Secretory phospholipase A2 modified HDL rapidly and potently suppresses platelet activation

Levels of secretory phospholipases A2 (sPLA2) highly increase under acute and chronic inflammatory conditions. sPLA2 is mainly associated with high-density lipoproteins (HDL) and generates bioactive lysophospholipids implicated in acute and chronic inflammatory processes. Unexpectedly, pharmacological inhibition of sPLA2 in patients with acute coronary syndrome was associated with an increased risk of myocardial infarction and stroke. Given that platelets are key players in thrombosis and inflammation, we hypothesized that sPLA2-induced hydrolysis of HDL-associated phospholipids (sPLA2-HDL) generates modified HDL particles that affect platelet function. We observed that sPLA2-HDL potently and rapidly inhibited platelet aggregation induced by several agonists, P-selectin expression, GPIIb/IIIa activation and superoxide production, whereas native HDL showed little effects. sPLA2-HDL suppressed the agonist-induced rise of intracellular Ca2+ levels and phosphorylation of Akt and ERK1/2, which trigger key steps in promoting platelet activation. Importantly, sPLA2 in the absence of HDL showed no effects, whereas enrichment of HDL with lysophosphatidylcholines containing saturated fatty acids (the main sPLA2 products) mimicked sPLA2-HDL activities. Our findings suggest that sPLA2 generates lysophosphatidylcholine-enriched HDL particles that modulate platelet function under inflammatory conditions.

suggesting that HDL is a principal substrate for sPLA 2 13, 16 . In addition, recombinant sPLA 2 -IIA and V have been shown to be more active on isolated HDL in comparison to other lipoproteins, such as LDL 17 .
Platelets have a key role in arterial thrombosis, the most common cause of myocardial infarction and stroke 18 . Accumulating evidence suggests that inflammatory responses are linked to increased platelet activation in the bloodstream of patients with systemic inflammation and sepsis 19 . It is well known that platelets substantially contribute to sepsis complications such as disseminated intravascular coagulation, thrombotic microangiopathy and multiple organ failure 20 . Platelet function has been shown to be very sensitive to the influence of plasma lipoproteins and cholesterol levels 21,22 . However, very little is known about the effects of inflammation-induced changes in HDL composition (especially lysophospholipid enrichment) on HDL-platelet interaction and functional responses of platelets. Pharmacological inhibition of sPLA 2 resulted in increased incidence of myocardial infarction and stroke 7 , raising the possibility that increased sPLA 2 activity during inflammation might modulate platelet activity. Based on these observations, we tested whether sPLA 2 -induced hydrolysis of HDL-associated phospholipids generates particles that affect platelet functionality. Our findings suggest sPLA 2 -modified HDL particles potently suppress agonist induced platelet activation.
Results sPLA 2 -modified HDL rapidly suppresses agonist induced platelet aggregation. In order to generate sPLA 2 -modified HDL, native HDL (nHDL) isolated from plasma of healthy human volunteers was treated with human recombinant sPLA 2 type V. The reaction was stopped by addition of the sPLA 2 type V inhibitor varespladib. Treatment of HDL with sPLA 2 for 90 min (sPLA 2 -HDL-low) and overnight treatment (sPLA 2 -HDLhigh) hydrolyzed ~30-40% and 70-80% of HDL-associated phosphatidylcholines, respectively (Supplementary Figure 1).
