10.4081/btvb.2023.46
Epidemiological data on the association between fibrinogen levels and mortality are scarse and controversial. Longitudinal analyses were performed, separately by sex, on 17,689 individuals from the Moli-sani study [53% women, ≥35 years, free from cardiovascular disease (CVD) or cancer at enrolment], to evaluate the association between plasma fibrinogen and all-cause and cause-specific mortality. Over a median follow-up of 11.2 years, 1,058 deaths (34.7% CVD, 36.3% cancer) were ascertained. Both in the lowest (1.12-2.64 g/L) and highest (≥3.62 g/L) fibrinogen quintiles, women had an increased all-cause mortality hazard, when compared with third quintile (2.97-3.23 g/L). Dose-response analyses showed a U-shaped relationship in women (P overall &lt;0.0001; P non-linear association &lt;0.0001), but a positive linear association for all-cause mortality in men (P overall 0.0038; P non-linear association 0.76). Similar trends for a U-shaped association were observed for CVD mortality, while no association was observed with cancer deaths. A U-shaped association of fibrinogen levels with other-cause mortality was also found in both sexes. This study shows that not only higher but also lower fibrinogen levels represent hazard for mortality when compared to normal levels; U-shaped curves being prevalently observed in women.
application/pdf
2785-5309
46
1
en
for the Moli-sani Study Investigators
https://creativecommons.org/licenses/by-nc/4.0
Bleeding, Thrombosis and Vascular Biology
atheroschlerosis
Plasma fibrinogen levels and all-cause and cause-specific mortality in an Italian adult population: results from the Moli-sani study
Text.Serial.Journal
https://www.btvb.org/btvb
2
Original Articles
2023-01-27
2022-06-21
2023-01-27
2023-03-21
Bleed Thromb Vascul Biol
for the Moli-sani Study Investigators
2023/01/27
10.4081/btvb.2023.46
Department of Data Analysis and Artificial Intelligence, Synapse Research Institute, Maastricht; Department of Functional Coagulation, Synapse Research Institute, Maastricht
2785-5309
1
atheroschlerosis
en
de Laat-Kremers R, Di Castelnuovo A, van der Vorm L, et al. In­creased BMI and Blood Lipids Are Associated With a Hyper­coagulable State in the Moli-sani Cohort. Front Cardiovasc Med 2022;9:897733.
Plasma fibrinogen levels and all-cause and cause-specific mortality in an Italian adult population: results from the Moli-sani study
https://www.btvb.org/btvb/article/download/46/53
2
                                                                                                           ORIGINAL ARTICLE

Correspondence: Simona Costanzo, Department of Epidemiology                Plasma fibrinogen levels and all-cause
and Prevention, IRCCS Neuromed, via dell’Elettronica, 86077
Pozzilli (IS), Italy.                                                      and cause-specific mortality in an
Tel.: +39.0865915243.
E-mail: simona.costanzo@moli-sani.org,                                     italian adult population: results from
simona.costanzo@neuromed.it
                                                                           the Moli-sani study
Citation: Parisi R, Costanzo S, de Laat-Kremers R, et al. Plasma fib-
rinogen levels and all-cause and cause-specific mortality in an italian    Roberta Parisi,1 Simona Costanzo,2
adult population: results from the Moli-sani study. Bleeding, Throm-
bosis, and Vascular Biology 2023;2:46.                                     Romy de Laat-Kremers,3 Augusto Di Castelnuovo,4
                                                                           Amalia De Curtis,2 Teresa Panzera,2
Key words: Fibrinogen, All-cause mortality, Cardiovascular mor-
tality, Atherosclerosis, Inflammation.
                                                                           Mariarosaria Persichillo,2 Chiara Cerletti,2
                                                                           Giovanni de Gaetano,2 Maria Benedetta Donati,2
Acknowledgements: The Authors wish to thank prof. H.C. Hemker,             Licia Iacoviello,2,5 Bas de Laat,3,6
University of Maastricht, for his pioneering inspiration in the field
of clotting biochemistry and thrombin generation. The Authors              for the Moli-sani Study Investigators*
thanks the Associazione Cuore Sano Onlus (Campobasso, Italy)
for its cultural support.                                                  *Moli-sani Study Investigators are listed in the
Contributions: RP and SC contributed equally. BdL, SC, and LI
                                                                           Supplementary Appendix S3
contributed to the conception and design of the work and interpre-




                                                                                             ly
tation of data; RP, and SC wrote the paper and performed the sta-          1
                                                                            Department of Medicine and Health Sciences, University of
tistical analyses; BdL and RdLK performed fibrinogen                       Molise, Campobasso, Italy; 2Department of Epidemiology and




                                                                                     on
measurements; SC, ADiC, ADeC, TP and MP, managed data col-
lection; CC, MBD, GdG, and LI, originally inspired the Moli-sani
                                                                           Prevention, IRCCS Neuromed, Pozzilli, IS, Italy; 3Department
Study and critically reviewed the manuscript. All Authors gave             of Data Analysis and Artificial Intelligence, Synapse Research
final approval and agree to be accountable for all aspects of the          Institute, Maastricht, the Netherlands; 4Mediterranea




                                                                               e
work ensuring integrity and accuracy.                                      Cardiocentro, Napoli, Italy; 5Research Center in Epidemiology
Conflict of interest: BdL and RdLK are employees of Synapse Re-
search Institute, part of Diagnostica Stago SAS. The other Authors
report no conflict of interest.
                                                                          us
                                                                           and Preventive Medicine (EPIMED), Department of Medicine
                                                                           and Surgery, University of Insubria, Varese, Italy; 6Department
                                                                           of Functional Coagulation, Synapse Research Institute,
                                                                          al
                                                                           Maastricht, the Netherlands
Data availability: The data underlying this article will be shared on
                                                       ci

reasonable request to the corresponding Author. The data are stored
in an institutional repository (https://repository.neuromed.it) and
                                                                           ABSTRACT
                                                     er


access is restricted by the ethical approvals and the legislation of
the European Union.                                                             Epidemiological data on the association between fibrinogen
                                                                           levels and mortality are scarse and controversial. Longitudinal
                                                    m



Funding: The analyses conducted in this study were partially sup-          analyses were performed, separately by sex, on 17,689 individuals
ported by the Italian Ministry of Health (Ricerca Corrente 2022-
                                        om




2024) and the following grants: AIRC 2021, Individual Grant to             from the Moli-sani study [53% women, ≥35 years, free from car-
LI (ID 25942) and AIRC 5xmille: the HYPERCAN Study (ID                     diovascular disease (CVD) or cancer at enrolment], to evaluate
12237). The enrolment phase of the Moli-sani Study was supported           the association between plasma fibrinogen and all-cause and
by research grants from the Pfizer Foundation (Rome, Italy), the           cause-specific mortality. Over a median follow-up of 11.2 years,
                                  -c




