10.4081/btvb.2022.50
Direct oral anticoagulants (DOACs) are currently recommended by European guidelines as the first line therapy for both stroke prevention in patients with atrial fibrillation (AF) and the prevention and the treatment of venous thromboembolism (VTE). Recently, it has been speculated that DOACs have anti-inflammatory capabilities in reducing the abnormal release of pro-inflammatory factors in addition to inhibiting the activation of factor X or factor II of the coagulation cascade. However, this hypothesis is based on limited pathophysiological data with small sample size, often on in vitro studies. Real-world, in vivo, and large clinical data are scarce. The aim of the present study was the evaluation of the possible anti-inflammatory and anti-proliferative effects of DOACs treatment in a cohort of patients affected by AF or VTE, by analyzing an extensive panel of cytokines and molecules involved in the process of vascular and tissue remodeling. Our data evidenced that DOACs treatment is associated with variations in systemic inflammation markers and in metalloproteinases. Further studies with larger number of patients are required to confirm these data.
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Bleeding, Thrombosis and Vascular Biology
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Pleiotropic effects of anti-thrombotic therapies: have direct oral anticoagulants any anti-inflammatory effect?
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Rossella Marcucci
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Department of Experimental and Clinical Medicine, University of Florence; Atherothrombotic Diseases Center, Careggi Hospital, Florence
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Zhang YB, Li W, Yao LQ, et al. Expression changes and roles of matrix metalloproteinases in a rat model of traumatic deep vein thrombosis. Chin J Traumatol 2010;13:188-92.
Pleiotropic effects of anti-thrombotic therapies: have direct oral anticoagulants any anti-inflammatory effect?
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                                                                                                           ORIGINAL ARTICLE

Pleiotropic effects of anti-thrombotic therapies: have direct oral anticoagulants
any anti-inflammatory effect?
Anna Maria Gori,1,2 Eleonora Camilleri,1 Alessia Bertelli,1 Angela Rogolino,2 Francesca Cesari,2 Elena Lotti,2
Tommaso Capobianco,1 Walther Iannotti,1 Betti Giusti,1,2 Rossella Marcucci1,2
1
 Department of Experimental and Clinical Medicine, University of Florence, Italy; 2Atherothrombotic Diseases Center, Careggi Hospital,
Florence, Italy



ABSTRACT
     Direct oral anticoagulants (DOACs) are currently recommended by European guidelines as the first line therapy for both stroke
prevention in patients with atrial fibrillation (AF) and the prevention and the treatment of venous thromboembolism (VTE). Recently,
it has been speculated that DOACs have anti-inflammatory capabilities in reducing the abnormal release of pro-inflammatory factors
in addition to inhibiting the activation of factor X or factor II of the coagulation cascade. However, this hypothesis is based on limited
pathophysiological data with small sample size, often on in vitro studies. Real-world, in vivo, and large clinical data are scarce. The aim
of the present study was the evaluation of the possible anti-inflammatory and anti-proliferative effects of DOACs treatment in a cohort
of patients affected by AF or VTE, by analyzing an extensive panel of cytokines and molecules involved in the process of vascular and
tissue remodeling. Our data evidenced that DOACs treatment is associated with variations in systemic inflammation markers and in
metalloproteinases. Further studies with larger number of patients are required to confirm these data.




    Correspondence: Anna Maria Gori, Department of Experimental               INTRODUCTION
    and Clinical Medicine, University of Florence, Largo Brambilla 3,
    50134 Florence, Italy.                                                        Direct oral anticoagulants (DOACs) represent a valid
    Tel.: +39.055794942.1                                                     substitute for vitamin K antagonists in the prevention and
    E-mail: annamaria.gori@unifi.it                                           the treatment of thromboembolic events due to their char-
    Key words: Direct oral anticoagulants, Atrial fibrillation, Venous
                                                                              acteristics that make them more manageable and safer.
    thromboembolism, Inflammatory markers, Metalloproteinases.                The European guidelines recommend DOACs as a first
                                                                              line therapy both for stroke prevention in patients with
    Contributions: The authors contributed equally.                           atrial fibrillation (AF) and for the prevention and the treat-
    Conflict of interest: The authors declare no potential conflict of in-
                                                                              ment of venous thromboembolism (VTE).1,2
    terest.                                                                       Their clinical efficacy and safety have been reported
                                                                              in various clinical trials,3-5 but few studies have evaluated
    Funding: None.                                                            their possible pleiotropic effects, particularly anti-inflam-
                                                                              matory and anti-proliferative effects.
    Ethical approval and consent to participate: Written informed con-
    sent was obtained from all patients.                                          The study of the modulation of inflammation by oral
                                                                              anticoagulants represents a relevant subject both from a
    Availability of data and material: Data and materials are available       pathophysiological and a clinical point of view, supported
    from the Authors.                                                         by the literature on inflammation as a common substrate
    Received for publication: 16 August 2022.                                 for AF and VTE. Inflammation is linked to a series of
    Accepted for publication: 28 November 2022.                               pathological processes, including oxidative stress, apop-
                                                                              tosis and fibrosis, that are capable of creating a patholog-
    Publisher’s note: All claims expressed in this article are solely those   ical substrate for AF, through their contribution to atrial
    of the authors and do not necessarily represent those of their affili-
    ated organizations, or those of the publisher, the editors and the re-
                                                                              remodelling.6 In the biomarker study of the ARISTOTLE
    viewers. Any product that may be evaluated in this article or claim       trial, which evaluated more than 14,000 AF patients on
    that may be made by its manufacturer is not guaranteed or endorsed        oral anticoagulation (warfarin or apixaban), the inflam-
    by the publisher.                                                         matory markers [C Reactive Protein (CRP) and inter-
                                                                              leukin-6] were significantly associated with an increased
    ©
     Copyright: the Author(s), 2022
    Licensee PAGEPress, Italy                                                 risk of mortality.7 Moreover, inflammation is also associ-
    Bleeding, Thrombosis and Vascular Biology 2022; 1:50                      ated with the occurrence of thromboembolic event, due
    doi:10.4081/btvb.2022.50                                                  to the capability of inflammatory markers to induce en-
                                                                              dothelial dysfunction, platelets and coagulation activation,
    This work is licensed under a Creative Commons Attribution-
    NonCommercial 4.0 International License (CC BY-NC 4.0).                   thus contributing to thrombus formation. Recently, evi-
                                                                              dence has emerged on the role of inflammation also in