To test whether sPLA 2 -HDL affects platelet aggregation, platelets isolated from plasma of healthy donors were exposed to different agonists including ADP, collagen and thrombin in the presence or absence of native HDL and sPLA 2 -HDL. Strikingly, sPLA 2 -HDL potently inhibited platelet aggregation induced by ADP, collagen and thrombin, whereas sPLA 2 in the absence of HDL and the sPLA 2 inhibitor varespladib showed no effect ( Fig. 1a-d). sPLA 2 -HDL-mediated inhibition of platelet aggregation was concentration dependent (Fig. 2a) and aggregation was significantly decreased already after one minute pre-treatment of platelets with sPLA 2 -HDL (Fig. 2b). Importantly, Annexin-V staining of phosphatidylserine surface expression confirmed that sPLA 2 -HDL did not affect platelet viability (Supplementary Figure 2). Furthermore, since it has been shown that HDL isolation procedure can have a significant impact on HDL composition 23 , we tested whether HDL isolated by a different method will have the same effects on platelets. Therefore, we isolated HDL from human plasma by dextran-sulfate precipitation 24 . We found that HDL isolated by dextran-sulfate precipitation also inhibited platelet activation after sPLA 2 -mediated modification (Supplementary Figure 3), whereas native HDL showed no effect. sPLA 2 -HDL inhibits P-selectin expression, GPIIb/IIIa activation and superoxide production in platelets. Furthermore, we assessed platelet functional responses involved in hemostatic and immune platelet functions, such as P-selectin expression, integrin activation, and reactive oxygen species production. Notably, sPLA 2 -HDL inhibited activation of the main platelet integrin GPIIb/IIIa (Fig. 3a) as well as surface exposure of the platelet activation marker P-selectin (Fig. 3b). Upon activation, platelets produce reactive oxygen species which promote their recruitment to a growing thrombus 25 . Importantly, sPLA 2 -HDL significantly attenuated superoxide anion production by platelets (Fig. 3c). sPLA 2 -generated lysophosphatidylcholines mediate effects of sPLA 2 -HDL on platelets. In comparison to native HDL, sPLA 2 -modified HDL contains significantly higher amounts of LPCs (Supplementary Figure 1b). In order to investigate whether HDL-bound LPCs are the active moiety of sPLA 2 -HDL, native HDL was enriched with different LPC species. Saturated LPC species (16:0 and 18:0) are the most abundant LPC species in sPLA 2 -HDL and account for about 70% of total LPCs (Supplementary Figure 1b). Importantly, when enriched in HDL, LPCs mimicked the effects of sPLA 2 -HDL, with LPC 16:0 and LPC 18:0 being the most potent mediators (Fig. 4a). In addition, other LPCs species (LPC 18:1 and LPC 18:2) as well as free fatty acid (FFA) 18:1 were able to inhibit platelet aggregation, but to a much weaker extent (Fig. 4a). These results indicate that several LPCs and some FFA species contribute to the effects of sPLA 2 -HDL on platelets. In sharp contrast, FFA 18:2 and FFA 20:4 induced platelet aggregation when enriched in HDL (Fig. 4b).
sPLA 2 -HDL suppresses agonist-induced Ca 2+ flux and inhibits kinase phosphorylation in platelets. To gain insights into the signaling pathways involved in sPLA 2 -HDL effects on platelets, we examined intracellular signaling events such as release of Ca 2+ (Ca 2+ flux) and phosphorylation of protein kinases, both of which play a crucial role in platelet activation cascades. First, we assessed agonist induced changes in Ca 2+ flux. ADP stimulation leads to a rise in intracellular Ca 2+ in platelets, which was significantly suppressed upon sPLA 2 -HDL treatment (Fig. 5a,b).
To identify sPLA 2 -HDL modulated signaling cascades, we screened the phosphorylation status of kinases (including p38 MAPK, Akt, eNOS, ERK1/2, p38 MAPK, members of the Src family kinases, GSK-3α/β, AMPK, STAT3) important in platelet activation. For that purpose, platelets were activated with the platelet agonists collagen or ADP in presence of sPLA 2 -HDL or control HDL. We observed that in comparison to control HDL-treated platelets, phosphorylation of Akt at Ser473 and ERK1/2 was significantly decreased in both collagen and ADP-stimulated platelets pre-treated with sPLA 2 -HDL (Fig. 6). Effects of sPLA 2 -HDL on Akt and ERK1/2 phosphorylation were further confirmed by Western blot analysis ( Fig. 7 and Supplementary Figure 4). These data suggest that inhibition of Akt and ERK1/2 phosphorylation by sPLA 2 -HDL is involved in the observed anti-aggregatory effects. Both, phosphorylation of Akt and ERK1/2 are well known to induce platelet activation [26][27][28] .

Discussion
Increasing evidence suggests that HDL exerts its cardioprotective, antithrombotic and anti-inflammatory roles via direct immunomodulatory effects on several cell types including macrophages, neutrophils and endothelial cells [29][30][31][32] . Furthermore, HDL can actively interact with platelets, which results in modulation of platelet function 21,[33][34][35] . Most investigations showed inhibitory effects of native HDL on platelets 22 . However, there are also studies showing no effect or even pro-aggregatory activity of native HDL 36,37 .
