Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: J Thromb Haemost. 2016 Feb 29;14(5):995–1007. doi: 10.1111/jth.13209

The Alzheimer’s disease peptide Aβ promotes thrombin generation through activation of coagulation factor XII

Daria Zamolodchikov 1, Thomas Renné 2,3,4, Sidney Strickland 1
PMCID: PMC4870142  NIHMSID: NIHMS741654  PMID: 26613657

Abstract

Background

Beta-amyloid (Aβ) is a key pathological element in Alzheimer’s disease (AD), but the mechanisms by which it disrupts neuronal function in vivo are not completely understood. AD is characterized by a prothrombotic state, which could contribute to neuronal dysfunction by affecting cerebral blood flow and inducing inflammation. The plasma protein factor XII (FXII) triggers clot formation via the intrinsic coagulation cascade and has been implicated in thrombosis.

Objectives

We investigated the potential for Aβ to contribute to a prothrombotic state.

Methods and Results

We show that Aβ activates FXII, resulting in factor XI (FXI) activation and thrombin generation in human plasma, thereby establishing Aβ as a possible driver of prothrombotic states. We provide evidence for this process in AD by demonstrating decreased levels of FXI and its inhibitor C1 esterase inhibitor in AD patient plasma, suggesting chronic activation, inhibition, and clearance of FXI in AD. Activation of the intrinsic coagulation pathway in AD is further supported by elevated fibrin levels in AD patient plasma.

Conclusions

The ability of Aβ to promote coagulation via the FXII-driven contact pathway suggests new mechanisms by which it could contribute to neuronal dysfunction and identifies potential new therapeutic targets in AD.

Keywords: Alzheimer disease, Amyloid beta-Peptides, Factor XI, Factor XII, Thrombin

INTRODUCTION

Alzheimer’s disease (AD) is a fatal cognitive disorder affecting ~26 million people worldwide. There is evidence that the AD-related peptide beta-amyloid (Aβ) is a primary driver of both early- and late-onset disease [1, 2]. While the direct neuronal toxicity of Aβ in vitro is well documented, the mechanism by which Aβ disrupts neuronal function in AD patients is still unclear [1].

It is unlikely that direct toxicity of Aβ to neurons is the sole factor responsible for eliciting AD. Indeed, cerebrovascular pathology is present in the majority of AD patient brains upon autopsy [3, 4], but whether it contributes to AD etiology or is simply a co-morbidity is debated. A causative role for vascular pathology in AD is suggested by the increased risk of AD in vascular disease states such as atherosclerosis, diabetes, hypertension, and hypercholesterolemia [5]. In keeping with a link between AD and vascular dysfunction, accumulating evidence suggests that AD patients and mouse models exhibit a prothrombotic state: AD patients have numerous prothrombotic markers in the circulation [6], AD mouse models have a propensity to form thrombi [7, 8], and AD patients are at a higher risk for microinfarcts [9, 10] and stroke [11]. Furthermore, prothrombotic conditions such as elevated levels of D-dimer, prothrombin fragment 1+2, coated platelets, plasma homocysteine, and fibrinogen, as well as the presence of factor V Leiden, silent brain infarcts, microinfarcts, and stroke all contribute to AD onset and progression [6, 10, 12–15]. The idea that a prothrombotic state plays a role in AD is further supported by improvements in AD pathology and memory in patients [16, 17] and mouse models [18, 19] following treatment with anticoagulants.

Coagulation factor XII (FXII) is a circulating protein which when activated (FXIIa), can initiate two pathways: the intrinsic coagulation pathway through activation of factor XI (FXI), and the kallikrein-kinin system through activation of prekallikrein. Since Aβ has been shown to interact with and activate FXII [20–23], and FXII has been implicated in thrombosis in mice [24], one possibility is that Aβ may contribute to the prothrombotic environment in AD through FXII-dependent activation of FXI. Although Aβ has not been shown to trigger FXII-dependent FXI activation in vitro or in vivo, it has been implicated in FXII-dependent activation of the kallikrein-kinin system in vitro [20–22] and in mouse models [25]. Furthermore, increased activation of the kallikrein-kinin system is found in AD patient plasma [25], brain [26], and CSF [27], suggesting a role for FXII-driven processes in AD pathology. In the circulation, FXII can encounter increased concentrations of Aβ at sites of potential thrombosis, including the luminal side of cerebral capillary walls (where Aβ enters the blood through the blood-brain barrier (BBB)) [28], atherosclerotic lesions [29], sites of platelet activation [30], and areas of erythrocyte accumulation [31, 32]. A role for circulating Aβ in AD is supported by increased levels of plasma Aβ in high-risk populations for AD (Down syndrome patients, familial AD patients, and family members of AD patients) [33, 34] as well as in sporadic AD patients prior to the onset of symptoms [33–35]. Moreover, elevated plasma Aβ levels increase AD risk [36, 37].