                                                Bleeding, Thrombosis and Vascular Biology 2022; 1:50
            Pleiotropic effects of anti-thrombotic therapies: have direct oral anticoagulants any anti-inflammatory effect?     61



VTE. Pro-inflammatory cytokines such as interleukin-1                  in patients on DOACs (apixaban, dabigatran and rivarox-
(IL-1), interleukin-6 (IL-6) and Tumor Necrosis Factor-                aban) treatment.
alpha (TNF-α) promote a pro-coagulant state, inducing
the occurrence and the progression of VTE and their com-
plication.8,9 The association between anti-inflammatory                MATERIALS AND METHODS
effect and cardiovascular disease has been supported by
                                                                           This is a prospective, observational cohort study with
the CANTOS study, in which the use of the anti-IL-1β an-
                                                                       the aim of evaluating pleiotropic effects of DOACs.
tibody canakinumab was associated with the reduction of
                                                                           Three hundred and fifty-eight patients were enrolled
cardiovascular mortality.10 Other cardiovascular drugs
                                                                       in the period between 10th October 2016 and 19th October
such as statins, heparins and anti-PCSK-9 antibodies are
                                                                       2017 (dabigatran n=90, rivaroxaban n=143 e apixaban
endowed with anti-inflammatory effects. The reduction
                                                                       n=125) at Careggi University Hospital. We present here
of the cardiovascular mortality observed with statin treat-
                                                                       data on the first 54 patients (dabigatran n=16, rivaroxaban
ment was ascribed not only to the LDL-lowering effect
                                                                       n=20 e apixaban n=18) without acute or chronic inflam-
but also to the anti-inflammatory effect of statins, which
                                                                       matory disease from whom two venous samples were
contributes to plaque stability and decrease endothelial
                                                                       available: one collected at one month after the beginning
dysfunction.11,12 In vitro and ex-vivo studies showed that
                                                                       of treatment (T1) and the second one after 6 months (T2).
heparins, in addition to their anticoagulant activity, pro-
                                                                       Inclusion criteria were the diagnosis of AF or of VTE,
vided anti-inflammatory effect.13,14 Recently, it has been
                                                                       therapy with DOACs (dabigatran, rivaroxaban or apixa-
suggested that also the novel lipid lowering therapy anti-
                                                                       ban), written informed consent for venous sample. Exclu-
PCSK-9 antibodies had anti-inflammatory properties.15
                                                                       sion criteria were the presence of acute or chronic
    The modulation of DOACs on inflammation, extra-
                                                                       inflammatory disease.
cellular matrix, cellular proliferation and angiogenesis
                                                                           The study design consisted of a clinical evaluation at
was studied by in vitro and animal models. In in vitro
                                                                       the time of the first contact, with venous blood sample
studies, the anti-inflammatory activities of DOACs may
                                                                       taken at one month and at six months after the beginning
be ascribed to their capability to inhibit the activation of
                                                                       of the treatment.
the protease-activated receptor (PAR)-1 or PAR-2 signal-
                                                                           Demographic data, clinical characteristics, risk factors
ing pathway,16-19 or to down-regulate platelet activation,
                                                                       and treatment modalities were collected at the time of first
by a mechanism involving Glycoprotein VI (GPVI) shed-
                                                                       contact.
ding reduction.20 Animal models showed that rivaroxaban
and edoxaban attenuated atrial fibrosis induced by trans-
                                                                       Blood samples
verse aortic constriction in mice and reduced vulnerability
to AF, suggesting that FXa inhibition might exert a car-                   Whole venous blood was collected in tubes without
dioprotective effect against atrial remodelling.21-23 Fur-             anticoagulant and with 3.2% trisodium citrate (9:1 v/v,
thermore, rivaroxaban significantly attenuated neointima               blood/anticoagulant). Blood was centrifuged within 1 h
formation in rat injured arteries, and reduced expression              from collection at 2000 × g for 15 min. Aliquots of serum
of cytokines (as IL-1β e TNF-β) both in injured arteries               or plasma were quickly frozen and stored at −80°C until
and in mouse peritoneal macrophages.24                                 testing. Blood samples were collected 1 and 6 months
    In humans, few and contrasting studies about the effect            after treatment with DOACs at C-trough level, obtained
of DOACs treatment on inflammation and atrial fibrosis                 at 12 h from the last dose intake for dabigatran and apix-
are available. In patients with AF dabigatran, rivaroxaban             aban, and at 24 h from the last dose intake for rivaroxa-
and apixaban administration was associated with significant            ban. Levels of different inflammatory markers [IL-1RA,
decrease of inflammatory markers.25,26 In the same clinical            IL-6, IL-8, IL-10, TNF-alpha, vascular endothelial growth
setting, it has been also observed that the anti-Xa drugs sig-         factor (VEGF), intercellular adhesion molecule-1 (ICAM-
nificantly reduce the urinary excretion of TxB2, and soluble           1), vascular cell adhesion molecule-1 (VCAM-1)] were
GPVI, a peptide derived from GPVI shedding, suggesting                 determined using BioPlex suspension array system and
that they possess an antiplatelet effect.27 In acute ischemic          Biorad Kits (Bio-Rad Laboratories Inc., Hercules, CA,
stroke patients both apixaban and dabigatran treatment re-             USA). Metalloproteinases (MMP1, MMP2, MMP3,
duced CRP and IL-6 levels.28 At variance, other studies                MMP7, MMP8, MMP9 and MMP10) were assessed in
failed to reveal any significant effect of DOACs treatment             the same patients using Bio-Plex suspension array system
on inflammation.29                                                     (Bio-Rad Laboratories Inc., Hercules, CA, USA) and
    The aim of the present study is to evaluate the anti-in-           R&D Kits (R&D System, Milan Italy) following manu-
flammatory and anti-proliferative effects of DOACs treat-              facturer’s instructions. The coefficient of variation of in-
ment through evaluation of cytokines and molecules                     flammatory markers, and MMPs assays was <6%.
involved in the process of vascular and tissue remodeling                  Apixaban, rivaroxaban and dabigatran plasma levels,

