10.4081/btvb.2022.45
The anticoagulant protein S (PS) binds phospholipids with very high affinity, but PS interaction with lipoproteins and lipidrich atherosclerotic plaques remains still poorly defined. We investigated PS in plasma lipoproteins and in atherosclerotic plaques from ten patients undergoing endarterectomy. PS was detected by Western blotting after exposure of the necrotic core to liposomes and was found to maintain its ability to bind phosphatidylserine micelles. The amounts of PS bound to low/very low-density lipoproteins in patient’ plasmas were higher and more variable than those detected in healthy subjects. A direct correlation between bound PS and low-density lipoproteins (LDL), plasma levels was found only in patients (r=0.921, p&lt;0.001), thereby leading to hypothesize that the PS-phospholipids binding may increase by oxidative processes of LDL in atherosclerotic patients. The presence of the PS into the necrotic core of atherosclerotic plaques and on the surface of lipoproteins, particularly the atherogenic LDL, suggests a LDL-based delivery of PS to the atherosclerotic plaques and emphasizes the deep link between plasma lipids and coagulation in cardiovascular diseases.
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Francesco Bernardi
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Bleeding, Thrombosis and Vascular Biology
Phospholipids
Protein S on the surface of plasma lipoproteins: a potential mechanism for protein S delivery to the atherosclerotic plaques?
Text.Serial.Journal
https://www.btvb.org/btvb
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Brief Reports
2022-10-17
2022-07-28
2022-10-17
2022-10-24
Bleed Thromb Vascul Biol
Francesco Bernardi
2022/10/17
10.4081/btvb.2022.45
Department of Life Sciences and Biotechnology, University of Ferrara
2785-5309
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Phospholipids
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Martinelli N, Baroni M, Castagna A, et al. Apolipoprotein C-III Strongly Correlates with Activated Factor VII-Anti-Thrombin Complex: An Additional Link between Plasma Lipids and Coagulation. Thromb Haemost. 2019;119:192-202.
Protein S on the surface of plasma lipoproteins: a potential mechanism for protein S delivery to the atherosclerotic plaques?
https://www.btvb.org/btvb/article/download/45/39
1
                                                                                                                       BRIEF REPORT

Protein S on the surface of plasma lipoproteins:
a potential mechanism for protein S delivery to the atherosclerotic plaques?
Marcello Baroni,1 Paolo Ferraresi,1 Nicole Ziliotto,2 Daria Bortolotti,3 Pierfilippo Acciarri,4 Nicola Martinelli,5
Giovanna Marchetti,6 Matteo Coen,7 Francesco Bernardi1
1
  Department of Life Sciences and Biotechnology, University of Ferrara, Italy; 2Department of Pharmacy, University of Pisa, Italy;
3
  Department of Chemistry, University of Ferrara, Italy; 4Unit of Vascular and Endovascular Surgery, S. Anna University-Hospital,
Ferrara, Italy; 5Department of Medicine, Unit of Internal Medicine, University of Verona, Policlinico G.B. Rossi, Verona, Italy;
6
  Department of Neurosciences and Rehabilitation, University of Ferrara, Italy; 7Division of General Internal Medicine, Department of
Medicine, Geneva University Hospital, Geneva, Switzerland



    Correspondence: Marcello Baroni, University of Ferrara, 74 Fos-            ABSTRACT
    sato di Mortara, 44121 Ferrara, Italy.                                         The anticoagulant protein S (PS) binds phospholipids with
    Tel. +39 0532 974423.                                                      very high affinity, but PS interaction with lipoproteins and lipid-
    E-mail: brnmcl@unife.it
                                                                               rich atherosclerotic plaques remains still poorly defined. We in-




                                                                                                 ly
    Key words: Protein S, Atherosclerotic plaques, Lipoproteins, Phos-         vestigated PS in plasma lipoproteins and in atherosclerotic
                                                                               plaques from ten patients undergoing endarterectomy. PS was de-




