Proprotein Convertase Subtilisin Kexin Type 9 Inhibitors Reduce Platelet Activation Modulating Ox-LDL Pathways

International Journal of Molecular Sciences

Article Proprotein Convertase Subtilisin Kexin Type 9 Inhibitors Reduce Platelet Activation Modulating ox-LDL Pathways

Vittoria Cammisotto 1,† , Francesco Baratta 2,† , Valentina Castellani 2, Simona Bartimoccia 3, Cristina Nocella 2, Laura D’Erasmo 4, Nicholas Cocomello 2 , Cristina Barale 5, Roberto Scicali 6 , Antonino Di Pino 6, Salvatore Piro 6 , Maria Del Ben 2 , Marcello Arca 4, Isabella Russo 5 , Francesco Purrello 6, Roberto Carnevale 7,8 , Francesco Violi 2,8, Daniele Pastori 2 and Pasquale Pignatelli 2,8,*

1 Department of General Surgery and Surgical Speciality “Paride Stefanini”, Sapienza University of Rome, 00161 Rome, Italy; [email protected] 2 I Clinica Medica, Department of Clinical Internal, Anaesthesiological and Cardiovascular Sciences, Sapienza University of Rome, 00161 Rome, Italy; [email protected] (F.B.); [email protected] (V.C.); [email protected] (C.N.); [email protected] (N.C.); [email protected] (M.D.B.); [email protected] (F.V.); [email protected] (D.P.) 3 Istituto Pasteur Italia-Fondazione Cenci Bolognetti and Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy; [email protected], 4 Department of Translational and Precision Medicine, Sapienza University of Rome, 00161 Rome, Italy; [email protected] (L.D.); [email protected] (M.A.) 5 Department of Clinical and Biological Sciences, University of Turin, 10154 Turin, Italy; [email protected] (C.B.); [email protected] (I.R.) 6 Department of Clinical and Experimental Medicine, Internal Medicine, University of Catania, Citation: Cammisotto, V.; Baratta, F.; 95122 Catania, Italy; [email protected] (R.S.); [email protected] (A.D.P.); [email protected] (S.P.); Castellani, V.; Bartimoccia, S.; Nocella, [email protected] (F.P.) 7 C.; D’Erasmo, L.; Cocomello, N.; Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy; [email protected] Barale, C.; Scicali, R.; Di Pino, A.; et al. 8 Mediterranea Cardiocentro, 80133 Napoli, Italy Proprotein Convertase Subtilisin * Correspondence: [email protected]; Tel./Fax: +39-06-4461933 Kexin Type 9 Inhibitors Reduce † Equal contribution at this work. Platelet Activation Modulating ox-LDL Pathways. Int. J. Mol. Sci. Abstract: Background: Proprotein convertase subtilisin kexin type 9 inhibitors (PCSK9i) lower 2021, 22, 7193. https://doi.org/ LDL-cholesterol and slow atherosclerosis preventing cardiovascular events. While it is known 10.3390/ijms22137193 that circulating PCSK9 enhances platelet activation (PA) and that PCSK9i reduce it, the underlying

Academic Editor: Denise Jackson mechanism is not still clariﬁed. Methods: In a multicenter before–after study in 80 heterozygous familial hypercholesterolemia (HeFH) patients on treatment with maximum tolerated statin dose Received: 3 June 2021 ± ezetimibe, PA, soluble-NOX2-derived peptide (sNOX2-dp), and oxidized-LDL (ox-LDL) were Accepted: 30 June 2021 measured before and after six months of PCSK9i treatment. In vitro study investigates the effects Published: 3 July 2021 of plasma from HeFH patients before and after PCK9i on PA in washed platelets (wPLTs) from healthy subjects. Results: Compared to baseline, PCSK9i reduced the serum levels of LDL-c, ox- Publisher’s Note: MDPI stays neutral LDL, Thromboxane (Tx) B2, sNOX2-dp, and PCSK9 (p < 0.001). The decrease of TxB2 correlates with regard to jurisdictional claims in with that of ox-LDL, while ox-LDL reduction correlated with PCSK9 and sNOX2-dp delta. In vitro published maps and institutional afﬁl- study demonstrated that wPLTs resuspended in plasma from HeFH after PCSK9i treatment induced iations. lower PA and sNOX2-dp release than those obtained using plasma before PCSK9i treatment. This reduction was vanished by adding ox-LDL. ox-LDL-induced PA was blunted by CD36, LOX1, and NOX2 inhibition. Conclusions: PCSK9i treatment reduces PA modulating NOX2 activity and in turn ox-LDL formation in HeFH patients. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: platelets; ox-LDL; PCSK9; familial hypercholesterolemia; NOX2 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Int. J. Mol. Sci. 2021, 22, 7193. https://doi.org/10.3390/ijms22137193 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 7193 2 of 12