                                       Bleeding, Thrombosis and Vascular Biology 2022; 1:50
62                                                       A.M. Gori et al.




expressed as drug concentration (ng/mL), were assessed              Table 1. Demographic and clinical characteristics.
by commercial kits (Werfen Milan, Italy), according to               Demographic characteristics and risk factor   All patients (n=54)
the manufacturer’s instructions.
                                                                     Age (years), mean and SD                            73 (15)
    Diluted thrombin, calibrated for dabigatran, and spe-            Men, n (%)                                        32 (59.3%)
cific anti-FXa assays, calibrated for apixaban and rivarox-          BMI (Kg/m2), mean and SD                           26.4 (4.0)
aban, were used to measure DOACs plasma levels.                      Creatinine (mg/dL), mean and SD                    0.9 (0.2)
                                                                     Creatinine Clearance (mL/min), median and IQR   74 (53.3-93.5)
Statistics                                                           Hb (g/dL), mean and SD                             13.5 (1.8)
                                                                     RBC (106 /μL), median and IQR                    4.7 (4.4-5.4)
     Discrete data are expressed as frequencies, and con-            Platelets (103 /μL), mean and SD                223.07 (64.1)
tinuous data as mean±SD or median and interquartile                  ALT (U/L), median and IQR                         23 (17-32)
range, as appropriate. The χ² test was used to compare cat-          AST (U/L), median and IQR                        20 (17-24.5)
egorical variables, and the unpaired two-tailed Student’s            Hypertension, n (%)                                38 (70.4)
                                                                     Smoking habit, n (%)                                4 (7.4)
t-test or Mann-Whitney rank-sum test was used to test dif-           Diabetes, n (%)                                     9 (16.7)
ferences between continuous variables.                               Family history for CVD, n (%)                      15 (27.8)
     In order to evaluate the degree of inflammation, patients       Medical history, n (%)
treated with DOACs were divided into three categories on              Non valvular atrial fibrillation (NVAF)           33 (61.1)
the basis of the grade of inflammation (low, moderate and             Cronic NVAF                                       15 (27.8)
elevated). For this purpose the levels of cytokines IL-1Ra,           Parossistic NVAF                                  18 (33.3)
                                                                      Venous thromboembolism                            21 (38.9)
IL-6, IL-8, IL-10 and TNFα were divided into tertiles and
                                                                     DOACs, n (%)
were used to calculate a global grading of inflammation.              Dabigatran                                        16 (29.6)
Patients with high levels of pro-inflammatory cytokines               Rivaroxaban                                       20 (37.0)
(IL-6, IL-8 and TNF-α) not balanced with level of the anti-           Apixaban                                          18 (33.3)
inflammatory ones (IL-1Ra, IL-10) were assigned as pa-               CHADSVASc score of NVAF patient (n=33), n (%)
tients with an elevated grade of inflammation; patients with          1                                                   2 (6.1)
                                                                      2                                                  5 (15.2)
moderate-high levels of pro-inflammatory cytokines par-
                                                                      3                                                 14 (42.4)
tially balanced with levels of anti-inflammatory ones were            4                                                  6 (18.2)
assigned as patients with a moderate grade of inflammation,           5                                                  5 (15.2)
whereas patients with balanced levels of pro- and anti-in-            6                                                   1 (3.0)
flammatory cytokines were assigned as patients with a low            Concomitant therapy, n (%)
level of inflammation.                                                Aspirin                                             2 (3.7)
     As for DOACs plasma concentrations, they were di-                Ticagrelor                                             0
                                                                      Clopidogrel                                        1 (1.9)
vided in two groups in relation to patients’ median level. In
                                                                      Prasugrel                                              0
particular, patients on apixaban therapy were divided in two          Antiarrhythmic drugs                               9 (16.7)
groups depending on if apixaban plasma levels were above              Antidiabetic drugs                                 7 (13.0)
or under 94 ng/mL after one month of treatment and 90                 Lipid lowering drugs                              24 (44.4)
ng/mL after 6 months of treatment. Similarly, patients on             Antihypertensive drugs                            35 (64.8)
rivaroxaban were divided in two groups depending if ri-               Ace inhibitors                                    14 (25.9)
                                                                      Beta blockers                                      20 (37)
varoxaban plasma levels were above or under 28 ng/mL
                                                                      Calcium channel blockers                          10 (18.5)
after 1 month of treatment and 24 ng/mL after six months              Diuretics                                         13 (24.1)
of treatment, and patients in treatment with dabigatran were          Sartans                                           14 (25.9)
divided in two groups on the basis of dabigatran plasma               Nitrate                                            2 (3.7)
levels (above or under 111 ng/mL after 1 month of treat-              Digoxin                                            4 (7.4)
ment, and 90 ng/ml after 6 month of treatment).                       Gastroprotectors                                  28 (51.9)
                                                                      Antiviral                                              0
                                                                      Immunosuppressive drugs                                0
                                                                      Steroids                                           2 (3.7)
RESULTS                                                               Drugs for thyroid disease                         8 (14.8)
                                                                      Antipsychotic drugs                                 7 (13)
    Demographic and clinical characteristics of enrolled
                                                                      Antiepileptic                                      2 (3.7)
patients are shown in Table 1. As shown in Table 2, no                Anxiolytics                                       8 (14.8)
significant difference in terms of cytokines and metallo-             Painkillers                                         3 (5.6)
proteinases levels was found among the different groups             SD, standard deviation; BMI, body mass index; IQR, interquartile
of DOACs , both one and six months after treatment. No              range; Hb, hemoglobin; RBC, red blood cell counts; ALT, alanine
significant difference in plasma levels of inflammatory             aminotransferase; AST, aspartate aminotransferase; CVD,
marker were found between patient with AF and TEV                   cardiovascular disease; DOACs, direct oral anticoagulants.


