than patients with only IgM aβ2GPI-positive antibodies (23.1 ± 7.2 nmol/min/ml vs. 20.1 ± 5.3 nmol/min/ml; p = 0.035), as well as than patients showing only isolated LAC, aCL or aPS/PT (16.9 ± 3.8 nmol/min/ml; p = 0.003). PAF-AH plasmatic activity is particularly up-regulated in LAC+ and in aβ2GPI IgG+ patients, possibly representing an alternative prognostic biomarker for the therapeutic management of APS patients.


Introduction
Anti-phospholipid syndrome (APS) is a hypercoagulable disorder clinically displayed by venous or arterial thrombosis and/or adverse obstetric events, accompanied by persistent and elevated levels of anti-phospholipid antibodies (aPL) [1]. According to the 2006 revised international classification criteria [2], patients with definite diagnosis of APS are those presenting positive lupus anticoagulant (LAC) and/or one among anti-cardiolipin (aCL) IgG or IgM or anti-beta2 glycoprotein I (ab2GPI) IgG or IgM antibodies. However, during the last international congress on aPL antibodies, the major experts defined the role of other so-called ''non criteria'' antibodies, contributing to assess the risk of thrombosis or the identification of potential seronegative APS, such as the antiprothrombin/phosphatidylserine antibodies (aPS/PT) [3]. Of note, the combination of ab2GPI, aPS/PT and LAC has demonstrated the best diagnostic accuracy for APS [4] and aPS/PT were recently recommended as a surrogate for LAC when specific inhibitors and/or analytical variables may affect its interpretation [5]. However, no definite recommendations are available to guide the therapeutic approach in patients positive only for aPS/PT antibodies.
Platelet-activating factor acetylhydrolase (PAF-AH) is a family of enzymes, the most abundant of which is the plasma form, also called lipoprotein-associated phospholipase A2 (Lp-PLA2) [6]. The plasmatic PAF-AH is constitutively active and circulates bound to LDL, HDL and other lipoproteins and catalyses the hydrolysis of the sn2 acetate of PAF and PAF mimetics, which are early mediators of inflammation [7]. PAF activates a variety of cells of the innate immune system promoting migration, adhesion and inflammatory effects. Thus, PAF-AH while inactivating PAF is considered an important factor in preventing an exaggerated inflammatory response and in protecting cells from uncontrolled oxidative damage [8].
Several studies reported a significant association between higher PAF-AH plasmatic activity and the severity of cardiovascular (CV) disease and identified PAF-AH as a marker of vascular inflammation and atherosclerotic plaque instability [9][10][11].
More and more papers in recent literature emphasize the relevant link between endothelial dysfunction, atherosclerosis and APS [12][13][14]. Chronic inflammation is involved in various stages of development of the atherosclerotic plaques. Among the key molecules involved in the atherosclerotic process are heat-shock proteins, oxidized LDL (oxLDL) and b2GPI. The latter is identified as an anti-atherogenic agent involved in the atheromatous plaque formation in APS patients, since it is targeted by the ab2GPI antibodies, typically associated with APS [15,16].
In this study we analysed PAF-AH plasmatic activity in a large series of unselected patients screened for aPL antibodies in a reference laboratory for the diagnosis of autoimmune diseases, investigating its association with different pattern of aPL positivity.

Patients
The study was conducted in 167 consecutive unselected patients (124 females and 69 males; mean age: 51 ± 16 years) who were screened for the presence of aPL at the Laboratory of Immunopathology of the University Hospital of Udine in the context of routine testing for thrombotic events, risk of thrombosis or obstetric complications. Patients were compared to 77 blood donors (BDs; 39 females and 38 males; mean age: 39 ± 13 years) enrolled at the Transfusion Unit of the same Hospital. All patients and controls gave their informed consent to the study according to the Declaration of Helsinki (1964) and to the Italian legislation (Authorization of the Privacy Guarantor No. 9, 12th of December 2013).
Anti-cardiolipin (aCL) and anti-beta2 glycoprotein I (ab2GPI) IgG/IgM antibodies were investigated in all patients, while 125 patients were tested for lupus anticoagulant and 125 for anti-phosphatidylserine/prothrombin (aPS/PT) IgG/IgM antibodies. aCL and ab2GpI antibodies were analysed by chemiluminescence (Zenit RA instrument by A. Menarini Diagnostics, Italy), while aPS/PT were assayed by Quanta Lite aPS/PT IgG/IgM ELISA kit (Inova Diagnostics Inc, San Diego, CA). Plasma samples were tested for the presence of LAC according to the recommended criteria from the ISTH Subcommittee on Lupus Anticoagulant-Phospholipid-dependent antibodies and optimized according to recently published standardization [17,18]. Total cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL) and triglycerides were analysed by diagnostic methods.

Statistic analyses
Quantitative variables were expressed as mean ± standard deviation and checked for normality distribution by the Shapiro-Wilk test. Statistical analyses were performed with GraphPad Prism software. To compare biomarker serum levels between patients and controls, either Mann-Whitney or unpaired t test was used when appropriate. Correlation analyses were performed using the Pearson's or the Spearman's rank correlation coefficient.