Italian Ministry of University and Research (MIUR, Rome, Italy
Programma Triennale di Ricerca, Decreto no.1588) and Instrumen-            1,058 deaths (34.7% CVD, 36.3% cancer) were ascertained. Both
                         on




tation Laboratory, Milan, Italy. Laboratory analyses of fibrinogen         in the lowest (1.12-2.64 g/L) and highest (≥3.62 g/L) fibrinogen
were supported by Diagnostica Stago, Paris, France and performed           quintiles, women had an increased all-cause mortality hazard,
at the Synapse Research Institute. Funders had no role in study de-        when compared with third quintile (2.97-3.23 g/L). Dose-response
                   N




sign, collection, analysis or interpretation of data, writing of the
manuscript, nor decision to submit the article for publication.
                                                                           analyses showed a U-shaped relationship in women (P overall
                                                                           <0.0001; P non-linear association <0.0001), but a positive linear
Informed consent: The manuscript does not contain any individual           association for all-cause mortality in men (P overall 0.0038; P non-
person’s data in any form.                                                 linear association 0.76). Similar trends for a U-shaped association
                                                                           were observed for CVD mortality, while no association was ob-
Received for publication: 21 June 2022.
Accepted for publication: 16 December 2022.                                served with cancer deaths. A U-shaped association of fibrinogen
                                                                           levels with other-cause mortality was also found in both sexes.
Publisher’s note: All claims expressed in this article are solely those    This study shows that not only higher but also lower fibrinogen
of the authors and do not necessarily represent those of their affili-     levels represent hazard for mortality when compared to normal
ated organizations, or those of the publisher, the editors and the re-
viewers. Any product that may be evaluated in this article or claim
                                                                           levels; U-shaped curves being prevalently observed in women.
that may be made by its manufacturer is not guaranteed or endorsed
by the publisher.
©
 Copyright: the Author(s), 2023                                            INTRODUCTION
Licensee PAGEPress, Italy
Bleeding, Thrombosis and Vascular Biology 2023; 2:46                           Fibrinogen is a glycoprotein synthesized by the liver
doi:10.4081/btvb.2023.46
                                                                           that plays a key role in the coagulation cascade. In healthy
This work is licensed under a Creative Commons Attribution-                individuals, the plasma levels of this protein range from
NonCommercial 4.0 International License (CC BY-NC 4.0).
                                                                           2 to 4 g/L, with a normal half-life of 3-5 days.1,2

                                            Bleeding, Thrombosis and Vascular Biology 2023; 2:46
2                                                           R. Parisi et al.




     Circulating fibrinogen promotes hemostasis as the sol-             the association between plasma levels of fibrinogen and
uble fibrin precursor, but also by bridging activated                   all-cause and cause-specific mortality in an Italian adult
platelets and enabling a correct location of erythrocytes,              population, apparently healthy at enrolment. Furthermore,
macrophages and fibroblasts at the site of a wound.2,3 Fib-             it was assessed if the mortality hazards associated to
rinogen thus contributes not only to stop bleeding, but also            plasma fibrinogen levels could be modified by inflamma-
to enhance wound healing and promote tissue regenera-                   tory and hemostatic biomarkers.
tion.3 In addition, fibrinogen levels play an important role
in atherosclerosis development and blood viscosity.4 The
potential causal role of increased fibrinogen levels in the             MATERIALS AND METHODS
pathophysiology of coronary artery and cardiovascular                   Study population and study design
disease (CVD) can be attributed to several mechanisms,
including increased blood viscosity and platelet aggrega-                   The Moli-sani study is an ongoing prospective cohort
tion, altered fibrin clot structure and enhanced red blood              study that enrolled 24,325 individuals (48.1% men, aged
cell attachment to thrombi.2,5-10 In one of the earliest meta-          ≥35 years, mean age ± SD: 55.8±12.0 years) randomly re-
analyses on this subject, high plasma fibrinogen levels                 cruited from the general population of Molise region in
were associated with an increased risk of CVD to the                    Southern-central Italy between 2005 and 2010.20 Thirty




                                                                                                ly
same extent in healthy subjects and high-risk individuals               percent of subjects refused to participate; these were gen-




                                                                                         on
with previous CVD events.11                                             erally older and had a higher prevalence of CVD and can-
     Fibrinogen is also involved in the inflammatory re-                cer than the Moli-sani participants.
sponse. Indeed, it is an acute phase reactant and, during                   The Moli-sani study complies with the Declaration of




                                                                                    e
episodes of inflammation, the synthesis of this protein is              Helsinki and was approved by the Catholic University
enhanced, making it a biomarker of systemic inflamma-
tion.12 Due to its inflammatory function, fibrinogen is also
                                                                               us
                                                                        Ethical Committee, Rome, Italy. All the participants en-
                                                                        rolled provided written informed consent. Details of the
                                                                        Moli-sani study have been previously described.20
implicated in the pathophysiology of several chronic dis-
                                                                      al
eases, including cancer.5,13,14 Recent studies, show that el-               For the purpose of this study, participants with missing
                                                                        measurements of plasma fibrinogen, as well as individuals
                                                        ci

evated plasma levels of this coagulation factor are
associated with an increased risk of colorectal, lung,                  with an un-known cause of death were excluded from the
                                                      er


breast,13 and digestive tract cancers.14                                analysis. In addition, participants with incomplete medical
     CVD and cancer are responsible for the majority of                 and dietary questionnaires and a previous history of can-
                                                     m




morbidity, mortality and disabilities worldwide.15 Since                cer or CVD were omitted. The final study sample con-
                                            om




traditional risk factors alone do not adequately identify               sisted of 17,689 individuals (53% women; mean age
individuals at risk for these disorders,16 fibrinogen has               54.2±11.3 years, Supplementary Figure S1).
been used as a predictive biomarker of fatal and non-fatal                  Supplementary Figure S2 reported the distribution of
                                      -c




CVD and of different types of cancer,4,5,8,9,11,13,14,17-19 even        fibrinogen levels (means and standard error) in individu-
if the majority of these studies have been performed in in-             als excluded according to history of cancer or CVD and
                               on




dividuals already affected by these disorders.                          in the final study cohort, separately by sex, showing that
     In contrast, the association between fibrinogen levels             men with a history of CVD or cancer had fibrinogen lev-
                          N




and all-cause and cause-specific mortality has been less                els significantly higher than those selected for the final
frequently studied in general populations and results are               study sample.
still controversial.5,17-19 In a Japanese-American general
population of elderly men (N=3,571, age range 71-93                     Plasma fibrinogen measurements
years), the rise of one standard deviation of fibrinogen                    Fibrinogen levels were measured in citrated plasma
levels (0.64 g/L) was associated with an increased risk of              samples by the Clauss method,21 using the STA Liquid
all-cause, cardiovascular and cancer mortality.19 Similar               FIB reagent on the STA-R Max (Diagnostica Stago,
findings were reported in a nationwide study in the US                  France) according to the manufacturers recommendations
(N=5,054, 47% men, mean age = 57 years), which                          at the Department of Functional Coagulation, Synapse
showed a positive association across quartiles of fibrino-              Research Institute, Maastricht, the Netherlands.22 Both
gen (from lower to higher) with all-cause mortality and                 intra- and inter-assay coefficients of variation were 2.1%
heterogeneous associations with cardiovascular                          for samples within the normal range of fibrinogen speci-
mortality.5 In addition, the potential effect of other inflam-          fied to be 2-4 g/L in healthy subjects.
matory and hemostasis biomarkers on the association of                      In Appendix S1 venous blood sample collection, stor-
fibrinogen levels and cause-specific mortality still re-                age, shipment and quality check procedures are reported.
mains poorly investigated.                                              Additionally, a detailed description of the common risk
     The main purpose of the present study was to evaluate              factors’ assessment is provided in Appendix S2.