1. Introduction Proprotein convertase subtilisin kexin type 9 (PCSK9) plays a key role in lipid metabolism and atherothrombotic process binding low-density lipoprotein cholesterol receptors (LDLRs) and addressing them to lysosomes where they are degraded [1,2]. PCSK9 inhibitors (PCSK9i) are monoclonal antibodies binding circulating PCSK9 and preventing PCSK9 binding to the LDLRs. In this way, PCSK9i increase LDLRs expression on cells membrane and LDL-cholesterol (LDL-c) scavenge from circulation [3,4]. In several clinical studies, the PCSK9i, alirocumab, and evolocumab, have been shown to improve atherosclerosis [5,6] and prevent cardiovascular events [7,8]. While the lipid- lowering effect of PCSK9 inhibitors is well established, pleiotropic effect of PCSK9i on cellu- lar mechanisms involved in atherogenesis and plaques complication is still debated [9–12]. A recent metanalysis evaluating a large cohort of patients (n = 28,319), showed that high circulating PCSK9 concentration significantly associated with increased risk of major adverse cardiovascular events (MACEs) [13]. Several studies demonstrated that PCSK9 circulating level directly enhances platelet activation (PA) and in vivo thrombosis suggesting a possible role of PA in inducing MACE in subjects with elevated PCSK9 levels [14,15]. Recently, Barale et al. [16] showed that up to 12 months treatment with PCSK9i impacts on platelet function in heterozygous familial hypercholesterolemia (HeFH). FH is a particularly attractive model as the genetic mutations result in persistent lifelong extremely raised LDL-c levels, premature coronary artery disease, and systemic atherosclerosis [17]. LDL-c play a key role in the thrombotic process. Indeed, LDL modification by phospholipid oxidation during the inflammatory and oxidative processes of plaque formation results in the generation of the prothrombotic oxidized-LDL (ox-LDL) [18][19]. Dyslipidaemia induces the generation of ox-LDL, which in turn, facilitate platelet activation by binding scavenger receptors on platelet’s surface including LOX-1 and CD36 [18]. Once activated, platelets can oxidize LDLs, generating a positive feedback of platelet activation through the activation of NOX2 [18]. This enzymatic system seems to play a key role in PA as PCSK9 per se can induce NOX2 activation and once produced, ox-LDL also amplify PA by inducing NOX2 activation [19]. This intricated process can be monitored in vivo by dosing sNOX2-dp, a soluble peptide released upon NOX2 activation [20], and TXB2 a marker of PA induced by arachidonic acid pathway [21]. Furthermore, these markers were reported to be correlated with atherosclerosis and cardiovascular disease in other settings [14,22–25]. However, NOX2 activity modulation after PCSK9i treatment was never investigated and, while the inhibition of PA in patients treated with PCSK9i was previously demonstrated by Barale et al., whether this depends on free circulating PCSK9 reduction or on lowering oxLDL is still to be defined [14–16,26]. For this purpose, we conducted a multicenter before–after study in HeFH patients to evaluate if six months of PCSK9i treatment could inhibit PA by modulating ox-LDL production and ox-LDL pathway.

2. Results 2.1. Patients’ Characteristics The median age of patients treated with PCSK9i was 57.7 ± 10.8 years and 36 out of 80 (45%) were female. The median BMI was 26.5 ± 4.2 kg/m2. Prior cardiovascular events were recorded in 51.5% of patients, 41 out of 41 had coronary heart disease and 2 had peripheral artery diseases in addition. Antiplatelet drugs were used by 42 out of 80 (52.5%) patients and the same number of smokers was recorded. As per inclusion criteria, none of the patients had diabetes and all the patients were on statin treatment (Table1). Int. J. Mol. Sci. 2021, 22, 7193 3 of 12

Table 1. Patients’ characteristics.

HeFH Patients Before Treatment (n = 80) Age (years) 57.7 ± 10.8 Female 45% BMI (kg/m2) 26.5 ± 4.2 Smokers (%) 27.5% Total Cholesterol (mg/dL) 246.0 ± 66.0 HDL cholesterol (mg/dL) 50.8 ± 11.3 LDL cholesterol (mg/dL) 169.2 ± 59.3 Triglycerides (mg/dL) 121.9 ± 53.5 Hypertension (%) 52.5% Prior Cardiovascular Events (%) 51.5% Antiplatelet drugs 52.5% Diabetes 0% Statins 100%

2.2. Effect of PCSK9i Treatment After 6 months treatment with PCSK9i, LDL-c changed from 169.2 ± 59.4 to 71.9 ± 42.0 mg/dl, p < 0.001 (Figure1, panel A). PA was signiﬁcantly inhibited. Thus, TxB2 lowered from 204.3 ± 62.6 to 116.2 ± 52.9 pg/mL, p < 0.001, (Figure1, panel B). There was also a reduction in circulating PCSK9 levels, changing from 121.0 ±24.1 to 64.3 ± 22.2 ng/mL (Figure1, panel C). Concerning oxidative stress, we found that sNOX2-

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWdp decreased from 29.5 ± 11.2 to 20.3 ± 7.4 pg/mL, p < 0.001 (Figure4 of 13 1, panel D) and ox-LDL from 28.6 ± 13.8 to 17.1 ±6.9 mU/mL, p < 0.001 (Figure1, panel E).