                                      Bleeding, Thrombosis and Vascular Biology 2022; 1:50
             Pleiotropic effects of anti-thrombotic therapies: have direct oral anticoagulants any anti-inflammatory effect?                         63



(data not shown), therefore we could analyze globally pa-                      VCAM-1 are significantly higher at T2 with respect T1
tients’ data as a single population.                                           (Table 3).
                                                                                   In Table 4 the ratios between pro-inflammatory and
Inflammatory markers and DOACs treatment                                       anti-inflammatory cytokines at T1 and at T2 are shown.
   In Table 3 inflammatory markers’ levels at one and six                      The ratios between IL-6/IL-10 and IL-8/IL-10 were sig-
months after treatment are shown. Treatment with                               nificantly lower at time T2 compared to the one at time
DOACs for six months is associated with significantly                          T1, whereas the ratio between TNF-α/IL-1RA was signif-
higher circulating levels of anti-inflammatory cytokine                        icantly higher at T2 compared to the one at T1 (Table 4).
IL-10, pro-inflammatory cytokine TNF-α and VEGF-α.                                 By using a global inflammatory score, we identify at
Furthermore, circulating levels of the adhesion molecule                       T1 30 patients (55.5%) in which the global inflammation


Table 2. Cytokines and metalloproteases levels according to DOACs treatment.
                                  Dabigatran (n=16)                  Rivaroxaban (n=20)                   Apixaban (n=18)                        P
IL-6 (pg/mL)                        1.70 (0.76-3.40)                    1.60 (0.52-2.36)                   1.35 (0.99-2.35)                    0.793
IL-8 (pg/mL)                        3.16 (1.94-4.43)                    3.85 (1.92-6.15)                   3.63 (2.30-6.28)                    0.760
TNF-α (pg/mL)                       0.11 (0.030-2.04)                  0.73 (0.030-3.61)                   1.99 (0.83-2.69)                    0.165
IL-1RA (pg/mL)                  276.54 (215.95-526.00)             362.19 (230.055-688.83)             389.79 (279.18-550.72)                  0.579
IL-10 (pg/mL)                       0.30 (0.20-7.65)                   1.07 (0.22-10.47)                  0.25 (0.20-11.90)                    0.463
VEGF-α (pg/mL)                     7.68 (0.030-21.80)                  6.37 (3.73-18.66)                  9.69 (5.26-16.23)                    0.823
ICAM-1 (ng/mL)                  184.54 (136.49-216.44)               132.61 (70.52-190.89)             117.72 (142.84-232.88)                  0.095
VCAM-1 (ng/mL)                  655.42 (462.18-844.55)              738.75 (419.43-922.61)            688.47 (561.07-1002.82)                  0.813
MMP-1 (ng/mL)                     0.287 (0.207-0.384)                 0.380 (0.265-1.070)                0.429 (0.194-1.130)                   0.298
MMP-2 (ng/mL)                   211.38 (164.59-619.19)              339.89 (156.13-583.01)             483.30 (123.05-744.96)                  0.783
MMP-3 (ng/mL)                     30.74 (19.61-56.20)                 33.41 (18.37-61.74)               49.98 (24.99-101.57)                   0.319
MMP-7 (ng/mL)                     1.762 (0.770-2.998)                 1.378 (1.050-1.678)                1.906 (1.043-3.114)                   0.423