                                       Bleeding, Thrombosis and Vascular Biology 2023; 2:46
                                                       Moli-sani study                                                            3



Outcome ascertainment                                             deaths (follow up time ≥2 years) and iii) stratifying by age
                                                                  classes (<65 years, ≥65 years).
    The subjects of the whole original Moli-sani cohort
                                                                       To assess the potential influence of sex hormones in
were monitored for mortality until December 31st 2018.
                                                                  the association between fibrinogen levels and mortality in
All-cause and cause-specific mortality were assessed by
                                                                  women, we also performed analyses stratified by
the Italian mortality registry (ReNCaM registry), vali-
                                                                  menopausal status. Finally, it has been also investigated
dated by Italian death certificates (ISTAT form), and
                                                                  if the studied association could be influenced by other in-
coded according to the 9th version of International Classi-
                                                                  flammatory and hemostasis biomarkers.
fication of Diseases (ICD-9).
                                                                       Multiplicative interaction between fibrinogen (contin-
    The primary outcome was all-cause mortality. Addi-            uous) and the potential effect modifiers was tested adding
tionally, cardiovascular mortality included deaths from           corresponding cross-product terms in the model. A two-
diseases of the circulatory system, when the underlying           sided P value <0.05 was considered as statistically signif-
cause of death included ICD-9 codes 390-459. Cancer               icant. Data analysis used SAS/STAT software, version 9.4
death was considered when the underlying cause of death           (SAS Institute Inc., Cary, NC, USA).23
included ICD-9 codes 140-208. Noncardiovascular/non-
cancer causes of death were included in the “other-cause




                                                                                    ly
mortality” group.                                                 RESULTS




                                                                            on
Statistical analysis                                                  In the Moli-sani cohort, the median (IQR) value of
                                                                  plasma fibrinogen levels was 3.09 (2.72-3.50) g/L in women
     Data are presented as median values and interquartile        and 2.80 (2.48-3.19) g/L in men (Supplementary Figure S3).




                                                                         e
range (IQR) or as frequencies in the case of categorical              Table 1 shows the distribution of the baseline charac-
variables. Plasma fibrinogen levels were classified into
quintiles.
                                                                usteristics according to quintiles of fibrinogen in women

     Differences in the distribution of baseline character-
                                                         al
istics in women (N=9,355) and in men (N=8,334), accord-
                                             ci

ing to plasma fibrinogen quintiles were calculated using
analysis of variance adjusted for age (CATMOD proce-
                                           er


dure for categorical variables and GLM procedure for
continuous variables in SAS software, Table 1).
                                          m




     To estimate the association between plasma fibrinogen
                                 om




quintiles and all-cause and cause-specific mortality haz-
ard, multiple imputation techniques were used (SAS
PROC MI, N=10 imputed datasets; and PROC MIANA-
                            -c




LYZE) to maximize data availability. Hazard ratios (HR)
and 95% CI for all-cause and cause-specific mortality ac-
                       on




cording to plasma fibrinogen quintiles were calculated
using Cox proportional hazard (unadjusted, age- and mul-
                N




tivariable) models with time-on-study on the time scale,
and considering the third quintile of fibrinogen as refer-
ence (Table 2). We conducted a cause-specific analysis of
competing risks of deaths from other causes. Final multi-
variable models included the variables associated with
both outcome and exposure with a P value (adjusted for
age) <0.20 (Supplementary Table S1). Adjusted survival
curves were constructed to show event rates during follow
up by plasma fibrinogen quintiles (Figure 1).
                                                                  Figure 1. Multivariable survival estimates for all-cause mortality
     Curvilinear relationships stratified by sex between          according to plasma fibrinogen quintiles in women (N= 9,355,
plasma fibrinogen levels and all-cause and cause-specific         panel A) and in men (N=8,334, panel B). Multivariable survival
mortality were tested via a multivariable Cox model using         curves were obtained from the multivariable model adjusted for
a restricted cubic spline with 3 knots at fixed percentiles       age, BMI, education, income, menopausal status, anti-
(5th, 50th, and 95th percentiles of the plasma fibrinogen         hypertensive and diabetes medications in women and for age,
distribution; Supplementary Figure S3). Sensitivity and           BMI, education, smoking habit, physical activity, and diabetes
sub-group analyses were performed: i) considering a case          medications in men, by using the first imputed dataset. The other
                                                                  imputed datasets are similar and thus omitted.
complete analysis for all variables; ii) excluding early

                                    Bleeding, Thrombosis and Vascular Biology 2023; 2:46
4                                                                      R. Parisi et al.




(N=9,355) and men (N=8,334) of the Moli-sani cohort. In                             tile were mostly non-smokers/former-smokers and were
both genders, individuals with higher levels of fibrinogen                          more frequently treated with medications for hyperten-
were older, with higher body mass index (BMI), D-dimer                              sion, diabetes and dyslipidaemia. Women with the higher
and hs-CRP levels, compared to those in the lower quin-                             levels of fibrinogen were mostly in menopausal status and
tiles. In addition, subjects in the highest fibrinogen quin-                        were less frequently treated with hormonal contraception.



Table 1. Baseline characteristics in women (N=9,355) and men (N=8,334) of the Moli-sani cohort, according to plasma fibrinogen
quintiles.
                                                           Women                                                          Men
                                                   Quintiles of Fibrinogen                                      Quintiles of Fibrinogen
                                    Q1            Q2         Q3            Q4             Q5      Q1          Q2          Q3          Q4          Q5
                                   1,870         1,872      1,871         1,871       1,871      1,667       1,666       1,672       1,664       1,665
Fibrinogen, g/L
 Range (min-max)                 1.12-2.64 2.65-2.96 2.97-3.23 3.24-3.61 3.62-6.38 1.10-2.41 2.42-2.68 2.69-2.94 2.95-3.30 3.31-7.17




                                                                                                                ly
 Median                             2.44      2.81      3.09      3.40      3.93      2.22      2.55      2.80      3.10      3.62




                                                                                                         on
 IQR range                       2.27-2.55 2.72-2.89 3.03-3.16 3.32-3.50 3.76-4.21 2.08-2.33 2.48-2.62 2.74-2.86 3.01-3.19 3.43-3.93
Baseline characteristics
Age, years                         46.1        50.1        52.7        56.0        59.0        49.2      50.8        53.1        57.7        58.2