Discussion
Increased PAF-AH expression demonstrated a predictive role for cardiovascular events in relation to the vulnerability of atherosclerotic plaques. Therefore, PAF-AH dosage has been proposed in the assessment of CV risk, to ensure a better stratification of at risk populations [10]. To date, PAF-AH has never been investigated in the context of APS patients, or, even less, in patients at risk to develop an overt APS (i.e. asymptomatic carriers of aPL antibodies, patients affected by systemic connective tissues diseases).
Our study was originally conducted in a context of patients routinely screened for APS, demonstrating a significant association between the presence of aPL antibodies, LAC and ab2GPI IgG in particular, and PAF-AH upregulation in plasma.
Atherosclerosis is definitely recognized as a chronic inflammatory response to the accumulation of lipoproteins in the walls of arteries [19]. PAF-AH, produced by monocytes, macrophages and T lymphocytes, and mainly associated with LDL, is predominantly expressed in the necrotic centre of the atherosclerotic plaques and in the macrophage-rich areas and releases pro-inflammatory mediators, such as lysophospholipids and oxidized fatty acids [8].
Besides the presence of LAC, several different targets of aPL could be determined by a number of analytical methods, with frequent discordant results, that could make the laboratory diagnosis of APS extremely complicated. The main role of the ab2GPI antibodies, especially those specifically targeting domain I [20], is widely accepted and present results seem to further confirm their importance with regard to CV risk stratification, since PAF-AH appeared particularly elevated in ab2GPI-positive patients and more so in those displaying LAC activity and carrying the IgG isotype. This particular association may be explained by the fact that IgG ab2GPI antibodies are able to recognize the stable complex between oxLDL and b2GPI, thus facilitating macrophage-derived foam cell formation in patients with APS [15]. The immunepathological mechanisms sustained by oxLDL/b2GPI complexes are not yet fully understood, but Toll-like receptor 4 (TLR4) was recently shown to be involved [15]. TLR4 could be the key player linking PAF-AH upregulation to ab2GPI IgG antibodies in APS, as evidenced by a mouse model of preterm delivery which demonstrated that PAF effects and signalling depend upon TLR4 stimulation [21].
Lp-PLA2 activity proved to be markedly reduced in vivo when the enzyme is bound to HDL [8], and this is in line with our observation that ab2GPI IgG? patients disclosed higher PAF-AH and lesser HDL than BDs. This is not true for other subgroups of patients, such as aPLnegative patients or those presenting only isolated LAC or aCL or aPS/PT antibodies. Compared to these patients, PAF-AH plasmatic activity up-regulation in ab2GPI IgG? cases appeared to be at least partially disconnected from the lipoprotein levels and specifically linked to the presence of such aPL antibodies.
Therefore, PAF-AH up-regulation arose as a specific thrombotic risk marker in patients carrying ab2GPI antibodies and is not generally associated with other aPL antibodies possibly implicated in APS manifestations, but further studies are needed to confirm this observation.
Unfortunately, in two large randomized clinical trials, an inhibitor of PAF-AH (darapladib) [22,23] failed to reduce the risk of major coronary events as compared to placebo. In addition, it was associated with significantly higher rates of drug discontinuation and adverse effects. These results suggested that PAF-AH may be a biomarker of vascular inflammation, rather than a causal pathway of CV diseases [23]. Therefore, high PAF-AH activity could reflect a response to pro-inflammatory stress characteristic both of atherosclerosis and APS [24].
The leading cause of death in primary and secondary APS patients are cardiovascular events due to accelerated atherosclerosis, which often progresses more rapidly, compared with the general population [14]. Some key proinflammatory proteins correlate with APS clinical manifestations [25] and common radiological markers of subclinical atherosclerosis and CV risk were often reported in such patients [13]. However, to date, besides the presence of aPL itself, no serological biomarkers specifically associated with aPL-related pathogenic mechanisms have been identified as useful to improve the classification of CV risk in aPL? patients, with and without overt clinical manifestations.
In this scenario, present findings on PAF-AH assume a relevant place, possibly representing a reliable and affordable biomarker useful to identify patients at higher risk in which to take a more cautious therapeutic attitude in the follow-up.
Moreover, studying PAF-AH metabolic pathway may help to better explain the pathogenesis of APS and to improve management and interpretation of aPL-related issues, from the analytical results, to the final therapeutic decision.
We and others recently demonstrated an important role of aPS/PT antibodies in the serological diagnosis of APS [5,26]; however, the therapeutic management of patients characterized by the presence of isolated aPS/PT remains an open issue. Patients with isolated aPS/PT antibodies disclosed lower, BDs-like, PAF-AH as compared to patients with positive ab2GPI antibodies. Nevertheless, aPS/PT antibodies may exert their distinct pathogenic role through pathways in which PAF-AH is not involved. Anyhow, in case aPS/PT IgG (but not IgM) are combined to ab2GPI IgG antibodies, PAF-AH tends to be further upregulated.
In conclusion, the prognostic information conveyed by plasmatic PAF-AH activity in patients with positive aPL antibodies appeared to be independent of common lipid metabolic markers (i.e. LDL), as previously reported by other authors in the context of patients with major coronary events [11]. The major international scientific societies of cardiologists have included the measurement of PAF-AH activity among the biomarkers useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk in their guidelines [27].
Our data are encouraging, but since some cohort comparison include partially overlapping data, a definite utility of PAF-AH plasmatic activity as a new prognostic biomarker also in patients with aPL antibodies and/or definite APS is actually precluded. For this reason further prospective studies on selected patients are ongoing.

Compliance with ethical standards
Conflict of interest All authors declare no conflict of interest with regard to this paper.
Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from all individual participants included in the study.
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