MMP-8 (ng/mL)                     0.677 (0.332-1.128)                 0.722 (0.400-1.013)                0.689 (0.371-1.464)                   0.914
MMP-10 (ng/mL)                    0.387 (0.210-0.748)                 0.543 (0.351-0.748)                0.367 (0.234-0.557)                   0.208
IL, interleukin; TNF, tumor necrosis factor; IL-1RA, interleukin -1 receptor antagonist; VEGF, vascular-endothelial growth factor; ICAM, intercellular
adhesion molecule; VCAM, Vascular cell adhesion protein; MMP, matrix metalloproteinases. Values are reported as median and interquartile ranges
(in parenthesis)



Table 3. Inflammatory markers levels after 1 and 6 months of DOACs treatment.
                                                   T1                                  T2                                  P
IL-6 (pg/mL)                                 1.58 (0.89-2.43)                   1.55 (0.80-2.60)                         0.652
IL-8 (pg/mL)                                 3.63 (2.10-5.79)                   3.23 (2.04- 5.67)                        0.959
TNF-α (pg/mL)                                1.05 (0.03-2.46)                   1.62 (0.65-3.23)                         0.033
IL-1RA (pg/mL)                           354.24 (248.92-572.93)              364.22 (269.55-544.60)                      0.997
IL-10 (pg/mL)                               0.30 (0.20-10.21)                    5.04 (0.3-11.71)                        0.005
VEGF-α (pg/mL)                              7.68 (3.34-18.89)                  16.19 (10.45-21.19)                      <0.001
ICAM-1 (ng/mL)                           143.51 (105.61-212.84)              149.93 (108.96-232.29)                      0.997
VCAM-1 (ng/mL)                           686.95 (487.44-9.21.43)            769.35 (651.96-1002.89)                      0.034
IL, interleukin; TNF, tumor necrosis factor; IL-1RA, interleukin -1 receptor antagonist; VEGF, vascular-endothelial growth factor; ICAM, intercellular
adhesion molecule; VCAM, Vascular cell adhesion protein. Values are reported as medians and interquartile ranges (in parenthesis); values statistically
significative are in italics.



Table 4. Pro- and anti-inflammatory cytokines ratios after 1 and 6 months of DOACs.
                                                   T1                                  T2                                  P
IL-6/IL-10                                  3.46 (0.20-8.77)                   0.36 (0.12-3.47)                          0.012
TNF-α/IL-10                                 0.42 (0.10 -5.15)                   0.45 (0.13-2.16)                         0.188
IL-8/IL-10                                  8.7 (0.55-18.30)                   0.90 (0.35-2.96)                         <0.001
IL-6/IL-1RA                              0.0044 (0.0023-0.0077)             0.0042 (0.0022-0.0067)                       0.887
TNF-α/ IL-1RA                            0.0028 (0.0001-0.0071)             0.0049 (0.0020-0.0106)                       0.001
IL-8/ IL-1RA                             0.0094 (0.0059-0.0154)             0.0113 (0.0052-0.0177)                       0.608
IL, interleukin; TNF, tumor necrosis factor; IL-1RA, interleukin -1 receptor antagonist. Values are reported as medians and interquartile ranges (in
parenthesis); values statistically significative are in italics.


                                            Bleeding, Thrombosis and Vascular Biology 2022; 1:50
64                                                                   A.M. Gori et al.