                                                                                                 e
                                (41.4-52.9) (43.5-58.0) (45.3-61.6) (47.8-64.5)(51.5-67.9)(43.0-58.2) (43.6-59.4) (45.2-61.6) (46.6-63.2) (49.2-67.4)
Residence
 Rural
 Urban
                                    33.0
                                    67.0
                                                 35.2
                                                 64.8
                                                             32.2
                                                             67.8
                                                                           31.5
                                                                           68.5
                                                                                      32.1
                                                                                      67.9
                                                                                            us    36.5
                                                                                                  63.5
                                                                                                              34.5
                                                                                                              65.5
                                                                                                                          35.1
                                                                                                                          64.9
                                                                                                                                      32.5
                                                                                                                                      67.5
                                                                                                                                                  34.6
                                                                                                                                                  65.4
                                                                                    al
Education
 Up to lower secondary school       42.8         48.4        53.2          57.2       60.8        49.1        46.9        49.2        50.1        51.3
                                                                ci

 High school or higher              57.2         51.6        46.8          42.8       39.2        50.9        53.1        50.8        49.9        48.7
Income
                                                              er


 <40000 €/year                      57.0         56.6        53.0          57.6       54.5        62.8        60.0        58.6        60.0        57.5
 ≥40000 €/year                      12.7         12.9        11.0          9.0        8.5         13.1        15.0        14.5        14.2        14.1
                                                             m



 Not responders                     30.3         30.5        36.0          33.4       37.0        24.1        25.0        26.9        25.8        28.4
Physical activity
                                                  om




 Low                                33.5         31.8        33.6          33.3       34.4        28.7        33.4        35.0        34.0        35.5
 Medium                             29.3         33.1        34.3          35.0       34.9        29.8        30.5        30.3        36.2        39.8
 High                               37.2         35.1        32.1          31.7       30.7        41.5        36.1        34.7        29.8        24.7
                                           -c




Smoking habit
 Never smoker                       62.0         64.3        64.0          66.2       67.8        37.9        37.2        33.6        30.0        27.1
                                  on




 Current smoker                     24.6         23.6        22.0          19.7       18.2        22.4        23.5        27.3        30.7        32.8
 Former smoker                      13.4         12.1        14.0          14.1       14.0        39.7        39.3        39.1        39.3        40.1
                             N




BMI, kg/m2
 Normal weight                      56.6         41.9        32.1          26.5       17.5        26.9        23.9        21.1        17.9        15.1
 Overweight                         30.3         37.5        40.8          37.7       34.8        50.5        52.8        50.3        51.9        48.0
 Obese                              13.1         20.6        27.1          35.8       47.7        22.6        23.3        28.6        30.2        36.9
D-dimer, ng/dl                      166       173       182       187       200       147       159       162       165       185
                                 (128-210) (134-216) (140-224) (143-232) (154-255) (110-185) (119-193) (123-200) (123-201) (142-233)
hs-CRP, mg/L                         0.7          1.1         1.5          2.1         3.6         0.8         1.0         1.4         1.8         3.2
                                  (0.4-1.3)    (0.6-2.0)   (0.8-2.6)    (1.1-3.8)   (1.8-6.9)   (0.5-1.4)   (0.6-1.7)   (0.8-2.2)   (1.0-3.1)   (1.7-6.3)
Antithrombotic medications          0.9           2.0        2.5           3.4            3.8     2.0         1.8         3.1         3.4         5.5
Liver disease                       3.2           3.8        3.9           3.2            3.8     5.1         4.5         4.6         4.9         4.6
Anti-hypertensive medications       13.4         18.8        24.7          29.2       40.8        18.6        19.0        23.3        25.4        34.8
Diabetes medications                1.0           2.1        2.7           3.2            6.0     2.9         3.8         4.1         5.4         7.9
Dyslipidaemia medications           2.7           3.8        5.6           7.0            9.8     3.2         3.3         4.5         4.8         7.1
Menopausal status                   30.8         44.4        55.3          65.3       75.5
Hormonal contraception              38.0         34.5        29.7          25.2       20.7
Hormonal replacement therapy        4.5           4.8        6.3           5.3            5.6
Median values and interquartile range (IQR) are reported for continuous variables and percentage for categorical variables. P adjusted for age. BMI:
body mass index; hs-CRP: high sensitivity C-reactive Protein.


                                              Bleeding, Thrombosis and Vascular Biology 2023; 2:46
                                                                       Moli-sani study                                                                               5



Plasma fibrinogen levels and all-cause mortality                                         Multivariable survival curves for all-cause mortality
    The cohort of 17,689 participants was followed-up for                            according to plasma fibrinogen quintiles, were well sep-
a median of 11.2 years (IQR=10.2-12.2 years; 196,668.38                              arated in both women and men (Figure 1, P=0.0001 in
person-years) during which 1,058 deaths were ascer-                                  women and P=0.042 in men).
tained; 367 (34.7%) subjects died from CVD and 384                                       Table 2 shows hazard ratio and 95% confidence inter-
(36.3%) from cancer.                                                                 vals for all-cause and cause-specific mortality according


Table 2. Hazard Ratios (95% confidence interval) for all-cause and casuse-specific mortality according to plasma fibrinogen quintiles, in
women and men of the Moli-sani cohort.
                                                          Women                                                                     Men
                                                 Quintiles of Fibrinogen                                            Quintiles of Fibrinogen
                                 Q1         Q2          Q3          Q4          Q5        Pvalue       Q1         Q2          Q3          Q4         Q5         Pvalue
Fibrinogen Range, g/L         1.12-2.64 2.65-2.96 2.97-3.23 3.24-3.61 3.62-6.38                    1.10-2.41 2.42-2.68 2.69-2.94 2.95-3.30 3.31-7.17
                                                                       All-cause mortality




                                                                                                             ly
N events/ N total             48/1,870 45/1,872       57/1,871 106/1,871 163/1,871                  64/1,667   96/1,666 107/1,672 142/1,664 230/1,665




                                                                                                   on
Death rate (95% CI)/            22.9       21.2        27.0        50.4        79.4                   34.4        51.7        58.0        77.8       128.6
10,000 Person Years          (17.3-30.4)(15.8-28.4) (20.8-35.0) (41.7-61.0) (68.1-92.6)            (26.9-43.9) (42.3-63.2) (48.0-70.1) (66.0-91.7)(113.0-146.3)
HR Crude (95% CI)                0.99       0.60                     1.40        2.92               0.57        0.88                      1.56        2.21
                                                        Ref.                             <.0001                              Ref.                             <.0001




                                                                                             e
                             (0.66-1.50)(0.38-0.94)              (1.01-1.95) (2.18-3.91)        (0.43-0.75) (0.68-1.14)               (1.22-2.00) (1.74-2.80)
HR1 (95% CI)

HR2 (95% CI)
                                 2.05       0.89
                             (1.36-3.11)(0.57-1.40)
                                 1.98       0.90
                                                        Ref.
                                                                     1.02
                                                                                  us
                                                                                 1.56
                                                                 (0.74-1.42) (1.16-2.09)
                                                                     1.00        1.45
                                                                                         <.0001
                                                                                                    0.87        1.01
                                                                                                (0.66-1.15) (0.78-1.30)
                                                                                                    0.94        1.07
                                                                                                                             Ref.
                                                                                                                                          1.22        1.39
                                                                                                                                      (0.95-1.56) (1.09-1.77)
                                                                                                                                          1.24        1.31
                                                                                                                                                              0.0026