                                                                                         on
    pholipids.
                                                                               tected by Western blotting after exposure of the necrotic core to
    Acknowledgments: The study has been carried out at the Depart-             liposomes and was found to maintain its ability to bind phos-
    ment of Life Sciences and Biotechnology, University of Ferrara,            phatidylserine micelles. The amounts of PS bound to low/very




                                                                                   e
    Ferrara, Italy. The study was financially supported by Grant from          low-density lipoproteins in patient’ plasmas were higher and more
    Italian Ministry of University and Research, University of Ferrara,
    Italy (FAR 2020, FAR 2021), 5x1000 University of Ferrara grant
    (2021_NAZ.A_B) and by “Bando FIRD 2022 – BARONI” Uni-
                                                                              us
                                                                               variable than those detected in healthy subjects. A direct correla-
                                                                               tion between bound PS and low-density lipoproteins (LDL),
                                                                               plasma levels was found only in patients (r=0.921, p<0.001),
                                                                              al
    versity of Ferrara grant.
                                                                               thereby leading to hypothesize that the PS-phospholipids binding
                                                                               may increase by oxidative processes of LDL in atherosclerotic
                                                           ci

    Contributions: AP, CM and MG characterized the patient material
    and clinical data. BM and FP were responsible for immunodeple-             patients. The presence of the PS into the necrotic core of athero-
                                                         er


    tion, ELISA and Western blotting analysis. ZN did the statistical          sclerotic plaques and on the surface of lipoproteins, particularly
    analyses and together with BD, MN and MG contributed to the in-            the atherogenic LDL, suggests a LDL-based delivery of PS to the
                                                        m



    terpretation of the data and drafting of the manuscript. BM and BF         atherosclerotic plaques and emphasizes the deep link between
    designed the study, interpreted the data and wrote the manuscript.
                                                                               plasma lipids and coagulation in cardiovascular diseases.
                                            om




    Conflict of interest: The Authors declare no potential conflict of
    interest.
                                      -c




    Funding: None.                                                             INTRODUCTION
                                                                                    Atherosclerosis is a lipoprotein-driven disease that
                             on




    Ethical approval: This study was approved by local Ethical Com-
    mittee (University-Hospital of Ferrara) with protocol number 7-            leads to plaque formation at specific sites of the arterial
    2010. All the recruited subjects signed an informed consent.               vessels through intima inflammation, necrosis, fibrosis,
                       N




                                                                               and calcification.1 Atherosclerosis is also a chronic low-
    Received for publication: 20 June 2022.
    Accepted for publication: 15 September 2022.                               grade inflammatory disease associated with increased ex-
                                                                               pression of the acute phase reactants, like the isoforms of
    Publisher’s note: All claims expressed in this article are solely those    Serum Amyloid A (SAA1 and SAA2) which circulate
    of the authors and do not necessarily represent those of their affili-
                                                                               linked with the high-density-lipoprotein (HDL) and may
    ated organizations, or those of the publisher, the editors and the re-
    viewers. Any product that may be evaluated in this article or claim        represent a plasma biomarker for future cardiovascular
    that may be made by its manufacturer is not guaranteed or endorsed         events.2,3 Atherosclerosis is initiated by the subendothelial
    by the publisher.                                                          retention of apolipoprotein B (apoB)-containing lipopro-
    ©
                                                                               teins, particularly the low-density lipoproteins (LDL), and
     Copyright: the Author(s), 2022
    Licensee PAGEPress, Italy                                                  continues with their oxidation (oxLDL) and with
    Bleeding, Thrombosis and Vascular Biology 2022; 1:45                       macrophage uptake of oxLDL.4 These processes are fol-
    doi:10.4081/btvb.2022.45                                                   lowed by macrophage transformation to foam cells that
    This work is licensed under a Creative Commons Attribution-
                                                                               play a critical role in the development of atherosclerosis.
    NonCommercial 4.0 International License (CC BY-NC 4.0).                         Protein S (PS) is a plasma vitamin K-dependent protein,
                                                                               produced by a variety of cell types (hepatocytes, endothe-
                                                                               lial cells, megakaryocytes, osteoblasts).5 PS acts as an an-
                                                                               ticoagulant, alone and as cofactor for activated protein C