level was low, 10 patients (18.5%) with moderate grade of                          Correlations between inflammatory markers,
inflammation and 14 (30%) with high level of inflamma-                             metalloproteinases and DOACs plasma concentrations
tion. Treatment for six months with DOACs is associated
                                                                                       We analyzed the correlation between DOACs plasma
with a significant reduction of patients classified in the
                                                                                   levels and circulating inflammatory markers after 1 and 6
group with high level of inflammation. In fact, 8 out of 14
                                                                                   months of treatment. After 1 month, apixaban and rivarox-
(57.1%) patients with high level of inflammation at T1
                                                                                   aban plasma levels were positively and significantly corre-
switched to the low level of inflammation group and 1
                                                                                   lated with anti-inflammatory cytokines IL-1RA and IL-10
(7.1%) into the moderate group. Similarly, 5 of 10 patients
                                                                                   levels. Furthermore, dabigatran plasma levels were posi-
classified as moderate level of inflammation at T1 after 6
                                                                                   tively related with circulating levels of TNF-α (Table 6).
months switched into the low-level category (50%), and in
                                                                                       After six months of treatment apixaban levels were in-
only one patient the grade of inflammation became higher.
                                                                                   versely correlated with anti-inflammatory cytokines IL-
Among 30 (73.3%) patients with low grade of inflamma-
                                                                                   10 and positively related with adhesive molecule
tion at T1 only 8 patients (26.7%) switched to a higher in-
                                                                                   ICAM-1, whereas dabigatran levels were positively cor-
flammation group. Overall, only 4 patients out of 54 (7.4%)
                                                                                   related with circulating levels of IL-1RA.
switched from a lower-moderate level of inflammation to
                                                                                       As MMPs levels are concerned, at T1 dabigatran
a higher one, instead 14 out of 54 patients (25.9%) with
                                                                                   plasma levels were inversely related with circulating lev-
high grade of inflammation switched to a lower grade.
                                                                                   els of MMP-8 (rho=-0.615; p=0.011) whereas at T2 cir-
                                                                                   culating levels of dabigratan were related with increasing
Metalloproteinases and DOACs treatment
                                                                                   levels of MMP-7 (rho=0.600; p=0.014).
   Treatment with DOACs (1 and 6 months) was associ-                                   By dividing DOACs plasma concentrations in relation
ated with significant increase in circulating levels of                            to patients’ median levels of drugs, patients with DOACs
MMP-7 and MMP-10 (Table 5).                                                        concentrations at T1 above the median values had signif-


Table 5. Circulating levels of metalloproteinases after 1 and 6 months of DOACs treatment.
                                                     T1                                    T2                                   P
MMP-1 (ng/mL)                               0.368 (0.207-1.003)                   0.697 (0.258-1.458)                         0.065
MMP-2 (ng/mL)                              313.88 (158.32-651.4)                  245.7 (165.3-714.5)                         0.689
MMP-3 (ng/mL)                               37.93 (20.33-62.58)                   47.60 (26.14-70.38)                         0.272
MMP-7 (ng/mL)                                 1.59 (1.01-2.58)                      2.16 (1.34-2.82)                          0.003
MMP-8 (ng/mL)                               0.693 (0.367-1.145)                   0.806 (0.426-1.471)                         0.183
MMP-9 (ng/mL)                               28.48 (21.05-43.71)                  29.41 (22.065-47.88)                         0.499
MMP-10 (ng/mL)                              0.464 (0.308-0.648)                   0.602 (0.463-0.780)                         0.015
MMP, matrix metalloproteinases. Values are reported as medians and interquartile ranges (in parenthesis); values statistically significative are in italics.



Table 6. Correlations between circulating levels of cytokines and DOACs levels after 1 and 6 months of treatment.
                                   Apixaban (ng/mL)                            Rivaroxaban (ng/mL)                             Dabigatran (ng/mL)
                                          (N=18)                                        (N=20)                                         (N=16)
                                   T1                 T2                         T1                 T2                          T1                T2
IL-8 (pg/mL)                     -0.013             -0.140                     0.023               0.351                      -0.026            -0.021
                                  0.958              0.578                     0.922              -0.129                       0.922             0.940
TNF-α (pg/mL)                    -0.240             -0.044                     -0.138             -0.005                       0.575             0.375
                                  0.338              0.861                     0.562               0.985                       0.020             0.152
IL-6 (pg/mL)                     -0.191             -0.160                      0.213             -0.221                       0.313             0.279
                                  0.447              0.526                     0.367               0.348                       0.237             0.295
IL-10 (pg/mL)                    -0.079             -0.537                      0.542              0.013                      -0.205             0.186
                                  0.757              0.022                     0.014               0.957                       0.447             0.490
IL-1RA (pg/mL)                    0.690              0.298                     -0.050              0.096                       0.235             0.556
                                  0.002              0.229                     0.835               0.686                       0.380             0.025
VEGF-α (pg/mL)                   -0.131              0.084                      0.226              0.151                      -0.032             0.397
                                  0.605              0.741                     0.339               0.526                       0.905             0.128
ICAM-1 (ng/mL)                    0.522              0.571                      0.123             -0.110                      -0.009            -0.135
                                  0.026              0.013                     0.605               0.645                       0.974             0.617
IL, interleukin; TNF, tumor necrosis factor; IL-1RA, interleukin -1 receptor antagonist; VEGF, vascular-endothelial growth factor; ICAM, intercellular
adhesion molecule. T1 and T2, levels after 1 and 6 months of treatment, respectively. Values statistically significative are in italics.