                                                        Ref.                             0.0001                              Ref.                             0.052
                                                                          al
                             (1.30-3.01)(0.57-1.41)              (0.72-1.39) (1.08-1.96)        (0.71-1.24) (0.83-1.38)               (0.97-1.58) (1.03-1.67)
                                                                   Cardiovascular mortality
                                                         ci

N events/ N total             13/1,870 15/1,872       18/1,871    46/1,871   69/1,871               14/1,667   28/1,666    32/1,672    53/1,664   79/1,665
                                                       er


Death rate (95% CI)/              6.2        7.1        8.5       21.9        33.6                     7.5       15.1        17.3        29.0        44.2
10,000 Person Years           (3.6-10.7) (4.3-11.7) (5.4-13.5) (16.4-29.2) (26.6-42.6)             (4.5-12.7) (10.4-21.8) (12.3-24.5) (22.2-38.0) (35.4-55.1)
                                                      m



HR Crude (95% CI)                0.74       0.95                     1.72        4.40               0.42        0.84                      1.70        2.44
                                                        Ref.                             <.0001                              Ref.                             <.0001
                             (0.32-1.70)(0.46-1.96)              (0.96-3.08) (2.62-7.39)        (0.25-0.72) (0.53-1.32)               (1.11- 2.61)(1.61-3.69)
                                         om




HR1 (95% CI)                     1.80       1.59                     1.15        2.04              0.70        0.98                       1.27        1.42
                                                        Ref.                             0.014                               Ref.                             0.043
                             (0.78-4.19)(0.77-3.28)              (0.64-2.07) (1.21-3.43)       (0.41-1.18) (0.62-1.55)                (0.83-1.95) (0.94-2.16)
HR2 (95% CI)                     1.87       1.68                     1.10        1.80              0.74        1.03                       1.28        1.33
                                   -c




                                                        Ref.                             0.056                               Ref.                             0.14
                             (0.80-4.36)(0.81-3.46)              (0.61-1.98) (1.06-3.04)       (0.43-1.26) (0.65-1.62)                (0.83-1.96) (0.88-2.02)
                                                                        Cancer mortality
                          on




N events/ N total             18/1,870 19/1,872       21/1,871    36/1,871   47/1,871               22/1,667   45/1,666    46/1,672    52/1,664   78/1,665
Death rate (95% CI)/              8.6        9.0        9.9       17.1        22.9                    11.8        24.2       24.9       28.5        43.6
                       N




10,000 Person Years           (5.4-13.6) (5.7-14.0) (6.5-15.3) (12.3-23.7) (17.2-30.5)             (7.8-17.9) (18.10-32.5)(18.7-33.3)(21.7-37.4) (34.9-54.4)
HR Crude (95% CI)                1.00       0.30                     1.27        1.92               0.43        0.83                      1.18        1.55
                                                        Ref.                             <.0001                              Ref.                             <.0001
                             (0.53-1.86)(0.12-0.73)              (0.76-2.12) (1.20-3.07)        (0.27-0.66) (0.57-1.21)               (0.80-1.74) (1.06-2.26)
HR1 (95% CI)                     1.51       0.37                     1.05        1.31              0.60        0.93                       0.97        1.05
                                                        Ref.                             0.032                               Ref.                             0.14
                             (0.80-2.84)(0.15-0.90)              (0.63-1.76) (0.81-2.10)       (0.39-0.94) (0.63-1.36)                (0.66-1.42) (0.72-1.54)
HR2 (95% CI)                     1.57       0.38                     1.03        1.17              0.69        1.00                       0.97        0.96
                                                        Ref.                             0.053                               Ref.                             0.48
                             (0.83-2.97)(0.16-0.93)              (0.62-1.72) (0.72-1.90)       (0.44-1.08) (0.68-1.46)                (0.66-1.43) (0.65-1.41)
                                                                     Other-cause mortality
N events/ N total             17/1,870 11/1,872       18/1,871    24/1,871   47/1,871               28/1,667   23/1,666    29/1,672    37/1,664   73/1,665
Death rate (95% CI)/              8.1       5.2        8.5       11.4       22.9                      15.0        12.4       15.7        20.3        40.8
10,000 Person Years           (5.0-13.0) (2.9-9.4) (5.4-13.5) (7.7-17.0) (17.2-30.5)               (10.4-21.8) (8.2-18.6) (10.9-22.6) (14.7-28.0) (32.4-51.3)
HR Crude (95% CI)                1.26       0.70                     1.27        2.90               1.09        1.06                      2.16        3.29
                                                        Ref.                             <.0001                              Ref.                             <.0001
                             (0.61-2.63)(0.31-1.59)              (0.67-2.41) (1.66-5.06)        (0.66-1.82) (0.62-1.80)               (1.31-3.56) (2.03-5.33)
HR1 (95% CI)                     2.85       1.09                     0.89        1.44              1.72        1.22                       1.66        2.02
                                                        Ref.                             0.010                               Ref.                             0.032
                             (1.37-5.96)(0.48-2.48)              (0.47-1.68) (0.82-2.51)       (1.03-2.86) (0.72-2.08)                (1.01-2.74) (1.24-3.28)
HR2 (95% CI)                     2.60       1.07                     0.88        1.43              1.74        1.28                       1.73        2.00
                                                        Ref.                             0.022                               Ref.                             0.042
                             (1.24-5.48)(0.47-2.43)              (0.46-1.66) (0.81-2.51)       (1.04-2.91) (0.75-2.18)                (1.05-2.85) (1.23-3.25)
Model 1: adjusted for age; Model 2 women: model 1 plus BMI, education, income, menopausal status, anti-hypertensive and diabetes medications; Model 2 men:
model 1 plus BMI, education, smoking habit, physical activity and diabetes medications. BMI: body mass index; CI: confidence interval and HR: Hazard ratio.


                                             Bleeding, Thrombosis and Vascular Biology 2023; 2:46
6                                                        R. Parisi et al.




to plasma fibrinogen quintiles stratified by sex. Women              ships between plasma fibrinogen levels and other-cause
in the lowest (Q1: 1.12-2.64 g/L) and in the highest (Q5:            mortality were found in both women and men (Table 2 and
≥3.62 g/L) fibrinogen quintiles had an increased hazard              Figure 3). The most frequent causes of death in this out-
for all-cause mortality, when compared with those in the             come were diseases of respiratory (26.5% in women and
third quintile (Q3: 2.97-3.23 g/L), (HRQ1vsQ3: 1.98; 95%             25.8% in men) and gastro-intestinal (14.5% in women and
CI 1.30-3.01 and HRQ5vsQ3: 1.45; 95% CI 1.08-1.96, re-               17.4% in men) systems (Supplementary Table S5). Unfor-
spectively; model 2, Table 2).                                       tunately, further analyses specific for the different causes
     Instead, only men in the highest (Q5: ≥3.31 g/L) quin-          of death were unreliable due to the small number of events.
tile had an increased hazard of all-cause mortality as com-
pared to those in the third (Q3: 2.69-2.94), (HRQ5vsQ3: 1.31;        Sub-group analyses by age
95% CI 1.03-1.67; model 2, Table 2).
                                                                        No difference in the association between fibrinogen
     Dose-response analyses between fibrinogen and all-
                                                                     and all-cause and cause-specific mortality according to
cause mortality showed a U-shaped relationship in women
                                                                     two age classes (<65 years and ≥65 years) was found
(P value for overall association <0.0001 and P value for non-
                                                                     (Supplementary Figure S5).
linear association <0.0001; Figure 2) and a positive linear
association in men (P value for overall association = 0.0038




                                                                                               ly
and P value for non-linear association = 0.76; Figure 2).