                                                Bleeding, Thrombosis and Vascular Biology 2022; 1:45
6                                                         M. Baroni et al.




and tissue factor pathway inhibitor.6 Beyond the historically        was collected from the homogenate, incubated in ice for 30
well recognized anticoagulant role, PS may influence other           min and centrifuged at 13,000 g at 4°C for 15 min. Total
crucial pathways. PS can activate TAM receptors, such as             protein was quantified by bicinchoninic acid kit (BCA Pro-
Mer,7 thereby favoring leukocyte extravasation, inflamma-            tein Assay Reagent Kit, Pierce).
tory activation and adhesion of platelets to endothelial cells.
By activation of Mer receptor, PS inhibits macrophage                Liposome-mediated extraction of PS
scavenger receptor A-mediated acetylated LDL uptake in               from atherosclerotic plaques
macrophages, thus reducing the formation of foam cells.8
                                                                         Two liposome preparations,16 phosphatidylserine/
PS is also associated to damaged endothelial surface, to             phosphatidylcoline 80/20% (Lip80/20) and 100/0% (Lip100),
very low-density lipoprotein (VLDL) rich in triglycerides,           or Hepes buffer (Hepes 20 mM, NaCl 150 mM, CaCl2 5
and localizes in atherosclerotic coronary lesions by im-             mM, pH 7.4) were used to draw PS from plaque samples,
munohistochemistry and Western blotting experiments.9 PS             followed by Western blotting analysis conducted by 4-
also promotes phagocytosis of apoptotic cells10 and mitosis
                                                                     12% SDS-PAGE separation and polyclonal anti-PS anti-
of vascular smooth muscle cells.11 Taking into account these
                                                                     body (Dako, Glostrup, Denmark), recognizing free and
multi-faceted features of PS and its ability to bind biologi-
                                                                     C4BP-bound PS.
cal membranes, particularly the oxidized phospholipid sur-




                                                                                              ly
faces,12 it appears plausible that PS may be present both in
                                                                     Lipoproteins separation and PS content analysis




                                                                                      on
the necrotic core of the atherosclerotic plaques and on the
surface of lipoproteins, including the atherogenic LDL.                  Apolipoprotein B100-bound VLDL and LDL
    Although the presence of PS in the carotid atheroscle-           (ApoB100VLDL/LDL) and SAA2-containing HDL




                                                                                  e
rotic plaques has been reported,9 several questions remain           (SAA2HDL) were obtained from fresh plasma samples of
open, particularly concerning the interaction and binding
of PS with lipoproteins. In the present study we investi-
                                                                             us
                                                                     fasting TEA-P and HS on microplates by using goat poly-
                                                                     clonal anti-ApoB100 (Sigma-Aldrich, Milan, Italy) and
gate these issues in a group of patients who underwent en-           mouse polyclonal anti- SAA2 (Abnova, Taipei, Taiwan)
                                                                    al
darterectomy, a surgical procedure which allows to                   antibodies, respectively. As a background study, plasma
                                                        ci

remove the atheromatous plaque material and, thus, to                samples of TEA-P and HS were in parallel incubated on
perform specific analyses ex vivo.                                   microplates with rabbit polyclonal anti-BSA antibody
                                                      er


                                                                     (Sigma-Aldrich, Milan, Italy). Microplate bound lipopro-
                                                     m



                                                                     teins (SAA2HDL and ApoB100VLDL/LD) were detached by
MATERIALS AND METHODS                                                LDS Sample Buffer (ThermoFisher, Milan, Italy) and an-
                                           om




                                                                     alyzed for the presence of PS by Western blotting. Den-
Subjects
                                                                     sitometric analysis of Western blots defined the amounts
    Ten male patients who underwent thrombo-en-                      of PS bound to SAA2HDL and to ApoB100VLDL/LDL (PS-
                                      -c




darterectomy (TEA-P) for severe arterial occlusion13 and             SAA2HDL and PS- ApoB100VLDL/LDL) after background
                              on