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             Pleiotropic effects of anti-thrombotic therapies: have direct oral anticoagulants any anti-inflammatory effect?                        65



icantly higher levels of IL-1RA compared to the levels                         increase in TNF-α circulating levels. This datum seems to
observed in patients DOACs concentration under median                          be inconsistent with the supposed anti-inflammatory ef-
value (Table 7). Similarly, after 6 months of treatment, pa-                   fect of DOACs. However, it has to be considered that, be-
tients with DOACs concentration had higher levels of the                       yond the prevalent effect on a single molecule, cytokines
anti-inflammatory cytokine IL-1RA than the ones with                           are pleiotropic and synergic and some of them can behave
lower concentrations of the drugs (Table 7).                                   as either antagonist or agonist of the other cytokines. The
                                                                               alteration of the expression of one cytokine can signifi-
                                                                               cantly modify the profile of many other cytokines. There-
DISCUSSION                                                                     fore, it is necessary to evaluate the whole cytokines
    Treatment with DOACs for six months was associated                         network and consider the global pattern of pro- and anti-
with significantly higher levels of IL-10, TNF-α, VEGF-α,                      inflammatory cytokines. In actual fact, it is the displace-
and VCAM-1. Furthermore, by analyzing the balance be-                          ment of pro- and anti-inflammatory ratios that determines
tween pro-inflammatory and anti-inflammatory cytokines,                        a pro-inflammatory phenotype, rather than the increase of
we found that after 6 months of DOACs treatment IL-6/IL-                       a single pro-inflammatory cytokine.
10 and IL-8/IL-10 ratios were significantly lower compared                         Using a global score of inflammation (low, moderate,
to ratios observed at one month. On the contrary, the ratio                    and elevated grade of inflammation according to the ter-
between TNF-α/IL1RA were significantly higher at six                           tiles of pro- and anti-inflammatory cytokines), almost
months compared to ratio at one month.                                         60% of patients, after six months of therapy, showed a re-
    Therefore, our results suggest that prolonged DOACs                        duction of the grade of inflammation compared to the first
treatment is able to influence the subtle inflammation,                        month of therapy. Considering the balance between pro-
which characterized both venous thromboembolism and                            and anti-inflammatory cytokines, this result indicates a
atrial fibrillation. Interestingly, our data reveal that                       net anti-inflammatory effect of DOACs, mainly driven by
DOACs treatment increased the anti-inflammatory cy-                            the increase of IL-10 levels at six months.
tokine IL-10, whose levels might effectively counterbal-                           In addition to the anti-inflammatory effect of DOACs
ance the inflammatory status present in the                                    treatment, our data show a time-dependent modification
above-mentioned diseases, suggesting that DOACs treat-                         of MMP-7 and MMP-10 levels, suggesting that this treat-
ment reduces the adverse outcomes occurrence not only                          ment can modulate, mirroring the effect of DOACs on cy-
through their capability of reducing thrombin formation,                       tokines patterns, the MMP plasma levels and
but also through their action on the inflammation.                             consequently the remodeling process. MMPs are a family
    In fact, IL-10, produced by B and T lymphocytes and                        of zinc-dependent proteases, that have a specific role in
NK cells, is able to inhibit pro-inflammatory cytokines                        pathophysiological proteolysis of extracellular matrix and
synthesis, therefore reducing leukocyte maturation and re-                     in tissue remodeling. Moreover, MMPs are able to mod-
cruitment during inflammation. It also has a role in sup-                      ulate growth factors and cytokines activities. The balance
pressing the capacity to display the antigen to                                between MMPs and their inhibitors control the extracel-
antigen-presenting cells and induces the production of an-                     lular matrix turnover. MMP-7 acts not only on extracel-
tibodies and lymphocytes B survival.                                           lular matrix components but also on pro-inflammatory
    In our study, DOACs treatment causes a significant                         molecules such as pro-α defensin, Fas-Ligand, TNF-α and


Table 7. Circulating levels of cytokines according to DOACs concentrations above or below the median levels after 1 and 6 months of
treatment (N=18).
                                                            T1                P                                               T2                P
                                DOACs above           DOACs below                                 DOACs above           DOACs below
                                median value         median valuen                                median value          median value
                                     n. %                  n. %                                        n. %                  n. %
IL-8 (pg/mL)                     3.2 (1.9-5.8)         3.7 (2.6-6.3)        0.331                  3.5 (2.1-6.0)         3.2 (2.0-5.0)        0.551
TNF-α (pg/mL)                   0.6 (0.03-2.2)        1.6 (0.03-2.7)        0.224                  1.4 (0.6-2.6)         2.0 (0.7-5.1)        0.337
IL-6 (pg/mL)                    1.2 (0.6-2.19          1.8 (0.9-3.5)        0.161                  1.6 (1.0-2.5)         1.5 (0.8-3.3)        0.883
IL-10 (pg/mL)                   0.3 (0.2-10.6)        0.3 (0.2-10.1)        0.929                 5.0 (2.9-14.2)        4.8 (0.3-11.2)        0.386
IL-1RA (pg/mL)                 307.5 (224-520)       491.4 (275-699)        0.046                 305 (245-464)         399 (334-594)         0.049
VEGF-α (pg/mL)                  7.1 (2.4-15.4)        7.9 (3.7-22.3)        0.411                 13.4 (7.3-18.3)      14.9 (12.0-22.2)       0.057
ICAM-1 (ng/mL)                  140 (92-191)          157 (124-215)         0.346                  149 (87-225)         151 (113-306)         0.574
IL, interleukin; TNF, tumor necrosis factor; IL-1RA, interleukin -1 receptor antagonist; VEGF, vascular-endothelial growth factor; ICAM, intercellular
adhesion molecule. Values statistically significative are in italics.