                                                                                       on
     Case-complete analysis restricted to data without
missing values for covariates (8,944 women and 8,006
men; Supplementary Table S2) and analysis performed




                                                                                 e
with the exclusion of early deaths (follow-up ≥ 2 years,
9,329 women and 8,281 men; Supplementary Table S3)
yielded very similar results.
     To assess whether the results in women were affected
                                                                            us
                                                                   al
by sex hormones, we performed the analyses also strati-
fying by menopausal status (Supplementary Table S4 and
                                                      ci

Figure S4).
                                                    er


     In pre-menopause women, only a trend of association
between fibrinogen levels and all-cause mortality was ob-
                                                   m




served. In post-menopause women, instead, both with fib-
                                          om




rinogen levels lower than 2.64 g/L (HRQ1vsQ3: 2.15; 95% CI
1.36-3.41) and higher than 3.61 g/L (HRQ5vsQ3: 1.40; 95%
CI 1.03-1.92, Supplementary Table S4), an increased haz-
                                     -c




ard for all-cause mortality was found.
                             on




Plasma fibrinogen levels and cause-specific mortality
     For cardiovascular mortality, we observed a signifi-
                         N




cantly increased hazard only in women with fibrinogen
levels higher than 3.61 g/L (HRQ5vsQ3:1.80; 95% CI 1.06-
3.04; model 2, Table 2), when compared with those in the
third one. A marginally curvilinear relationship was found
between plasma fibrinogen levels and cardiovascular mor-
tality in women (P value for overall association = 0.15 and
P value for non-linear association = 0.12; Figure 3). In ad-         Figure 2. Dose-response curve of all-cause mortality according
                                                                     to plasma fibrinogen levels in women (N= 9,355) and in men (N=
dition to previous results, the linear association was con-
                                                                     8,334). The dose-response curves were obtained from
firmed in men (P value for overall association = 0.016 and           multivariable model adjusted for age, BMI, education, income,
P value for non-linear association = 0.24; Figure 3).                menopausal status, anti-hypertensive and diabetes medications in
     No relevant associations were found between fibrino-            women and for age, BMI, education, smoking habit, physical
gen levels and cancer mortality in either women or men               activity, and diabetes medications in men, by using the first
(Table 2 and Figure 3). However, dose-response analyses              imputed dataset. The other imputed datasets are similar and thus
showed a U-shaped trend in women and a sex-interaction               omitted. The reference value of the dose response association is
(P=0.045, Figure 3) describing different sex-effect of fib-          the median value of fibrinogen distribution in women and men
rinogen on cancer mortality.                                         (median 3.09 g/L and 2.80 g/L, respectively). CI: confidence
                                                                     interval and HR: Hazard ratio.
     On the other hand, non-linear but U shaped relation-

                                     Bleeding, Thrombosis and Vascular Biology 2023; 2:46
                                                          Moli-sani study                                                          7



Inflammatory and hemostasis biomarkers                                had an increased hazard for all-cause mortality, when
role assessment                                                       compared with the median quintile.
                                                                           Considering the wide range of normal values for fib-
    Inclusion in the final adjusted model of a marker of
                                                                      rinogen (2-4 g/L), the median quintile of our cohort,
inflammation (hs-CRP) and/or one of the hemostatic sys-
                                                                      which includes the median value found in healthy sub-
tem (D-dimer) only marginally affected the associations
                                                                      jects, was used as reference group for our analyses. In this
of fibrinogen with all-cause mortality rates (Supplemen-
                                                                      way it was possible to explore whether not only higher
tary Table S6). According to a cut off that distinguishes
                                                                      levels of fibrinogen but also lower ones could be associ-
between low and high hazard of all-cause mortality,24 a
                                                                      ated with mortality hazard.
modification of effect by D-dimer levels was found only
                                                                           In particular, in women, but not in men, a U-shaped re-
in men (P for interaction = 0.022) showing that low and
                                                                      lationship between plasma fibrinogen levels and the hazard
high fibrinogen levels were associated with increased haz-
                                                                      of all-cause mortality was observed. This finding in women
ard of all-cause mortality only in individuals with D-
                                                                      is difficult to explain, but could be linked in some way to
dimer ≥221 ng/dL (Figure 4).
                                                                      the influence of sex hormones on the hemostasis balance.
    Additionally, Supplementary Table S6 shows that the
                                                                      It is widely known indeed that sex hormones in women can
associations of fibrinogen levels with cardiovascular, can-
                                                                      affect many physiological pathways, directly influencing
cer or other-cause mortality were independent from in-




                                                                                        ly
                                                                      basal levels of inflammatory and hemostasis biomarkers.25-
flammatory (hs-CRP) and hemostasis (D-Dimer) markers.




                                                                                on
                                                                      29
                                                                         Of note, different associations have been observed con-
                                                                      sidering endogenous or exogenous hormones and also
                                                                      according to the type of hormonal therapy used.28,29 In this
DISCUSSION




                                                                            e
                                                                      context, to assess whether results in women were affected
   The main findings of our study conducted in an Italian
adult apparently healthy general population, show that
both women and men in the highest quintile of fibrinogen,
                                                                    usby sex hormones, we stratified the analysis by menopausal
                                                                      status and found that the U shaped association was more
                                                                      evident in postmenopausal women.
                                                             al
                                               ci
                                             er
                                            m
                                   om
                              -c
                      on
                 N




Figure 3. Dose-response curve of cause-specific mortality according to plasma fibrinogen levels in women (N= 9,355) and in men (N=
8,334). The dose-response curves were obtained from multivariable model in women adjusted for age, BMI, education, income,
menopausal status, anti-hypertensive and diabetes medications and in men adjusted for age, BMI, education,smoking habit, physical
activity, and diabetes medications; by using the first imputed dataset. The other imputed datasets are similar and thus omitted. The
reference value of the dose response association is the median value of fibrinogen distribution in women and men (median 3.09 g/L and
2.80 g/L, respectively). CI: confidence interval and HR: Hazard ratio.


                                      Bleeding, Thrombosis and Vascular Biology 2023; 2:46
8                                                           R. Parisi et al.




