Alzheimer’s disease patients were recruited from Chongqing Daping Hospital from December 2018 to May 2020. Age- and gender-matched controls with normal cognition were randomly recruited from the hospital at the same time. Subjects were excluded for the following reasons: (1) a family history of dementia; (2) a concomitant neurologic disorder that could potentially affect the cognitive function or other types of dementia; (3) severe cardiac, pulmonary, hepatic, or renal diseases or any type of tumor; (4) enduring mental illness (e.g., schizophrenia); (5) Diseases that may affect fibrinogen levels (e.g., bleeding disorders, hereditary abnormal fibrinogenemia, etc.); (6) Recently used treatments that affect fibrinogen levels (e.g., blood transfusion); (7) an allergy to the ¹¹C-Pittsburgh compound.

The diagnosis of AD was made according to the criteria of the National Institute of Neurological and Communicative Diseases and Stroke/AD and Related Disorders Association following the protocols we used before (Li et al., 2011). Besides, the patients who collected blood all received Aβ positron emission tomography (PET) examination of Pittsburgh compound B (PiB), and the diagnostic criteria were PiB-PET positive. The demographic data and medical history (such as hypertension, coronary heart disease, and diabetes mellitus) were collected and the cognitive and functional status was assessed based on a neuropsychological battery. Fasting blood was collected between 07:00 and 09:00 to avoid the potential circadian rhythm influence. The blood samples were centrifuged within an hour of collection and EDTA plasma was aliquoted in 0.5 mL polypropylene tubes and stored at −80°C until used. The cerebrospinal fluid (CSF) samples were centrifuged at 2,000g at 4°C for 10 min, and the aliquots were then immediately frozen and stored at −80°C until use. The informed consent was obtained before the acquisition of the blood and CSF samples.

Fibrinogen levels were measured using standard laboratory methods in the Clinical Laboratory, Daping Hospital, Chongqing, China. Fibrinogen−C is the test to measure fibrinogen by the Clasus method and is carried out with the commercial kit HemosIL Fibrinogen assay (Instrumentation Laboratory Company, United States) on ACL-TOP (Instrumentation Laboratory Company, United States). The kit uses an excess of thrombin to convert fibrinogen to fibrin in diluted plasma. Plasma levels of Aβ42, Aβ40 were measured using the commercially available single-molecule array (SIMOA) Human Neurology 3-Plex A assay kit (Quanterix, United States) on-board of the automated SIMOA HD-1 analyzer (Quanterix, United States). CSF levels of Aβ40, Aβ42, total tau (t-tau), and phosphorylated tau-181 (p-tau) were measured using the human Aβ and tau enzyme-linked immunosorbent assay (ELISA) kits (Innotest, United States). All of the measurements were performed according to the manufacturer’s instructions (Wilke et al., 2018).

The differences in demographic characteristics and fibrinogen levels between the groups were assessed with two-tailed independent t-tests, Mann–Whitney U test, or Chi-square test. Spearman correlation analyses were used to examine the correlations between fibrinogen levels and Aβ levels. The data are expressed as the mean ± standard deviation (SD). All hypothesis testing was two-sided, and p < 0.05 was defined as statistically significant. The computations were performed with SPSS version 20.0 (SPSS Inc., United States).

The characteristics of the subjects are shown in Tables 1, 2. The study consisted of 59 AD patients diagnosed by ¹¹C-PiB PET and 76 age- and gender-matched cognitively normal controls. There were no significant differences in age, sex, education level, or the comorbidity of hypertension, diabetes mellitus, cardiovascular disease, and hyperlipidemia between AD patients and cognitively normal controls. AD patients consisted of a higher proportion of APOE ε4 carriers (p = 0.004) and showed lower MMSE scores (p < 0.001). The AD patients had lower levels of both plasma Aβ40 (219.2 ± 107.1 pg/mL vs. 284.4 ± 71.67 pg/mL, p < 0.001) and Aβ42 (9.915 ± 5.126 pg/mL vs. 15.42 ± 4.598 pg/mL, p < 0.001) than the control group. The AD patients with CSF had lower levels of CSF Aβ40 (9150 ± 3926 pg/mL vs. 12190 ± 4482 pg/mL, p = 0.001) and Aβ42 (629.5 ± 286.5 pg/mL vs. 1508 ± 673.2 pg/mL, p < 0.001), and higher levels of CSF t-tau (402.3 ± 183.6 pg/mL vs. 184.0 ± 61.38 pg/mL, p < 0.001), CSF p-tau (66.09 ± 28.38 pg/mL vs. 42.84 ± 18.18 pg/mL, p < 0.001), CSF t-tau/Aβ42 (0.809 ± 0.511 pg/mL vs. 0.146 ± 0.080 pg/mL, p < 0.001), and CSF t-tau/Aβ42 (0.1317 ± 0.0844 pg/mL vs. 0.0334 ± 0.0206 pg/mL, p < 0.001) than the control group.