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Figure 1. Ex vivo markers changes after PCSK9i treatment in HeFH patients. Plasma levels of Figure 1. Ex vivo markers changes after PCSK9i treatment in HeFH patients. Plasma levels of LDL- LDL-cc (A), TxB (A2 (),B), TxB PCSK92 ( B(C),), PCSK9sNOX2-dp (C (D),), sNOX2-dp and ox-LDL (E (D) production), and ox-LDL in 80 HeFH (E) patients. production (Data in 80 HeFH patients. (Dataare represented are represented as median and as medianSD; * p < 0.0001). and SD; Abbreviation: * p < 0.0001). PCSK9 Abbreviation:inhibitors (PCSK9i); PCSK9 LDL- inhibitors (PCSK9i); LDL-cholesterolcholesterol (LDL-c); Thromboxane (LDL-c);Thromboxane B2 (TxB2); soluble NOX2 B (TxB-derived); peptide soluble (sNOX2 NOX2-derived-dp), oxidized- peptide (sNOX2-dp), LDL (ox-LDL), heterozygous familial hypercholesterolemia2 (HeFH)2 . oxidized-LDL (ox-LDL), heterozygous familial hypercholesterolemia (HeFH). A multivariate linear regression analysis showed that TxB2 reduction positively correlated with LDL-c (Beta:0.316; p = 0.007) and ox-LDL (Beta:0.233; p = 0.044). No correlations were found with PCSK9 and sNOX2-dp. Furthermore, ox-LDL reduction positively correlated with PCSK9 (Beta:0.277; p = 0.016) and sNOX2-dp variation (Beta:0.231; p = 0.045) (Table 2).

Table 2. Multivariate linear regression analyses. Δ TxB2 Δ ox-LDL B E.S Beta p B E.S Beta p Δ PCSK9 −0.025 0.343 −0.008 0.942 Δ PCSK9 0.153 0.062 0.277 0.016 Δ ox-LDL 1.246 0.607 0.233 0.044 - Δ LDL-c 0.387 0.140 0.316 0.007 Δ LDL-c 0.044 0.027 0.191 0.113 Δ sNOX2- Δ sNOX2- 0.315 0.595 0.061 0.597 0.223 0.109 0.231 0.045 dp dp After adjustment for age, sex, body mass index, antiplatelet drugs, hypertension, smoking habits.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 13

2.3. In Vitro Study Int. J. Mol. Sci. 2021, 22, 7193 The results showed that plasma from HeFH patients enhanced PA and oxidative4 of 12 stress, as assessed by platelet aggregation, TxB2, sNOX2-dp, and H2O2, compared to plasma from HS (Figures 2 and 3). Of note, PA and oxidative stress parameters were higher in wPLTs mixed with plasma taken from HeFH patients before treatment then A multivariate linear regression analysis showed that TxB reduction positively corre- wPLTs resuspended in plasma taken from HeFH patients after2 treatment. (Figures 2 and lated with LDL-c (Beta:0.316; p = 0.007) and ox-LDL (Beta:0.233; p = 0.044). No correlations 3, column D, panels A–D). were found with PCSK9 and sNOX2-dp. Furthermore, ox-LDL reduction positively cor- ox-LDL was 15.3 mU/mL and 26.4 mU/mL in plasma taken from HS and HeFH, re- related with PCSK9 (Beta:0.277; p = 0.016) and sNOX2-dp variation (Beta:0.231; p = 0.045) spectively. Furthermore, to prove the role of NOX2, wPLTs were pretreated with (Table2). NOX2ds-tat, a selective inhibitor of NOX2 activation, before being resuspended in plasma from HeFHTable patients 2. Multivariate before lineartreatment regression. TxB2, analyses. sNOX2-dp, and H2O2 production were significantly reduced (Figure 2, columns E, Panels A–D). Similarly, we observed the same ∆ TxB2results incubating wPLTS mixed with plasma taken from∆ ox-LDLHeFH patients before treatment Band E.S incubated Beta with CD36p inhibitor. This evidence Bsupports the E.S hypothesis Beta that NOX2p ac- ∆ PCSK9 −0.025tivation 0.343 was−0.008 due to CD36 0.942 pathway,∆ PCSK9 which we previously 0.153 demonstrated 0.062 0.277 to be activated 0.016 by ∆ ox-LDL 1.246PCSK9 0.607 [14] 0.233(Figure 2, columns 0.044 F, - panels A–D). CD36 is activated also by oxLDL. To prove ∆ LDL-c 0.387a 0.140 role of ox 0.316-LDL in the 0.007 process,∆ weLDL-c pre-incubated 0.044 wPLTS also 0.027 with LOX1, 0.191 observing 0.113 the ∆ sNOX2-dp 0.315same 0.595 results 0.061 again. (Figure 0.597 2, columns∆ sNOX2-dp G-H, panels 0.223 A–D) 0.109 0.231 0.045 After adjustmentAfter for age, PCSK9i sex, body treatment mass index,, the antiplatelet enhancement drugs, hypertension, of PA by plasma smoking habits.from HeFH was no longer detected (Figure 3, columns C vs. D, Panels A). To assess the role ox-LDL, we added ex- ogenous2.3. In Vitro ox- StudyLDL to plasma from patients after treatment. Platelet aggregation, TxB2, sNOXThe-dp resultslevels, showedand H2O that2 production plasma from were HeFH restored patients (Figure enhanced 3, columns PA and E, oxidative Panels A stress,–D). Asas assessed further confirm by plateletation aggregation,, pre-incubation TxB2, sNOX2-dp,with anti-CD36 and H2O2, and LOX1 compared reduced to plasma all marker from levelsHS (Figures (Figure2 and3, columns3). Of note, F-G, PA Panels and oxidativeA–D). Of stressnote, the parameters incubation were with higher both ininhibitors wPLTs inducedmixed with a further plasma decrease taken from of PA HeFH and oxidative patients before stresstreatment markers. then(Figure wPLTs 3, columns resuspended H, Pan- in elsplasma A–D) taken from HeFH patients after treatment. (Figures2 and3, column D, panels A–D).