Here, we show that Aβ oligomers promote coagulation by inducing FXII-mediated thrombin generation through the intrinsic coagulation pathway. We demonstrate the relevance of these findings to AD by showing decreased plasma levels of FXI and its inhibitor C1 esterase inhibitor (C1inh) as well as increased levels of fibrin in AD patient plasma, suggesting activation of FXI and the intrinsic coagulation pathway. The promotion of coagulation by Aβ42 provides a possible new mechanism for the prothrombotic state observed in AD patients and suggests new mechanisms by which it could contribute to neuronal dysfunction. Inhibition of the activation or activity of FXII in AD patients might therefore prove to be effective in treating the disease.

METHODS

Aβ preparation

Aβ42, Aβ40, and Aβ42 E22Q Dutch (Anaspec) monomers and oligomers were prepared as in [38]. For fibrils, Aβ42 was dissolved in 60 µl 1% NH4OH, adjusted to 200 µM with 50 mM Tris pH 7.4, 150 mM NaCl and incubated at 37°C, shaking, for 7 days. Aβ preparations were confirmed by transmission electron microscopy (TEM) at Rockefeller University’s Electron Microscopy Resource Center.

Blood collection and plasma preparation

Experiments with human plasma were approved by Rockefeller’s Institutional Review Board. Blood was drawn from healthy volunteers giving informed, written consent using 21 gauge 0.75 inch butterfly needles (BD) with a multi-adapter for S-Monovette (Sarstedt) into S-Monovette tubes containing 1/10 volume 0.106 mM trisodium citrate solution at Rockefeller University Hospital and Karolinska Institute Hospital. To obtain platelet rich plasma (PRP), blood was centrifuged at 130 × g for 10 min, and the top 1/2 of the PRP removed. To obtain platelet poor plasma (PPP), blood was centrifuged twice at 2000 × g for 10 min. PPP was frozen immediately at −80°C. Microparticle-free plasma was prepared by ultracentrifugation at 100,000 × g for 30 min at 4°C.

Mouse lines

Animal care and experimental procedures complied with the principles of laboratory and animal care established by the National Society for Medical Research and were approved by the Stockholms Norra Djurförsöksetiska Nämnd. FXII−/− [39] and FXI−/− [40] mice backcrossed to C57BL/6 mice for >10 generations and age-matched C57BL/6 control mice (Charles River) were used. Blood was collected using repel-gel (Sigma) coated glass capillary tubes into citrated Eppendorf tubes. PPP was prepared by centrifugation at 1500 × g for 15 min.

Thrombin generation in plasma

Thrombin generation in normal or FXII-deficient human plasma (George King Biomedical) was measured by Calibrated Automated Thrombogram (CAT) as described in [41]. In some cases, plasma was pre-incubated for 30 min with a FXIIa antibody [42] or active-site inhibited factor VII (ASIS; Novo Nordisk). Some reactions also contained FXIa (Haematologic Technologies; 3 pM) or phospholipids (Thrombinoscope BV; 4 µM). Thrombin generation in FXII−/−, FXI−/−, and C57BL/6 mouse plasma was measured as described above with modifications as in [43].

Aβ42-FXII binding

Human plasma diluted 1:5 in PBS containing 0.01% NP-40 and protease inhibitor cocktail (Roche) was incubated with 500 nM biotinylated Aβ42 or amylin (Anaspec) for 2 hrs at RT followed by pulldown with streptavidin Dynabeads M-280 (Life Technologies), then analysis by Western blot using monoclonal antibody against FXII (Haematologic Technologies).