There was no significant difference in plasma fibrinogen levels between AD patients and the control group [PiB-PET (+)-AD vs controls: 3.13 ± 0.563 g/L vs. 3.03 ± 0.433 g/L, p = 0.256] (Figure 1). There was also no significant difference in fibrinogen levels between APOE ε4 carriers and APOE ε4 non-carriers (Supplementary Figure 1A). Besides, there was no significant correlation between fibrinogen levels and MMSE scores (Supplementary Figure 1B).

Fibrinogen levels in AD patients diagnosed by positive PiB-PET had a significantly positive correlation with plasma Aβ42 levels (γ = 0.263, p = 0.045) and Aβ40 levels (γ = 0.327, p = 0.011). There was no correlation between fibrinogen levels and plasma Aβ42 levels (γ = 0.094, p = 0.421) and Aβ40 levels (γ = 0.111, p = 0.340) in controls. In all subjects, fibrinogen levels had a significantly positive correlation with plasma Aβ40 levels (γ = 0.189, p = 0.028) but not with Aβ42 levels (γ = 0.106, p = 0.220) (Figure 2). There was no correlation between fibrinogen levels in both AD and controls with plasma t-tau levels, Aβ42/Aβ40 levels, and t-tau/Aβ42 levels (Supplementary Figure 2).

Of all the clinical AD patients, 35 people had CSF collected to further analyze the correlation between fibrinogen levels with CSF Aβ and tau levels. As shown in Table 2, there were no significant differences in the comorbidity of hypertension, diabetes mellitus, cardiovascular disease, and hyperlipidemia between the two groups. Also, no significant difference was found in the fibrinogen between these two groups (AD vs controls: 2.97 ± 0.510 g/L vs. 3.03 ± 0.433 g/L, p = 0.541). Fibrinogen levels in AD patients had significantly positive correlation with CSF Aβ42 levels (γ = −0.339, p = 0.049), but no correlation with CSF Aβ40 levels (γ = −0.204, p = 0.271). There was no correlation between fibrinogen levels in controls with CSF Aβ42 levels (γ = −0.074, p = 0.536) and Aβ40 levels (γ = −0.121, p = 0.298). In all subjects, there was no correlation between fibrinogen levels with CSF Aβ42 levels (γ = −0.053, p = 0.591) and Aβ40 levels (γ = −0.115, p = 0.240) (Figures 3A,B).

To further reveal the relationship between fibrinogen and AD pathological changes, we then detected the t-tau and phosphorylated tau in CSF and analyzed their correlation. Fibrinogen levels in AD patients had significantly positive correlation with CSF t-tau levels (γ = 0.356, p = 0.042) and p-tau levels (γ = 0.426, p = 0.012). There was no correlation between fibrinogen levels in controls with CSF t-tau levels (γ = −0.004, p = 0.974) and p-tau levels (γ = 0.024, p = 0.837). In all subjects, there was no correlation between fibrinogen levels with CSF t-tau levels (γ = 0.086, p = 0.373) and p-tau levels (γ = 0.157, p = 0.102) (Figures 3C,D).

Compared with a single marker, recent studies have found that the ratio of tau and Aβ42, including t-tau/Aβ42 and p-tau/Aβ42, has a higher correlation with the PiB-PET cortical standard uptake ratio (SUVR), which can better reflect the pathology of Aβ deposition in the brain (Hansson et al., 2018; Schindler et al., 2018). Based on this, we calculated the correlation between these two ratios and fibrinogen to explore the relationship between fibrinogen and the pathological process in the brain. We found that fibrinogen levels in AD patients had significantly positive correlation with CSF t-tau/Aβ42 levels (γ = 0.524, p = 0.002) and p-tau/Aβ42 levels (γ = 0.427, p = 0.013). There was no correlation between fibrinogen levels in controls with CSF t-tau/Aβ42 levels (γ = 0.081, p = 0.494) and p-tau/Aβ42 levels (γ = 0.074, p = 0.538). In all subjects, fibrinogen levels had a significantly positive correlation with CSF t-tau/Aβ42 levels (γ = 0.206, p = 0.034) but not with p-tau/Aβ42 levels (γ = 0.161, p = 0.102) (Figures 3E,F).