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FigureFigure 2. PlateletPlatelet aggregation aggregation ( (AA)),, TxB TxB22 formationformation ( (BB),), sNOX2 sNOX2-dp-dp release release (C)),, and H2OO22 productionproduction ( (DD)) evaluated evaluated in in washedwashed platelets from HS ( n = 5 5)) resuspended resuspended in in plasma plasma derived derived from from HS HS or or HeFH HeFH patients patients before before treatment treatment with with PCSK9i PCSK9i andand stimulated stimulated with with STC STC of of collagen collagen (0.25 (0.25 µg/mL)µg/mL) in presence in presence or less or less of Nox2ds of Nox2ds-tat-tat or anti or anti-CD36-CD36 or anti or- anti-LOX1LOX1 or NC. or ** NC. p <** 0p.001).< 0.001 Abbreviation:). Abbreviation: PCSK9 PCSK9 inhibitors inhibitors (PCSK9i); (PCSK9i); Thromboxane Thromboxane B2 (TxB2); B2 (TxB2); soluble soluble NOX2 NOX2-derived-derived peptide peptide (sNOX2 (sNOX2--dp), hydrogen peroxide (H2O2); oxidized-LDL (ox-LDL), heterozygous familial hypercholesterolemia (HeFH), subthreshold dp), hydrogen peroxide (H O ); oxidized-LDL (ox-LDL), heterozygous familial hypercholesterolemia (HeFH), subthreshold concentration (STC), negative2 2 control (NC). concentration (STC), negative control (NC).

Int. J. Mol. Sci. 2021Int., 22 J., Mol. x FOR Sci. PEER2021 ,REVIEW22, 7193 6 of 13 5 of 12

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Figure 3. PlateletFigure aggregation 3. Platelet aggregation (A), TxB2 formation (A), TxB2 formation (B), sNOX2-dp (B), sNOX2 release-dp (C release), and H(C2)O, and2 production H2O2 production (D) evaluated in washed platelets(D) evaluated from HS (inn =washed 5) resuspended platelets from in plasma HS (n derived= 5) resuspended from HS orin HeFHplasma patients derived after from treatment HS or HeFH with PCSK9i and stimulatedpatients with after STC treatment of collagen with (0.25 PCSK9iµg/mL) and in stimulated presence orwith less STC of exogenousof collagen ox-LDL(0.25 µg/mL) (25 mU/mL), in presence Nox2ds-tat, or less of exogenous ox-LDL (25 mU/mL), Nox2ds-tat, anti-CD36, anti-LOX1, or NC (* p < 0.05; ** p anti-CD36, anti-LOX1, or NC (* p < 0.05; ** p < 0.001). Abbreviation: PCSK9 inhibitors (PCSK9i); Thromboxane B2 (TxB2); < 0.001). Abbreviation: PCSK9 inhibitors (PCSK9i); Thromboxane B2 (TxB2); soluble NOX2-derived soluble NOX2-derived peptide (sNOX2-dp), hydrogen peroxide (H2O2); oxidized-LDL (ox-LDL), heterozygous familial peptide (sNOX2-dp), hydrogen peroxide (H2O2); oxidized-LDL (ox-LDL), heterozygous familial hy- hypercholesterolemiapercholesterolemia (HeFH), subthreshold (HeFH), subthreshold concentration concentration (STC), negative (STC), control negative (NC). control (NC). ox-LDL was 15.3 mU/mL and 26.4 mU/mL in plasma taken from HS and HeFH, 3. Discussion respectively. Furthermore, to prove the role of NOX2, wPLTs were pretreated with NOX2ds- This studytat, demonstrates a selective inhibitor for the first of NOX2 time that activation, six months before of beingPCSK9i resuspended treatment re- in plasma from duces plateletHeFH activation patients lowering before circulating treatment. TxB2,levels sNOX2-dp,of ox-LDL and and NOX2 H2O2 production activity and were in significantly patients with reducedHeFH. (Figure2, columns E, Panels A–D). Similarly, we observed the same results incubat- PCSK9i representing wPLTS an mixedimportant with new plasma tool takenin the frommanagement HeFH patients of hypercholesterolemia before treatment and incubated and cardiovascularwith CD36prevention. inhibitor. Beyon Thisd the evidence effect on supports LDL reduction, the hypothesis ranging thatfrom NOX2 50–60% activation was [4,27] when useddue toon CD36top to pathway, statin ± whichezetimibe we previouslytherapy, PCSK9i demonstrated achieved to a be reduction activated of by PCSK9 [14] about 15% of the(Figure risk 2to, columnsdevelop a F, cardiovascular panels A–D). event CD36 in is secondary activated alsoprevention by oxLDL. [7,8] To. This prove a role of may suggest ancillaryox-LDL inposit theive process, effectswe of PCSK9i pre-incubated on the wPLTSthrombotic also cascade with LOX1, involved observing in the same acute coronaryresults events. again. (Figure2, columns G-H, panels A–D) Evidence of interplayAfter PCSK9i between treatment, PCSK9 theand enhancement platelet activation of PA bywere plasma found from in patients HeFH was no longer affected by atrialdetected fibrillation (Figure (AF)3, columns[28] and recently C vs. D, confirmed Panels A). by To Barale assess et theal. [16] role. In ox-LDL,deed, we added they found in exogenousa small population ox-LDL of to HeFH plasma a correlation from patients between after circulating treatment. levels Platelet of plate- aggregation, TxB2, let activation markerssNOX-dp and levels, PCSK9. and HNoteworthy,2O2 production Barale were and restored colleagues (Figure demonstrated3, columns E, that Panels A–D). As 6 months of PCSK9ifurther administration confirmation, pre-incubationreduced platelet with activation. anti-CD36 Nevertheles and LOX1s, they reduced did not all marker levels investigate the(Figure underling3, columns mechanism. F-G, Panels A–D). Of note, the incubation with both inhibitors induced Here, wea confirm, further decreasein a multicenter of PA and before oxidative–after stressstudy markers.conducted (Figure on a larger3, columns popula- H, Panels A–D). tion of HeFH patients, that PCSK9i reduces platelet activation and that this reduction correlated with oxLDL3. Discussion decrease. Moreover, oxLDL changes were significantly correlated to sNOX2-dp. As PCSK9This is study able demonstrates per se to activate for the NOX2, first time the thatconcomitant six months decrease of PCSK9i of both treatment reduces oxLDL and sNOX2platelet-dp activation suggests lowering that inhibiting circulating circulating levels oflevels ox-LDL of free and-PCSK9 NOX2 activitycould re- and in patients sult in reducedwith PA HeFH.via NOX2 activity modulation and ox-LDL formation. To support our hypothesis, we performedPCSK9i representan in vitro an study important using new platelets tool in from the managementHS mixed with of hypercholesterolemiaplasma from HeFH beforeand cardiovascular and after PCSK9i prevention. treatment. Beyond Platelet the activation, effect on in LDL presence reduction, of post ranging from ± treatment plasma50–60% results [4,27 in] whenlowerused platelet on topaggregation to statin andezetimibe TxB2 production. therapy, PCSK9i At the achieved same a reduction of about 15% of the risk to develop a cardiovascular event in secondary prevention [7,8].