10 healthy subjects (HS) - mean age 71.5±5.9 vs 28.8±5.3             subtraction and comparison with purified PS standard.
years, respectively, p<0.0001 - were studied.
                                                                     Statistics
                         N




PS and lipoproteins plasma assays
                                                                         Unpaired t test analysis (Welch's correction indicated
    Total plasma PS was measured by ELISA with poly-                 by *) and Pearson’s correlation were made by Graph Pad 8
clonal anti-PS antibodies.14 LDL and HDL levels were                 (Prism) and SPSS statistics 25 (IBM). Data were analyzed
evaluated by discontinuous gradient density and flotation            before and after logarithmic transformations of values. A p
ultracentrifugation from fresh plasma samples.15                     value <0.05 was considered statistically significant.

Atherosclerotic plaques processing
                                                                     RESULTS AND DISCUSSION
    A total of 10 TEA specimens, which were used for the
different experiments, were obtained at surgery from con-               We investigated the presence of PS in the carotid ath-
secutive patients who underwent conventional TEA. 2 mg               erosclerotic plaque of patients that underwent endarterec-
of necrotic core from carotid artery plaques were used for           tomy (TEA-P), a surgical procedure to remove the
protein extraction. The samples were homogenized, under              atheromatous plaque material.
cryogenic conditions, in a mortar pestle and proteins ex-               Plasma levels of PS and lipoproteins were compared
tracted using a lysis buffer (50 mM Tris-HCl, 150 mM                 between TEA-P and a group of healthy subjects (HS).
NaCl, 1% SDS, 0.1% NP-40, 1 mM DTT, 0.5% Sodium de-                     TEA-P did not differ from HS in PS plasma levels
oxycholate, 10 mM Benzamidine, pH 8.0). The supernatant              (TEA-P, 55.2±10.0 mg/L; HS, 52.5±14.9 mg/L) and HDL

                                       Bleeding, Thrombosis and Vascular Biology 2022; 1:45
     Protein S on the surface of plasma lipoproteins: a potential mechanism for protein S delivery to the atherosclerotic plaques?     7



concentration (TEA-P, 57.2±13.4 mg/dL; HS, 49.4±14.5                         deposition into the plaque, the presence of PS on lipopro-
mg/dL, p=0.228), and showed higher LDL concentration                         teins from fresh plasma was investigated. Lipoproteins
as a trend (TEA-P, 134.9±90.5 mg/dL; HS, 81.0±33.2                           were recovered by immunodepletion of plasma and sub-
mg/dL, p*=0.104). An inverse, although not significant,                      jected to Western blotting for PS (Figure 2A, lanes 1-4;
correlation between PS concentration and HDL levels was                      Figure 2C, lanes 1-4). To support proper quantification of
observed in HS (r=-0.609, p=0.061), but not in TEA-P                         PS specifically bound to lipoproteins, we also measured
(r=0.021, p=0.955).                                                          the amount of PS not specifically bound to the microplate.
    The analysis of the lysed necrotic core of atheroscle-                   This background, as reported in figures 2A (lanes 5-8) and
rotic plaque, tested via Western blotting, clearly showed                    2C (lanes 5-8), was considered to quantify the amounts of
the presence of increasing amounts of PS (Figure 1A) in                      bound PS. Comparison by densitometric analysis of sam-
direct relation to the increasing amounts of lysed tissue                    ples from TEA- P and, as a reference, from HS is reported
(lanes 1-3). Band size and pattern were comparable to that                   in figures 2B and 2D. Large variations in PS concentration
of PS in plasma (lane 4), albeit the larger and uncleaved                    were observed in VLDL/LDL captured by an anti-
form was more represented in plasma than in plaque.                          ApoB100 antibody in plasma from TEA-P (Figure 2B,
    Taking into account that the PS anticoagulant activities                 2.49±3.19 ng/µL PS-ApoB100VLDL/LDL, black bars) as
cannot be evaluated in lysed tissue, we investigated as sur-                 compared with that from HS (0.87±0.59 ng/µL, grey