    On the other hand, in dose-response analysis for car-               sociated with increased risk of fatal and not fatal colorec-
diovascular mortality we observed only a marginally U-                  tal, breast and lung cancers.13,32
shaped association in women. However, the results                            In the present study, no relevant associations were
confirmed the high hazard for CVD mortality in both gen-                found with cancer mortality in either gender.
ders. These findings for CVD mortality are consistent                        Concerning the absence of correlation of fibrinogen lev-
with the current data in the literature which reported that             els with cancer mortality, the latter is a heterogeneous out-
higher levels of this hemostatic factor are related both to             come that included deaths for different types of cancer (solid
the development of atherosclerosis and to increased risk                tumours and blood cancers). Unfortunately, analyses for dif-
of coronary artery and CVD.4,8,11                                       ferent types of specific cancer related death were unreliable
    Furthermore, the role of this coagulation protein has               due to the small number of events in the present study.
been widely investigated in cancer clinical settings, show-                  A curvilinear relationships between plasma fibrinogen
ing that high pre-operative levels of fibrinogen were as-               levels and other-cause mortality were found in both women
sociated with a poor prognosis of overall/disease free                  and men. Similarly to cancer mortality, also this outcome
survival in gastrointestinal and, in particular, colorectal             is not specific and includes all the noncardiovascular/non-
cancer.14,30,31 In addition, few studies from general adult             cancer deaths. Therefore, we don’t know which one of the
population reported that high levels of fibrinogen were as-             other-cause deaths is responsible for our findings.




                                                                                                 ly
                                                                                         on
                                                                                    e
                                                                               us
                                                                      al
                                                        ci
                                                      er
                                                     m
                                            om
                                      -c
                               on
                          N




Figure 4. Dose-response curve of all-cause mortality according to plasma fibrinogen levels separated by sex and stratified by D-dimer
levels (<221 ng/dL; ≥221 ng/dL). The dose-response curves were obtained from multivariable model in women adjusted for age, BMI,
education, income, menopausal status, anti-hypertensive and diabetes medications and in men adjusted for age, BMI, education, smoking
habit, physical activity, and diabetes medications; by using the first imputed dataset. The other imputed datasets are similar and thus
omitted. The reference value of the dose response association is the median value of fibrinogen distribution (median 2.96 g/L). CI:
confidence interval and HR: Hazard ratio.


                                       Bleeding, Thrombosis and Vascular Biology 2023; 2:46
                                                           Moli-sani study                                                            9



    For all these reasons, future studies are needed to bet-          pometric and biological variables, allowing for a better un-
ter define the role of fibrinogen in relation to cause-spe-           derstanding of the predictive role of fibrinogen levels.
cific mortality in the field of different types of tumors and             On the other side, a limitation is represented by the
non-cardiovascular deaths.                                            fact that fibrinogen levels have been assessed, for each
    Concerning previous evidence from literature, in par-             participant, only in a single plasma sample obtained at the
ticular, our findings are in line with the study by Liu et al.,       moment of recruitment of the cohort; therefore, indica-
that did not report any association with cancer mortality             tions on possible long-term variation of its baseline levels
and partially with the EPIC-Norfolk study (N=16,850, 55%              are lacking. Since the measurements were all performed
women, mean age 60 years)5 showing that men, but not                  on frozen samples thawed several years after their storage
women, in the highest quintile of fibrinogen had increased            in liquid nitrogen in a dedicated biobank, this could have
risk for all-cause, CVD and other-cause mortality, com-               theoretically influenced the measurements. However,
pared to those in the lowest quintiles.18                             freezing and long-term storage appears to have little effect
    When the potential effect of inflammatory and hemo-               on fibrinogen levels. The changes observed in the study
stasis biomarkers was investigated, it was found that the             were minimal and are irrelevant for clinical
association between fibrinogen plasma levels and all-                 interpretation.34 Indeed, the plasma quality was assured
cause or cause-specific mortality was independent from                experimentally by measuring the levels of coagulation




                                                                                        ly
any of these biomarkers. However, a modification of ef-               factors FV and FVIII, known to be sensitive to freezing




                                                                                on
fect by D-dimer was found in men since curvilinear rela-              and thawing, which were found within the normal range.35
tionship between fibrinogen levels and all-cause mortality
was observed at D-dimer levels ≥221 ng/dL. As demon-




                                                                             e
strated in a previous study,24 values of D-dimer ≥ 221                CONCLUSIONS
ng/dL, represent a cut off that distinguishes between low
and high hazard of all-cause mortality. For this reason we
selected this parameter for our analyses.
                                                                    us    This study confirms that higher levels of fibrinogen
                                                                      represent a hazard for all-cause mortality in a general ap-
                                                             al
    In individuals with both high levels of fibrinogen and            parently healthy population. However, different results
D-dimer, the increased hazard of all-cause mortality could            were obtained when considering the lowest fibrinogen
                                                ci

be explained by the inflammatory condition underlying a               levels separately between women and men, suggesting
                                              er


wide range of chronic disorders.33 In contrast, the high              that further investigations are needed to better define the
hazard observed in individuals with low levels of fibrino-            biological mechanisms explaining the fibrinogen dualistic
                                             m




gen and high levels of D-dimers could be explained by a               role (biomarker of both inflammation and hemostasis) in
                                                                      mortality prediction.
                                   om




possible condition of subclinical activation of the coagu-
lation, as suggested by the high levels of D-dimer with
subsequent fibrinogen consumption. Therefore, an in-
                              -c




creased hazard of all-cause mortality may be revealed not             REFERENCES
only by high, but also by low levels of fibrinogen, when
                      on




                                                                       1. Mosesson MW. Fibrinogen and fibrin structure and functions.
combined with high D-dimers levels.                                       J Thromb Haemost 2005;3:1894-904.
    In conclusion, the present study emphasizes the con-               2. Vilar R, Fish RJ, Casini A, Neerman-Arbez M. Fibrin(ogen)
                  N




cept that inflammation and hemostasis are intermingled                    in human disease: both friend and foe. Haematologica
pathways and should be considered together in risk as-                    2020;105:284-96.
sessment; indeed fibrinogen, due to its dual role in inflam-           3. Weisel JW, Litvinov RI. Fibrin Formation, Structure and
mation and hemostasis, could be considered as an ideal                    Properties. Subcell Biochem 2017;82:405-56.
candidate biomarker of mortality.                                      4. Peng Y, Wang H, Li YM, et al. Relation between admission
                                                                          plasma fibrinogen levels and mortality in Chinese patients
    Of note, a public health implication of our findings
                                                                          with coronary artery disease. Sci Rep 2016;6:30506.
could be to consider plasma fibrinogen as a marker, easy
                                                                       5. Liu J, Zhang Y, Lavie CJ, et al. Associations of C-reactive
to test, to identify high-risk individuals in terms of sur-               protein and fibrinogen with mortality from all-causes, cardio-
vival in a general adult population. In particular, major at-             vascular disease and cancer among U.S. adults. Prev Med.
tention should be paid to women for which, not only the                   2020;139:106044.
highest but also the lowest levels of fibrinogen represent             6. Ariëns RA. Fibrin(ogen) and thrombotic disease. J Thromb
an increased mortality hazard.                                            Haemost 2013;11:294-305.
                                                                       7. Luyendyk JP, Schoenecker JG, Flick MJ. The multifaceted
Strengths and limitations                                                 role of fibrinogen in tissue injury and inflammation. Blood
                                                                          2019;133:511-20.
    Major strengths of our study are its prospective design,           8. Fibrinogen Studies Collaboration, Danesh J, Lewington S,
the great number of apparently healthy individuals included               et al. Plasma fibrinogen level and the risk of major cardio-
and the availability of a wide number of lifestyle, anthro-               vascular diseases and nonvascular mortality: an individual