Int. J. Mol. Sci. 2021, 22, 7193 6 of 12

This may suggest ancillary positive effects of PCSK9i on the thrombotic cascade involved in acute coronary events. Evidence of interplay between PCSK9 and platelet activation were found in patients affected by atrial fibrillation (AF) [28] and recently confirmed by Barale et al. [16]. Indeed, they found in a small population of HeFH a correlation between circulating levels of platelet activation markers and PCSK9. Noteworthy, Barale and colleagues demonstrated that 6 months of PCSK9i administration reduced platelet activation. Nevertheless, they did not investigate the underling mechanism. Here, we confirm, in a multicenter before–after study conducted on a larger population of HeFH patients, that PCSK9i reduces platelet activation and that this reduction correlated with oxLDL decrease. Moreover, oxLDL changes were significantly correlated to sNOX2- dp. As PCSK9 is able per se to activate NOX2, the concomitant decrease of both oxLDL and sNOX2-dp suggests that inhibiting circulating levels of free-PCSK9 could result in reduced PA via NOX2 activity modulation and ox-LDL formation. To support our hypothesis, we performed an in vitro study using platelets from HS mixed with plasma from HeFH before and after PCSK9i treatment. Platelet activation, in presence of post treatment plasma results in lower platelet aggregation and TxB2 production. At the same time, NOX2 activity was downregulated as shown by decreased sNOX2-dp release and formation of H2O2, a stable marker of NOX activity [20]. LDL oxidation plays a critical role in PA [18] by binding specific receptors on platelet surface, namely, CD36 and LOX1 [18,19]. Importantly, hyperlipidemic mice and humans with high LDL cholesterol have detectable levels of circulating oxidized lipids [18]. Al- though circulating levels of ox-LDL are not well defined, studies mixing “normal” platelets with plasma isolated from hyperlipidemic humans or mice (which contain detectable oxidized lipids) show platelet activation in a CD36 and LOX1-dependent manner. [18,19]. In our in vitro model, we confirm that ox-LDL both native from HeFH patients at baseline or added to samples post PCSK9i, were able to increase PA, a phenomenon that was counteracted by pretreating the samples with the specific ox-LDL receptor CD36 and LOX1 inhibitors. Of note, we obtain a similar inhibition by blocking the downstream pathways with a NOX2 inhibitor confirming that once bent to cell surface, ox-LDL need a pathway involving NOX2 activation in order to amplify PA. We consider a limitation of our study that the reduction of ox-LDL concentration after PCSK9i treatment could be at least in part dependent on its ability to lower native LDL. However, at the multivariate linear regression analyses the delta reduction of ox-LDL did not significantly correlate to the delta reduction of native LDL, but only with that of sNOX2-dp and TxB2. Based on this, while recognizing the role of LDL reduction in reducing oxLDL formation and subsequent PA, the free-PCSK9 reduction after PCSK9i treatment might further decrease oxLDL formation, also stopping the known mechanism of PCSK9-dependent NOX2 activation [14]. Another possible limitation is the small sample size as well as the duration of the study to establish if this evidence might be persistent during a longer PCKS9i treatment. We found a decrease of PCSK9 circulating levels after treatment, differently from what was observed in other studies [29–31]. In fact, in three different prior studies, using an ELISA kit dosing both free PCSK9 and PCSK9-PCSK9i complex, an increase in PCSK9 levels was found after treatment. The evidence found in our study be due to the specify of our assay. probably, the assay we used measures the free-bound form of PCSK9. In fact, in a pooled analysis of clinical trials conducted on 3000 patients treated with evolocumab, using an assay built to detect free-PCSK9, authors found that PCSK9 decreased after treatment [32].