                                                                                             ly
rogate the lipid binding properties of PS, experimentally                    bars). The highest PS concentrations (>2 ng/µL) were de-




                                                                                      on
released from the plaque after exposure of the necrotic core                 tectable in VLDL/LDL from TEA-P. Noticeably, a posi-
fraction to liposomes with different lipid composition and,                  tive correlation between PS-ApoB100VLDL/LDL and LDL
as negative control, in the absence of liposomes (Figure 1B,                 levels was detectable in TEA-P (r=0.921, p<0.001, Figure




                                                                                 e
lane 6). The amounts of PS recovered from the necrotic                       3A, black dots), but not in HS (grey dots). The wide dif-
core in the presence of liposomes rich in phosphatidylserine
(lane 4, Lip80/20), for which PS affinity is well known, were
much higher than those obtained with phosphatidylcholine
                                                                             us
                                                                             ferences between TEA-P and HS in patterns of PS bound
                                                                             to lipoproteins are not based on different concentration
                                                                             and distribution of LDL, which were similar in the groups.
                                                                        al
liposomes (lane 5, Lip100), which in turn were similar to                        The analysis of PS was extended to HDL by the im-
those obtained with buffer (lane 6).
                                                        ci

                                                                             munodepletion approach with an anti-SAA2 antibody,
    These experiments, which confirm the presence of                         aimed at capturing the inflammatory HDL fraction. This
                                                      er


measurable amounts of PS forms in atherosclerotic                            approach could allow to evaluate the PS bound to these
plaques, indicate that a portion of PS protein still main-
                                                     m



                                                                             lipoproteins (Figure 2C and D). Densitometric analysis of
tains the ability to bind phosphatidylserine micelles.                       Western blots showed higher amounts (p*=0.024) of bound
                                          om




    Given the main role of plasma lipoproteins in lipid                      PS in TEA-P (3.19±2.37 ng/µL) than in HS (1.13±0.81
                                                                             ng/µL), with the highest concentrations present in patients.
                                                                             PS bound to SAA2, and thus to inflammatory HDL, corre-
                                   -c




                                                                             lated as a trend with HDL concentration in plasma in TEA-
                          on




                                                                             P (r=0.547, p=0.102, Figure 3B), but not in HS (r=-0.105
                                                                             p=0.773, Figure 3B, gray dots)). PS-SAA2HDL in patients
                                                                             and HS did not correlate with PS plasma levels (r=0.032,
                     N




                                                                             p=0.929 and r=0.016, p=0.965, respectively).
                                                                                 Interestingly, a positive correlation (r=0.650, p=0.042)
                                                                             was observed between levels of PS-SAA2HDL and PS-
                                                                             ApoB100VLDL/LDL in patients but not in HS (r=-0.031,
                                                                             p=0.933). Correlation between plasma PS concentration
Western blotting of PS in the necrotic core from carotid artery plaques      and PS-ApoB100VLDL/LDL was not detectable in either
were conducted by 4-12% SDS-PAGE separation and polyclonal anti-             TEA-P (r=-0.241, p=0.502) or HS (r=0.479, p=0.161).
PS antibody. (A) Reducing conditions. Lanes 1, 2 and 3, increasing
amounts (3, 10 and 30 µg of total protein extract, respectively) of lysate       The positive correlation between PS-bound and
necrotic core from plaque. Lane 4: human pooled normal plasma (PNP)          lipoprotein levels, detectable only in patients, suggested
diluted (1:5) to contain 100 ng of PS in 20 µL. Migration is compatible      that specific features of lipoproteins in this group could
with the entire (upper band) and cleaved (lower band) PS forms. (B)
Non-reducing conditions. Equal portions (2 mg) of necrotic core were
                                                                             be a key factor for the PS interaction. We are tempting to
incubated overnight with liposomes (Lip80/20, lane 4 and Lip100, lane 5)     speculate that the significant correlation between PS-
or Hepes buffer (lane 6) at 4°C, and supernatants tested for the presence    ApoB100VLDL/LDL and LDL may reflect the specific lipid
of PS. Lane 1, 30 ng of purified PS (HTI); lane 3, diluted (1:5) human       composition/oxidation of LDL in atherosclerotic patients.
PNP. Lane 2: 20 µL of 10 mM Lip80/20 as a negative control.
                                                                             The undetectable correlation between amounts of PS
Figure 1. PS in atherosclerotic plaques by Western blotting                  bound to lipoproteins and plasmatic PS levels may sup-
analysis.                                                                    port this hypothesis.