Previous studies reported that agonist-induced platelet aggregation is suppressed when platelets are exposed to mildly oxidized HDL [38][39][40] . In line with these previous studies, we show that sPLA 2 -mediated modification of HDL does not impair its anti-aggregatory activities towards platelets, but rather generates particles with increased ability to rapidly and potently suppress platelet activation. It is well known that mild oxidation generates LPCs 41,42 . Therefore, it is likely that the anti-aggregatory activities observed with oxidized HDL [38][39][40] are mediated, at least in part, by HDL associated LPCs. sPLA 2 -HDL inhibited platelet activation induced by a wide range of physiological agonists including ADP, collagen and thrombin in a concentration-dependent manner. Interestingly, in our experiments, native HDL showed only minimal effects and sPLA 2 in the absence of HDL showed no effect on agonist-induced activation of platelets. Therefore, our results clearly suggest that a certain threshold of HDL-associated LPC is required to suppress platelet activation. To keep the LPC content of control HDL low, we used HDL that was rapidly isolated within a few hours by using gradient ultracentrifugation 43-45 from blood obtained from blood donors within one day. We obtained very similar results when HDL was isolated by dextran sulfate precipitation, clearly suggesting that the rapid gradient ultracentrifugation procedure to isolate HDL does not markedly affect the LPC content of HDL. A rapid isolation procedure and usage of HDL is of critical importance, given that HDL-associated lecithin-cholesterol acyltransferase and lipoprotein-associated phospholipase A 2 constantly produce LPC. Therefore, it is reasonable to assume that the LPC content of native HDL used in different studies can determine the anti-aggregatory activities of HDL [46][47][48] .
Beside aggregation, sPLA 2 -HDL inhibited other platelet functional responses such as integrin GPIIb/IIIa activation and P-selectin expression. This is of importance given that these proteins not only mediate hemostatic and prothrombotic platelet functions, but also their immune interactions, such as activation of endothelium, formation of platelet-leukocyte complexes and priming of neutrophils to produce neutrophil extracellular traps [49][50][51] . In addition, activated platelets produce reactive oxygens species which are involved in platelet intracellular signaling, as well as platelet recruitment and thrombus growth when released from platelets 25,52 . Importantly, sPLA 2 -HDL was able to significantly suppress platelet superoxide anion production.
Physiological levels of LPCs in the circulation are high (around 190 µM) 53 and can reach even mM levels in some pathophysiological states, such as hyperlipidemia 54 . LPCs have been regarded to be involved in the etiology of several chronic inflammatory diseases including autoimmune diseases and atherosclerosis 55 . On the other hand, administration of LPC significantly reduced sepsis-induced mortality in mice 56 . In line with this study, septic patients show positive association between serum LPC levels and survival 57 . Biological activities of free LPC on endothelial and immune cells have been shown in many studies 55,58 . Although LPC might be transiently found in its free form, it is predominantly bound to albumin, other serum proteins and lipoproteins 55 . The albumin-bound fraction represents an inactive form of LPC since albumin abolishes most of LPC cellular effects 59 . Interestingly, physiological activity of lipoprotein-bound LPC is not well investigated. In comparison to low-density lipoproteins, HDL carries higher amounts of LPC 60, 61 because HDL-associated lecithin-cholesterol acyltransferase constantly produces LPC. Moreover, HDL has the ability to efficiently remove saturated and monounsaturated LPCs from low-density lipoproteins 62 . Of particular interest, deficiency in lecithin-cholesterol acyltransferase, which is the main enzyme involved in formation of HDL-associated lysophospholipids under physiological conditions 63 , decreased LPS-neutralizing capacity of HDL and enhanced LPS-induced inflammation in mice 64 . In line with these observations, we previously showed that sPLA 2 -HDL has potent anti-inflammatory effects on neutrophils 65 , indicating that HDL-bound LPCs represent a biologically active LPC fraction.