4. Materials and Methods We conducted a prospective, multicenter before-after study in 80 non-diabetic HeFH patients treated with maximum tolerated statin dose ± ezetimibe who undergo treatment with PCSK9i. HeFH diagnosis was performed according to Dutch Lipid Clinic Network (DLCN) score [33]. PCKS9i were prescribed according to the Italian regulations [34] in Int. J. Mol. Sci. 2021, 22, 7193 7 of 12

patients with DLCN greater than 8 or with a molecular confirmation of HeFH. All patients have been included in the LIPIGEN study and had confirmed molecular diagnosis of HeFH [35,36]. Diabetic patients are known to be affected by impaired PA [37]. For this reason, diabetic patients were excluded to reduce confounding factors in PA evaluation. Chronic infection, systemic autoimmune disease, diabetes, history of alcohol or drug abuse, cancer or liver insufficiency were criteria of exclusion for enrolment in the study. Anamnestic, demographic, anthropometric, and routinary laboratory data were collected at baseline (defined as the visit when PCKS9i were prescribed), and after 6 months of PCSK9i treatment. A blood sample was collected at baseline and after 6 months of therapy. Samples were collected by antecubital vein after a 12 h overnight fast. Plasma was stored at −70 ◦C until the analysis. Experimental marker analyses was performed centrally in Sapienza–University of Rome laboratory. For the in vitro study, blood samples obtained from HeFH patients were taken into tubes with 3.8% sodium citrate and centrifuged at 300 g for 10 min to obtain supernatant, then immediately stored at −80 ◦C. All subjects gave written informed consent to be included in the study. The study protocol was approved by the local ethical board of Sapienza University of Rome (ref n◦ 3939) and was conducted according to the principles of the Declaration of Helsinki. Plasma and platelets sNOX2-dp production Plasma and platelets NOX2 activation was measured as a soluble NOX2-derived peptide (sNOX2-dp) with an ELISA method as previously reported [38]. Briefly, the peptide is recognized by binding to a specific monoclonal antibody against the amino acid sequence (224–268) that corresponds to the extracellular membrane part of NOX2 (catalytic core of NADPH oxidase), which was released following platelet activation. The enzyme activity is measured spectrophotometrically by the increased absorbance at 450 nm. Values were expressed as pg/mL; intra-assay and inter-assay coefficients of variation were 8.95 and 9.01%, respectively. Plasma Detection of ox-LDL Ox-LDL levels were measured by commercially available immunoassays (Cusabio, Houston, TX, USA). The optical density of samples was read at 450 nm and was expressed in mU/L. Intra-assay and inter-assay coefficients of variation were <8 and <10%, respectively.

Plasma and platelets TxB2 Assay

Plasma and platelets Thromboxane (Tx) A2 were analyzed by its stable metabolite named TxB2 in the supernatant by an ELISA commercial kit (Cusabio, Houston, TX, USA), according to manufacturer instructions. The values were expressed as pg/mL × 108 cells and pg/mL, respectively. Intra- and inter-assay coefﬁcients of variation for TxB2 were <8 and <10%, respectively.

Plasma and platelets H2O2 production

The Hydrogen Peroxide (H2O2) was measured by using a colorimetric assay as previously described [39]. The samples were incubated at room temperature for 20 min, and the reaction product was spectrophotometrically measured at 450 nm and expressed as µM. Plasma PCSK9 detection Plasma levels of PCSK9 were assessed by a commercial ELISA kit (Abcam, Cambridge, UK). Values were expressed in ng/mL. The intra- and inter-assay coefﬁcients of variation were estimated at 4.4 and 4.6%, respectively.

4.1. In Vitro Study 4.1.1. Platelet Preparation Blood samples added with citrated (3.8%, 1/10 (v:v) were taken between 8 and 9 a.m. from healthy subjects (HS, n = 5, males 3, females 2, age 37.2 ± 7.0 years) in fasting conditions. Blood was centrifuged for 15 min at 180 g at room temperature (RT) and the supernatant obtained was separated (2 × 105 platelets/µL) and represents platelet-rich plasma Int. J. Mol. Sci. 2021, 22, 7193 8 of 12

(PRP). Washed platelets (wPLT) were isolated from PRP by consecutive centrifugation steps (10 min at 300× g at RT) and resuspended in Tyrode’s buffer (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, 20 mM HEPES, 0.35% w/v bovine serum albumin (BSA), and 5.6 mM glucose, pH 7.35; Sigma Aldrich, St. Louis MO, USA). Finally, we added prostaglandin E1 (PGE1,1 µM) to prevent platelet activation. Platelet test function was carried out in 3 h.