                                             Bleeding, Thrombosis and Vascular Biology 2022; 1:45
8                                                                M. Baroni et al.




                                                                                                           ly
                                                                                                   on
                                                                                            e
                                                                                     us
                                                                             al
                                                              ci
                                                            er


(A,C) Representative Western blots by polyclonal anti-PS on lipoproteins ApoB100VLDL/LDL (A) or SAA2HDL (C), captured in microplate from fresh
                                                           m



plasma of fasting TEA-P (lanes 1-4, A and C). The non-specific binding of plasma PS to microplate (background, lanes 5-8, A and C), was subtracted in
the densitometric analysis. (B, D) Densitometric analysis of PS-Western blots of ApoB100VLDL/LDL (B) or SAA2HDL (D) from plasma of HS (grey bars)
                                                om




or TEA-P (black bars), which showed large variation in PS concentration.
Figure 2. Western blotting analysis on lipoprotein-bound PS, in patients (TEA-P) and in healthy subjects (HS).
                                          -c
                                  on
                            N




Correlation between lipoprotein-bound (A) PS-ApoB100VLDL/LDL or (B) PS-SAA2HDL with plasma levels of LDL or HDL, respectively, in TEA-P (black
circles) and in HS (grey circles). Significant and positive correlation occurs only between PS-ApoB100VLDL/LDL and plasmatic LDL levels in TEA-P (A).
No correlation occurs between PS-SAA2HDL and HDL levels, both in TEA-P and HS (B).

Figure 3. Correlation analysis between lipoprotein-bound PS and lipoprotein levels in plasma.


                                           Bleeding, Thrombosis and Vascular Biology 2022; 1:45
    Protein S on the surface of plasma lipoproteins: a potential mechanism for protein S delivery to the atherosclerotic plaques?     9



     Overall data support the presence of PS protein in                4. Hartley A, Haskard D, Khamis R. Oxidized LDL and anti-
highly - one order of magnitude - variable amounts on the                 oxidized LDL antibodies in atherosclerosis - Novel insights
surface of lipoproteins, in particular on LDL from pa-                    and future directions in diagnosis and therapy. Trends Car-
tients. The positive correlation with the plasma concen-                  diovasc Med 2019;29:22-6.
                                                                       5. Di Scipio RG, Hermodson MA, Yates SG, Davie EW. A
tration of these micelles, and not with PS plasma
                                                                          comparison of human prothrombin, factor IX (Christmas
concentration, suggests a role of the lipoprotein features                factor), factor X (Stuart factor), and protein S. Biochemistry
for binding. On the other hand, it may be hypothesized                    1977;16:698-706.
that the PS-phospholipids binding may increase by oxida-               6. Gierula M, Ahnström J. J Thromb Haemost 2020;18:2801-11.
tive processes.12 The role of oxidized-LDL in atheroscle-              7. van der Meer JH, van der Poll T, van't Veer C. TAM recep-
rotic plaque formation and growth is well described,17                    tors, Gas6, and protein S: roles in inflammation and hemo-
together with the differentiation into foam cells of                      stasis. Blood 2014;123:2460-9.
macrophage and smooth muscle cells (SMCs) after LDL                    8. Liao D, Wang X, Li M, et al. Human protein S inhibits the
interactions.18 Furthermore SMCs, which are the major                     uptake of AcLDL and expression of SR-A through Mer re-
                                                                          ceptor tyrosine kinase in human macrophages. Blood
cell type in arterial wall with a pivotal role in plaque de-
                                                                          2009;113:165-74.
velopment, are able to synthesize and secrete PS with an-
                                                                       9. Hurtado B, Muñoz X, Recarte-Pelz P, et al. Expression of
ticoagulant function in vitro.19 The results of the present