Importantly, HDL enriched with LPC 16:0 and LPC 18:0, which are the most abundant LPC species in sPLA 2 -HDL, mimicked the effects of sPLA 2 -treated HDL on platelets. Other LPC species (LPC 18:1 and LPC 18:2) and some FFAs (FFA 18:1), showed weaker inhibitory activity when enriched in HDL. This suggests that other sPLA 2 -generated products contribute to the sPLA 2 -HDL activity on platelets. In addition, sPLA 2 -HDL carries a certain amount of pro-aggregatory mediators such as arachidonic acid 66 , which induced platelet aggregation when enriched in HDL. However, their activity seems to be completely counteracted by HDL-associated LPCs. Given that the sPLA 2 group of enzymes comprises several subtypes with different substrate specifities 67 , various LPC and FFA species are expected to be formed und inflammatory conditions that might generate sPLA 2 -HDL with diverse effects on platelets.  Of particular interest, we observed that sPLA 2 -HDL effectively suppressed agonist-induced rise in intracellular Ca 2+ as well as phosphorylation of Akt at Ser473 and ERK1/2. Importantly, Akt and ERK play a significant role in platelet secretory pathways leading to granule release. Additionally they are involved in regulation of integrin GPIIb/IIIa function [26][27][28] . ERK has been shown to phosphorylate and activate cytoplasmic phospholipase A 2 , an enzyme responsible for thromboxane A 2 synthesis 68 . Furthermore, phosphorylation of certain kinases, such as Akt, depends on the interaction with membrane cholesterol-rich microdomains 69 . We have recently shown that sPLA 2 -HDL depicts an increased cholesterol-mobilizing capacity and disrupts membrane cholesterol-rich microdomains in neutrophils 65 . Given that changes in membrane cholesterol content highly influence platelet functionality 70 , we expect that sPLA 2 -HDL-mediated cholesterol depletion might also be involved in observed inhibitory effects on agonist-induced platelet activation.
A potential limitation of the present study is that we only assessed the impact of sPLA 2 on HDL functionality, while under inflammatory conditions HDL particles are subjected to an array of complex proteomic and lipidomic changes that affect HDL function 60,61 . However, our study clearly demonstrates that upon modification by sPLA 2 , HDL is transformed into a particle with a strong ability to modulate platelet activation via suppression of  intracellular Ca 2+ increase and inhibition of Akt and ERK1/2 kinase phosphorylation. Our results may, therefore, contribute to a better understanding of the role of sPLA 2 in atherosclerosis and inflammation.

Materials and Methods
All reagents were from Sigma (Vienna, Austria), unless otherwise specified. Human recombinant secretory phospholipase A 2 type V was purchased from Cayman Europe (Tallin, Estonia). Varespladib was purchased from Eubio (Vienna, Austria). Fluo-3-AM was from Life Technologies (Vienna, Austria). ADP, collagen and thrombin were Blood collection. The study was approved by the Ethics Committee of the Medical University of Graz. All volunteers signed an informed consent form in agreement with the Institutional Review Board of the Medical University of Graz. All methods were carried out in accordance with the approved guidelines. Blood was collected into tubes with 3.8% (w/v) sodium citrate and platelet rich plasma (PRP) was obtained by centrifugation at 400 × g for 20 min at RT as described 71,72 . Subsequently, plasma was used for HDL and platelet isolation. . sPLA 2 -HDL inhibits Akt and ERK1/2 phosphorylation in platelets. Platelets were pretreated with vehicle, nHDL (50 µg/mL), sPLA 2 -HDL-low (50 µg/mL) or sPLA 2 -HDL-high (50 µg/mL). Subsequently cells were stimulated with (a,c) ADP (10 µM) or (b,d) collagen (5 µg/mL). (a,b) Akt (Ser473) and (c,d) ERK1/2 phosphorylation was assessed by Western blot. β-actin was used as the loading control. Blots have been cropped from full-length blots shown in Supplementary Figure 4. ChemiDoc Touch Imaging System and ECL Blotting Substrate (both Bio-Rad, Vienna, Austria) were used to visualize protein bands. Immunoblot images were quantified using Image Lab 5.2 software (Bio-Rad). Statistical analysis of phospho-Akt (p-Akt)/total Akt (t-Akt) and phospho-ERK1/2 (p-ERK)/total ERK1/2 (t-ERK) ratios were derived from 3 independent experiments which were run under the same conditions. Vehicle treated (unstimulated) control was set as baseline and values are expressed as % over baseline. Results are shown as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Dunnett's post hoc test. *p < 0.05, **p < 0.01 versus vehicle-pretreated and (a,c) ADP or (b,d) collagen-stimulated platelets.