4.1.2. oxLDL Preparation Human LDL was obtained from healthy subjects and blood was collected into tubes containing 7.2 mg EDTA. Brieﬂy, blood was centrifuged at 1500 g for 10 min at 4 ◦C. Next, 250 µL of PBS containing 0.25 mM EDTA were stratiﬁed on 750 µL of plasma and tubes were centrifuged at 100,000 rpm for 7 min. The upper 250 µL were removed to eliminate chylomicrons and another 250 µL of PBS with 0.25 mM EDTA were added. Thus, samples were centrifuged at 100,000 rpm for 2.5 h. Next, 250 µL of the upper layer were removed and 150 µL of potassium bromide (KBr) (50%, w/v) were added to obtain a density of 1.063 g/mL. Samples were centrifuged at 100,000 rpm for 5 h and the fraction containing LDL was recovered. Lastly, 200 µL of the fraction of LDL was dialyzed with PBS containing EDTA. Once the LDLs were isolated, they were oxidized by copper sulfate (CuSO4). Partic- ularly, LDL (50 µg/mL) was incubated with 10 µL of CuSO4 (1.65 µM). Finally, oxLDL concentration was determinate in cell supernatant after oxidation by ELISA kit as reported above.

4.1.3. Platelet Aggregation wPLTs were resuspended in a Tyrode solution containing 20% of plasma from 5 HeFH patients at center for ox-LDL distribution. Resuspension were obtained using (1) plasma before PCSK9i treatment (ox-LDL median: 24.58 [12.53–17.98–38.04] mU/mL); (2) plasma after PCSK9i treatment (ox-LDL median: 15.99 [11.28–22.21] mU/mL); as negative control was used plasma from 5 healthy subject (ox-LDL median: 8.28 [5.39–8.49] mU/mL). wPLTs were stimulated with a subthreshold concentration (STC, 0.25 µg/mL), the concentration of agonists was deﬁned as the highest concentration that elicited <20% platelet aggregation of collagen as a primer. STC was used to start aggregation test. To evaluate the role of ox-LDL, before activation, samples were pre-incubated 20 min at 37 ◦C with or without NOX2ds-tat, that is a selective inhibitor of NOX2 particularly, prevents the translocation of cytosolic p47phox in the membrane and inhibits its interactions with NOX2 [40] (10 µM, AnaSpec) or anti-CD36 (5 µM; Cayman, Ann Arbor, MI, USA) or LOX1 (5 µM; Cayman, Ann Arbor, MI, USA), the receptors on the platelet surface that binds and internalizes ox-LDL or negative control (NC). As negative control of all inhibitors, platelets were incubated with 1% Phosphate-buffered saline (PBS). After incubation, platelets were treated with exogenous ox-LDL (25mU/mL), for 5 min before activation. Platelet aggregation was performed with 2 dual-channel modules Chrono Log Model 700 LT aggregometer (Chrono Log, PA, USA), using siliconized glass cuvettes in constant stirring condition, using Born method [41]. Finally, all samples were centrifuged for 3 min at 3000 rpm and ◦ supernatants and pellets were separated and stored at −80 C for analysis of TXB2, and sNOX2-dp as reported below.

4.2. Statistical Analysis All 80 patients completed the study and there was no missing data. Continuous variables were expressed as mean, categorical variables as percentage. Before–after treatment comparisons were performed using a paired t-test. Treatment effect on LDL, ox-LDL, plasma TxB2, sNOX2-dp, and plasma PCSK9 were expressed as absolute variation. Two different stepwise multivariate linear regression analysis models were performed. The ﬁrst ones were conducted to investigate factors associated with TxB2 variation; LDL, Int. J. Mol. Sci. 2021, 22, 7193 9 of 12

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW ox-LDL, sNOX2-dp, and plasma PCSK9 change were alternatively tested10 of after 13 correction for age, sex, BMI, smoking habits, antiplatelet drugs use, hypertension. The second ones were conducted to test factors associated with ox-LDL change; LDL, sNOX2-dp, and plasma PCSK9 variation were one by one tested after correction for all other variables. “Prior iables. “Prior cardiovascular events” variable was not included in the multivariate anal- cardiovascular events” variable was not included in the multivariate analyses due to the yses due to the collinearity with “antiplatelet drugs” variable. Results were considered collinearity with “antiplatelet drugs” variable. Results were considered signiﬁcant when significant when p < 0.05. p < 0.05. All statistical analyses were performed using SPSS 25.0 for Windows (SPSS Institute, All statistical analyses were performed using SPSS 25.0 for Windows (SPSS Institute, Chicago, IL). Chicago, IL).