                                                                                        ly
                                                                          the vitamin K-dependent proteins GAS6 and protein S and
study are also consistent with the concept of a deep, and                 the TAM receptor tyrosine kinases in human atherosclerotic




                                                                                on
still uncharted, link between plasma lipids/lipoproteins                  carotid plaques. Thromb Haemost 2011;105:873-82.
and the molecular players of coagulation pathway in pa-               10. Anderson HA, Maylock CA, Williams JA, et al. Serum-de-
tients with cardiovascular diseases.20                                    rived protein S binds to phosphatidylserine and stimulates the




                                                                           e
                                                                          phagocytosis of apoptotic cells. Nat Immunol 2003;4:87-91.

CONCLUSIONS                                                         us11. Gasic GP, Arenas CP, Gasic TB, Gasic GJ. Coagulation fac-
                                                                          tors X, Xa, and protein S as potent mitogens of cultured aor-
                                                                          tic smooth muscle cells. Proc Natl Acad Sci USA
                                                             al
    Our study has some limitations. A small number of pa-                 1992;89:2317-20.
tients and healthy subjects, not matched for age, was com-            12. Baroni M, Pavani G, Marescotti D, et al. Membrane binding
                                               ci

pared, also related to the need of using fresh plasma and                 and anticoagulant properties of protein S natural variants.
                                                                          Thromb Res 2010;125:e33-9.
                                             er


plaque samples for optimal lipoprotein isolation and PS
extraction, respectively, in parallel assays. In addition, PS         13. Marchetti G, Girelli D, Zerbinati C, et al. An integrated ge-
                                            m



                                                                          nomic-transcriptomic approach supports a role for the proto-
analysis was not performed in all plasma HDLs, but only
                                                                          oncogene BCL3 in atherosclerosis. Thromb Haemost
on the SAA2-bound fraction, which anyway represents
                                   om




                                                                          2015;113:655-63.
the major HDL subfraction during inflammation.2 Finally,              14. Ziliotto N, Meneghetti S, Menegatti E, et al. Expression pro-
our data do not allow to establish whether a major part of                files of the internal jugular and saphenous veins: Focus on
the PS into atherosclerotic plaques derives from plasma
                             -c




                                                                          hemostasis genes. Thromb Res 2020;191:113-24.
or SMCs near to the necrotic core of the plaque, which                15. Wang Y, Qiao M, Mieyal JJ, et al. Molecular mechanism of
                      on




deserves further investigation.                                           glutathione-mediated protection from oxidized low-density
    Despite these limitations, the delivery of the plasma                 lipoprotein-induced cell injury in human macrophages: role
                                                                          of glutathione reductase and glutaredoxin. Free Radic Biol
                 N




PS into the necrotic core of the atherosclerotic plaques
                                                                          Med 2006;41:775-85.
through lipoproteins, particularly the LDL, could be a
                                                                      16. Baroni M, Mazzola G, Kaabache T, et al. Molecular bases
physiopathological process with a plausible protective                    of type II protein S deficiency: the I203-D204 deletion in
role during atherosclerosis progression and complica-                     the EGF4 domain alters GLA domain function. J Thromb
tions, like unstable plaque rupture, in which the antico-                 Haemost 2006;4:186-91.
agulant properties of PS may potentially limit the                    17. Steinberg D, Witztum JL. Oxidized low-density lipoprotein
superimposed thrombosis. However, the ability of PS to                    and atherosclerosis. Arterioscler Thromb Vasc Biol
influence and limit plaque related thromboembolic events                  2010;30:2311-6.
requires further investigation.                                       18. Pryma CS, Ortega C, Dubland JA, Francis GA. Pathways of
                                                                          smooth muscle foam cell formation in atherosclerosis. Curr
                                                                          Opin Lipidol 2019;30:117-24.
                                                                      19. Benzakour O, Kanthou C. The anticoagulant factor, protein
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