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66                                                      A.M. Gori et al.




E-cadherin. MMP-10 can directly lyse extracellular ma-             of the fibrinolysis and anti-fibrinolytic system during the
trix and act on several proMMPs, activating other MMPs.            course of thrombosis and thrombus resolution.
    Our data are not consistent with those obtained in an              Data on DOACs plasma concentrations indicate that
experimental animal study, in which rivaroxaban admin-             there was no selection bias in patients’ population, since
istration was able to inhibit MMP-9 expression on a                DOACs plasma concentrations observed in our patients
model of catheter thrombosis in mice external jugular              were similar to those reported in literature. Furthermore,
vein.30 However, some differences between the two stud-            levels of anticoagulation were stable between one month
ies must be taken into account. First, the animal model            and six months of treatment.
of catheter thrombosis is rather different from human ve-              Interestingly, our results showed, after one month of
nous thrombosis. Second, rivaroxaban dosage is quite               treatment, a positive and significant correlation between
different as animals were administered 5mg/kg of ri-               IL-10 and rivaroxaban plasma levels and between IL-1RA
varoxaban, a dose higher than the one used in humans               and apixaban plasma levels. After six months of treatment
(0.25 mg/kg). Therefore, we cannot compare the two                 only dabigatran plasma levels showed a positive and sig-
studies, as a higher dose of rivaroxaban (5mg/kg) cannot           nificant correlation with anti-inflammatory IL-1RA, sug-
be administered to humans.                                         gesting that dabigatran could exert a slower
    Until now, only one study about the effect of DOACs            anti-inflammatory effect as compared to apixaban and ri-
on metalloproteinases in human is available.31 In that             varoxaban. However, these preliminary data need to be
study, Japanese AF patients were randomized to receive             evaluated in a larger number of patients in order to find
either rivaroxaban (n=93) or warfarin (n=94) for 24                possible confounding factors that could have influenced
weeks. Rivaroxaban administration was associated with              our results.
a slight but significant reduction of MMP-9 levels. How-               Surprisingly, our data showed an inverse relationship
ever, that study enrolled only Japanese patients, so it is         between apixaban plasma levels and IL-10 after six months
likely that different genetic backgrounds might determine          of treatment, implying the possibility that, after an initial
different effect on MMPs. In addition, in the Japanese             beneficial effect, there was an inversion in the trend in the
study the authors evaluated the difference between the             following months. Nevertheless, several factors have to be
pre-treatment sample and the 24-week sample, whereas               taken into account. First of all, in our study pre-treatment
in our study we evaluated variation of MMP plasma levels           blood samples were not available, so we were unable to
between samples obtained after one and six months of               evaluate, after several months of treatment, the real degree
treatment.                                                         of inflammation inhibition. Secondly, the small number of
    Moreover, we can’t exclude that the small number of            patients in treatment with apixaban could have biased the
patients treated with rivaroxaban in our study could have          results. Thirdly, the possible noncompliance of patients
biased the results.                                                with treatment procedures has to be taken into account. In
    The correlation between atrial fibrosis and AF is well         addition, it is likely that after an initial decrease in IL-10
known.32,33 MMPs play a central role in AF remodeling              levels associated with apixaban intake, a steady state is
process and thus in the development of atrial fibrosis, sug-       achieved in which IL-10 is no longer inhibited.
gesting a possible pathogenetic role in the occurrence and             Dividing our population in two groups, patients with
the progression of AF.34,35 The possible pathogenetic role         DOACs plasma concentration above or below median
of MMP-7 in AF is suggested by an animal model and a               value, the former patients had significantly higher levels
clinical study.36-38 Consistent with these studies, we found       of IL-1RA, both after one and six months of treatment.
that MMP-7 increased according to the duration of anti-                Our study presents several limits, such as the lack of
coagulant therapy and of AF. This increase could be                a control group (without anticoagulant therapy or in ther-
linked to the disease natural history and might not be in-         apy with VKA anticoagulants) and the lack of pre-treat-
fluenced by oral anticoagulation.                                  ment blood samples, collected before the beginning of the
    Regarding venous thromboembolism, MMPs are in-                 therapy. Most importantly, the low number of investigated
volved in numerous vascular diseases such as deep vein             patients does not allow us to extrapolate the statistical
thrombosis (DVT) and post thrombotic syndrome (PTS).               analysis results to the overall population.
Increased levels of MMP, strictly correlated to the inflam-
mation grade, was observed into vein walls after an
                                                                   CONCLUSIONS
episode of DVT.39,40 Some reviews reported that MMP are
increased both in the acute phase of DVT and in patients               In conclusion, treatment with DOACs is associated
with PTS.41,42 In a murine model of DVT, MMP-7 and                 with variations in systemic inflammation markers, and in
MMP-10 expression in femoral vein was significantly                metalloproteinases, suggesting that, beyond the well-
higher than in the murine control group;43 MMPs may af-            known anticoagulant activities, DOACs may also modu-
fect the process of DVT through transcription regulation           late the inflammatory and remodeling processes and that

                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:50
            Pleiotropic effects of anti-thrombotic therapies: have direct oral anticoagulants any anti-inflammatory effect?              67



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