                                       Bleeding, Thrombosis and Vascular Biology 2023; 2:46
10                                                              R. Parisi et al.




      participant meta-analysis [published correction appears in            23. SAS Institute Inc, Base SAS® 9.4 Procedures Guide: Statis-
      JAMA. 2005 Dec 14;294(22):2848]. JAMA. 2005;294:                          tical Procedures, second ed., SAS Institute, Inc, Cary, NC,
      1799-809.                                                                 2013.
 9.   Woodward M, Lowe GD, Rumley A, Tunstall-Pedoe H. Fib-                 24. Di Castelnuovo A, De Curtis A, Costanzo S, et al. Association
      rinogen as a risk factor for coronary heart disease and mor-              of D-dimer levels with all-cause mortality in a healthy adult
      tality in middle-aged men and women. The Scottish Heart                   population: findings from the MOLI-SANI study. Haemato-
      Health Study. Eur Heart J 1998;19:55-62.                                  logica 2013;98:1476-80.
10.   Lind P, Hedblad B, Stavenow L, et al. Influence of plasma             25. Folsom AR, Golden SH, Boland LL, Szklo M. Association of
      fibrinogen levels on the incidence of myocardial infarction               endogenous hormones with C-reactive protein, fibrinogen,
      and death is modified by other inflammation-sensitive pro-                and white blood count in post-menopausal women. Eur J Epi-
      teins: a long-term cohort study. Arterioscler Thromb Vasc Biol            demiol 2005;20:1015-22.
      2001;21:452-8.                                                        26. Farré AL, Modrego J, Zamorano-León JJ. Effects of hor-
11.   Maresca G, Di Blasio A, Marchioli R, Di Minno G. Measuring                mones on platelet aggregation. Horm Mol Biol Clin Investig
      plasma fibrinogen to predict stroke and myocardial infarction:            2014;18:27-36.
      an update. Arterioscler Thromb Vasc Biol 1999;19:1368-77.             27. Williams MS, Cushman M, Ouyang P, et al. Association of
12.   Herrick S, Blanc-Brude O, Gray A, Laurent G. Fibrinogen.                  Serum Sex Hormones with Hemostatic Factors in Women On
      Int J Biochem Cell Biol. 1999;31:741-6.                                   and Off Hormone Therapy: The Multiethnic Study of Ather-
13.   Allin KH, Bojesen SE, Nordestgaard BG. Inflammatory bio-                  osclerosis. J Womens Health (Larchmt) 2016;25:166-72.




                                                                                                       ly
      markers and risk of cancer in 84,000 individuals from the gen-        28. Zegura B, Guzic-Salobir B, Sebestjen M, Keber I. The effect




                                                                                               on
      eral population. Int J Cancer 2016;139:1493-500.                          of various menopausal hormone therapies on markers of in-
14.   Lin Y, Liu Z, Qiu Y, et al. Clinical significance of plasma D-            flammation, coagulation, fibrinolysis, lipids, and lipoproteins
      dimer and fibrinogen in digestive cancer: A systematic review             in healthy postmenopausal women. Menopause 2006;13:643-




                                                                                         e
      and meta-analysis. Eur J Surg Oncol 2018;44:1494-503.                     50.
15.   WHO. Global Health Estimates: Life expectancy and leading             29. Bonnar J. Coagulation effects of oral contraception. Am J
      causes of death and disability. Available from: https://www.
      who.int/data/gho/data/themes/mortality-and-global-health-es-
                                                                                   us
                                                                                Obstet Gynecol 1987;157:1042-8.
                                                                            30. Son HJ, Park JW, Chang HJ, et al. Preoperative plasma hy-
      timates.                                                                  perfibrinogenemia is predictive of poor prognosis in patients
                                                                          al
16.   Averna M, Noto D. Clinical utility of novel biomarkers for                with nonmetastatic colon cancer. Ann Surg Oncol
                                                            ci

      cardiovascular disease risk stratification. Intern Emerg Med              2013;20:2908-13.
      2012;7:S263-70.                                                       31. Giaccherini C, Marchetti M, Verzeroli C, et al. Biomarkers of
                                                          er


17.   Pieters M, Ferreira M, de Maat MPM, Ricci C. Biomarker as-                hypercoagulability and prediction for early disease progres-
      sociation with cardiovascular disease and mortality - The role            sion and mortality in patients with metastatica gastrointestinal
                                                         m



      of fibrinogen. A report from the NHANES study. Thromb                     patients with metastatic gastrointestinal cancer. Abstract
      Res. 2021;198:182-9.                                                      Book of the 27th SISET National Congress. Bleeding, Throm-
                                               om




18.   Ahmadi-Abhari S, Luben RN, Wareham NJ, Khaw KT. Sev-                      bosis, and Vascular Biology 2022;1(s1).
      enteen year risk of all-cause and cause-specific mortality as-        32. Parisi R, Panzera T, Russo L, et al. Fibrinogen levels in rela-
      sociated with C-reactive protein, fibrinogen and leukocyte                tion to colorectal cancer onset: A nested case-cohort study
                                         -c




      count in men and women: the EPIC-Norfolk study. Eur J Epi-                from the Moli-sani cohort. Front Cardiovasc Med
      demiol 2013;28:541-50.                                                    2022;9:1009926.
                                  on




19.   Yano K, Grove JS, Chen R, et al. Plasma fibrinogen as a pre-          33. Pearson TA, Mensah GA, Alexander RW, et al. Markers of
      dictor of total and cause-specific mortality in elderly Japan-            inflammation and cardiovascular disease: application to clin-
                             N




      ese-American men. Arterioscler Thromb Vasc Biol                           ical and public health practice: A statement for healthcare pro-
      2001;21:1065-70.                                                          fessionals from the Centers for Disease Control and
20.   Di Castelnuovo A, Costanzo S, Persichillo M, et al. Distribu-             Prevention and the American Heart Association. Circulation
      tion of short and lifetime risks for cardiovascular disease in            2003;107:499-11.
      Italians. Eur J Prev Cardiol 2012;19:723-30.                          34. Alesci S, Borggrefe M, Dempfle CE. Effect of freezing
21.   Donati MB, Vermylen J, Verstraete M. Fibrinogen degrada-                  method and storage at -20 degrees C and -70 degrees C on
      tion products and a fibrinogen assay based on clotting kinet-             prothrombin time, aPTT and plasma fibrinogen levels.
      ics. Scand J Haematol Suppl 1971;13:255-6.                                Thromb Res 2009;124:121-6.
22.   Cupaiolo R, Govaerts D, Blauwaert M, Cauchie P. Perform-              35. de Laat-Kremers R, Di Castelnuovo A, van der Vorm L, et al.
      ance evaluation of a new Stago® automated haemostasis                     Increased BMI and Blood Lipids Are Associated With a
      analyser: The STA R Max® 2. Int J Lab Hematol                             Hypercoagulable State in the Moli-sani Cohort. Front Cardio-
      2019;41:731-7.                                                            vasc Med 2022;9:897733.




                                          Bleeding, Thrombosis and Vascular Biology 2023; 2:46