SCIEnTIfIC REPORTS | 7: 8030 | DOI:10.1038/s41598-017-08136-1 HDL isolation. HDL was isolated by density gradient ultracentrifugation as described [43][44][45] . Plasma density was adjusted with potassium bromide to 1.24 g/mL and a two-step density gradient was generated in centrifuge tubes (16 × 76 mm, Beckman) by layering the density-adjusted plasma (1.24 g/mL) underneath a NaCl-density solution (1.063 g/mL). Tubes were sealed and centrifuged at 90,000 rpm for 4 h in a 90Ti fixed angle rotor (Beckman Instruments, Krefeld, Germany). After centrifugation, the HDL-containing band was collected and desalted via PD10 columns (GE Healthcare, Vienna, Austria) and immediately used for experiments. sPLA 2 treatment of HDL. HDL was incubated in the presence of 400 ng/mL human recombinant type V sPLA 2 in PBS containing Ca 2+ and Mg 2+ at 37 °C for either 90 min (low modification) or overnight (high modification) in order to hydrolyse HDL-associated phospholipids. The reaction was stopped by addition of sPLA 2 inhibitor varespladib (1 µM). Platelet aggregation. Platelet aggregation was performed at 37 °C with constant stirring (1000 rpm) using the four-channel platelet aggregometer APACT4004 (LABiTec, Ahrensburg, Germany), which works on the principle of light transmission, as previously described 71,73 . Platelet response to a concentration range of ADP (5-20 µM), collagen (2.5-10 µg/mL) or thrombin (0.05-0.1 U/mL) was tested before each experiment. ADP, collagen and thrombin concentrations which induced 70-90% aggregation were used for platelet stimulation. Platelets were preincubated with vehicle or HDL samples for 5 min at 37 °C. Aggregation was induced with ADP in the presence of 1 µg/mL fibrinogen, collagen or thrombin and measured for 5 min. Data were expressed as percentage of maximum light transmission, with non-stimulated washed platelets being 0% and Tyrode's buffer 100% aggregation 71, 72 . P-selectin (CD62P) expression. Washed platelets resuspended in Tyrode's buffer were preincubated with vehicle, HDL samples (50 µg/mL), sPLA 2 (400 ng/mL) or varespladib (1 µM) for 10 min at RT. Subsequently, cells were stimulated with ADP (3 µM) in the presence of cytochalasin B (5 µg/mL) for 30 min at RT in the presence of anti-CD62P-FITC conjugated antibody. Cytochalasin B was used to facilitate translocation of P-selectin from granules to the cell surface. The samples were fixed and P-selectin upregulation was detected by flow cytometry.

Lysophosphatidylcholine (LPC
GPIIb/IIIa (PAC-1) activation. PRP was suspended in Tyrode's buffer and pretreated with vehicle, HDL samples (50 µg/mL), sPLA 2 (400 ng/mL) or varespladib (1 µM) for 10 min at RT. The activation of glycoprotein receptor GPIIb/IIIa was induced with ADP (3 µM) for 30 min at RT in the presence of the FITC-conjugated anti-PAC-1 antibody that recognizes a conformation-dependent determinant on the GPIIb/IIIa complex. The samples were fixed and GPIIb/IIIa activation was detected by flow cytometry. Superoxide production. Washed platelets (1 × 10 7 per sample) resuspended in Tyrode's buffer were pretreated with vehicle, HDL samples (50 µg/mL), sPLA 2 (400 ng/mL) or varespladib (1 µM) for 10 min at 37 °C and stimulated with collagen (5 µg/mL) for 20 min, at 37 °C. Superoxide production was detected with Superoxide Detection Kit according to manufacturer's protocol. Briefly, after the treatment platelets were washed once, resuspended in superoxide detection reagent and incubated for 30 min at 37 °C in the dark. Superoxide production was measured immediately by flow cytometry as an increase in fluorescence intensity (FL-2 channel).
Ca 2+ Flux. Platelet-rich plasma was loaded with the cell membrane permeable Ca 2+ -sensitive dye Fluo-3-AM Phosphokinase array. Analysis of phosphorylation profiles of kinases and their protein substrates was done by the Human Phospho-Kinase Array (R&D Systems, Minneapolis, MN). Washed platelets were pretreated with nHDL (50 µg/mL) or sPLA 2 -HDL-high (50 µg/mL) for 5 min at RT, stimulated with collagen (5 µg/mL) or ADP (10 µM) for 5 min at RT and analyzed according to manufacturer's protocol. The membranes were imaged using ChemiDoc Touch Imaging System and ECL Blotting Substrate (both Bio-Rad, Vienna, Austria). The average signal (pixel density) analysis of duplicate spots representing each protein was performed using Image Lab 5.2 software (Bio-Rad). A twofold increase/decrease in phosphorylation was considered to be significant.
Statistical analysis. All data are shown as mean ± SEM for n separate experiments. Experiments were repeated three to six times using platelets from different donors. Statistical analyses were performed with GraphPad Prism Version 5. Comparisons of groups were performed using one-way ANOVA with Dunnett's post-hoc test or two-way ANOVA with Bonferroni post-hoc test. Significances were accepted at *p < 0.05, **p < 0.01 and ***p < 0.001.