5. Conclusions 5. Conclusions To summarize, Towe summarize, found impaired we found oxidative impaired stress oxidativeand PA in stress patients and with PA in HeFH, patients with HeFH, which decreasewhich after treatment decrease afterwith treatmentPCSK9i. Moreover, with PCSK9i. we demonstrated Moreover, we the demonstrated key role of the key role of NOX2 in this process.NOX2 Indeed, in this process. blocking Indeed, NOX2 blockingactivity by NOX2 a specific activity inhibitor, by a specific the Nox2ds inhibitor,- the Nox2ds- tat, a reductiontat, of PA a reduction and oxidative of PA stress and oxidative was observed. stress wasAdditionally, observed. we Additionally, obtained the we obtained the same results blockingsame results CD36, blocking an upstream CD36, receptor an upstream activating receptor NOX2 activating cascade. NOX2As previ- cascade. As previously demonstrated,ously CD36 demonstrated, activity is CD36 modulated activity by is both modulated PCSK9 byitself both and PCSK9 ox-LDL itself [14,42] and. ox-LDL [14,42]. Supporting the Supportinghypothesis, theexogenous hypothesis, ox-LDL exogenous added to ox-LDL after treatment added to afterplasma treatment restored plasma restored the pre-treatmentthe conditions. pre-treatment Instead, conditions. the inhibition Instead, of the both inhibition receptors of of both ox- receptorsLDL, CD36, of ox-LDL, CD36, and LOX-1, inducedand LOX-1, a stronger induced decrease a stronger of PA decreaseand oxidative of PA stress and oxidative confirming stress their confirming in- their in- volvement in thevolvement process. Putting in the process. together Putting these findings, together we these can findings, conclude we that can in conclude vivo that in vivo treatment with treatment PCSK9i modulates with PCSK9i platelet modulates function platelet and function oxidative and oxidative stress: 1) stress: reducing (1) reducing NOX2 NOX2 activationactivation via CD36 via by CD36 lowering by lowering circulating circulating free-PCSK9 free-PCSK9 and 2) reducing and (2) reducingox-LDL ox-LDL effect effect on CD36 andon CD36 LOX1. and Thus, LOX1. the reduction Thus, the of reduction ox-LDL ofmight ox-LDL be due might by both be due the byreduc- both the reduction tion of LDL oxidation,of LDL oxidation,induced by induced circulating by circulating PCSK9, and PCSK9, the LDL and substrate the LDL reduction substrate reduction after after PCSK9i (FigurePCSK9i 4).(Figure 4).

Platelet Aggregation PCSK9 LDLs inhibitors

oxLDLs

H2O2 sNOX2-dp PCSK9 H2O2 H2O2 TxA2

NOX2 LOX1 CD36

phox p47 p67phox Platelet p40phox

TxB2

H2O2

H2O2 H2O2 Figure 4. CirculatingFigure 4. Circulating PCSK9 can PCSK9 bind thecan CD36 bind the receptor CD36 onreceptor platelets on andplatelets activates and activates the downstream the downstream signaling including NOX2 activation,signaling ROS including production, NOX2 and activation, plateletactivation. ROS production, PCSK9i and modulate platelet platelet activation. function PCSK9i and modulate oxidative stress both reducing NOX2platelet activation function byand preventing oxidative thestress interplay both reducing between NOX2 free PCSK9 activation molecule by preventing and CD36 the receptor interplay and reducing oxLDL effectbetween on CD36 free and PCSK9 LOX1. molecu Greenle arrow:and CD36 PCSK9i receptor pathway. and reducing Red lines: oxLDL PCSK9 effect and on ox-LDL CD36 pathway. and LOX1. Abbreviation: Green arrow: PCSK9i pathway. Red lines: PCSK9 and ox-LDL pathway. Abbreviation: PCSK9 in- PCSK9 inhibitors (PCSK9i); Thromboxane A2 (TxA2); soluble NOX2-derived peptide (sNOX2-dp), hydrogen peroxide hibitors (PCSK9i); Thromboxane A2 (TxA2); soluble NOX2-derived peptide (sNOX2-dp), hydrogen (H2O2); low-density lipoprotein (LDL), oxidized-LDL (ox-LDL). peroxide (H2O2); low-density lipoprotein (LDL), oxidized-LDL (ox-LDL).

Author Contributions: Investigation, data curation and writing—original draft preparation, conceptualization, and critical revision, V.C. (Vittoria Cammisotto); Investigation, statistical analysis and writing—original draft preparation, conceptualization, and critical revision F.B.; methodology and critical revision, V.C. (Valentina Castellani), S.B., and C.N.; patients recruitment and critical revision, L.D., N.C., C.B., R.S., A.D.P., and S.P.; writing—review and editing, critical revision, and

Int. J. Mol. Sci. 2021, 22, 7193 10 of 12

Author Contributions: Investigation, data curation and writing—original draft preparation, conceptualization, and critical revision, V.C. (Vittoria Cammisotto); Investigation, statistical analysis and writing—original draft preparation, conceptualization, and critical revision F.B.; methodology and critical revision, V.C. (Valentina Castellani), S.B., and C.N.; patients recruitment and critical revision, L.D., N.C., C.B., R.S., A.D.P., and S.P.; writing—review and editing, critical revision, and supervision, M.D.B., M.A., I.R., F.P., R.C., F.V., and D.P.; project administration, original draft preparation, critical revision, conceptualization and supervision, P.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grant from PhD course: Fisiopatologia Ed Imaging Cardio- Toraco-Vascolare, Sapienza University of Rome. Institutional Review Board Statement: The study protocol was approved by the local ethical board of Sapienza University of Rome (ref n◦ 3939) and was conducted according to the principles of the Declaration of Helsinki. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations Proprotein convertase subtilisin kexin type 9 (PCSK9); low-density lipoprotein cholesterol receptors (LDLRs); LDL-cholesterol (LDL-c); major adverse cardiovascular events (MACEs); platelet activation (PA); heterozygous familial hypercholesterolemia (HeFH); oxidized-LDL (ox-LDL); Dutch Lipid Clinic Network (DLCN); soluble NOX2-derived peptide (sNOX2-dp); Thromboxane (Tx); hydrogen peroxide (H2O2); healthy subjects (HS); room temperature (RT); platelet-rich plasma (PRP); washed platelets (wPLT); bovine serum albumin (BSA); prostaglandin E1 (PGE1); negative control (NC). phosphate-buffered saline (PBS).

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