FXII and FXI activation in vitro and in plasma

Chromogenic substrate

For FXII activation, 0.8 mM Pefachrome FXIIa (Centerchem), was added to 100 nM FXII (Haematologic Technologies) and 3 µM Aβ or vehicle. For FXII-dependent FXI activation, 0.8 mM Pefachrome FXIa (Centerchem) was added to 5 nM FXII, 15 nM FXI (Haematologic Technologies), and 3 µM Aβ or vehicle. Activity was monitored at 405 nm using a Molecular Devices Spectramax Plus 384 reader at 37°C in 96-well polystyrene plates (Fisher Scientific) pre-coated with 1% Polyethylene glycol 20,000 in 20 mM HEPES containing 140 mM NaCl (HEPES-buffered saline; HBS).

Western blot

For FXII activation, FXII (200 nM) and prekallikrein (150 nM) were incubated with Aβ42 (3 µM), dextran sulfate 500 kDa (DS500; Sigma; 10 µg/ml), or vehicle for 30 min at 37°C. For FXI activation, FXII (200 nM), prekallikrein (150 nM), HK (Molecular Innovations; 300 nM), and FXI (150 nM) were incubated with Aβ (3 µM), kaolin (Fisher; 100 µg/ml),,or vehicle for 30 min at 37°C. Plasma from healthy volunteers was diluted 1:10 in HBS and incubated with Aβ42 (20 µM) for 1 hour at 37°C. Reactions were stopped by adding reducing sample buffer and heating for 5 min at 85°C. Blots were probed with antibodies against FXII and FXI (Hematologic Technologies; HTI). FXI activation was quantified using densitometric analysis.

ELISA measuring FXIa-inhibitor complex formation

Normal or FXII-deficient human plasma was diluted 1:10 in HBS and incubated with Aβ42, Aβ42 Dutch (20 µM), kaolin (10 µg/ml), or vehicle at 37°C for 1 hr. Reactions were transferred to a plate pre-coated with a FXI monoclonal antibody (3 µg/ml; HTI) and blocked with PBS containing 2% milk (blocking buffer) for 1 hr. Following 1 hr incubation, wells were washed 3 × 5 min with PBS containing 0.05% Tween-20. A polyclonal C1 inhibitor (3 µg/ml; Cedarlane) or alpha-1-antitrypsin (3 µg/ml; Thermo Scientific) antibody in blocking buffer was applied for 1 hr. After washing, an HRP-conjugated anti-goat antibody (Jackson; 1:2000) in blocking buffer was applied for 1 hr. The ELISA was developed with TMB peroxidase substrate (Thermo Scientific).

FXI, C1 esterase inhibitor, and fibrin levels in human plasma

Plasma from AD patients and non-demented (ND) controls was obtained from the University of Kentucky Sanders-Brown Center on Aging (Group 1) and Washington University Knight Alzheimer’s Disease Research Center (Group 2). Group 1 AD cases were defined by clinical diagnosis of AD as well as postmortem Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) neuritic plaque score [44] of B or C, corresponding to probable or definite AD, respectively. ND cases had CERAD score 0 and no clinical diagnosis of AD (Supplemental Table 1). Group 2 AD cases had a Clinical Dementia Rating score (CDR; measuring cognitive function) [45] of ≥0.5 and CSF Aβ42 levels <500 pg/ml, and ND cases had a CDR score of 0 and CSF Aβ42 levels >500 pg/ml (Supplemental Table 2). For Group 1, blood was drawn into heparinized plastic Vacutainer tubes. For Group 2, blood was drawn using EDTA-coated syringes into polypropylene tubes containing a final concentration of 5 mM EDTA.

Equal amounts of total protein from each sample (as determined by BCA) were analyzed by Western blot with antibodies against FXI (HTI), C1 esterase inhibitor (Proteintech), fibrin beta chain (59D8 [46]), D-dimer (AbD Serotec) and transferrin (Abcam). Purified FXI, C1 esterase inhibitor (Athens Research and Technology), and FXI-deficient plasma (George King Biomedical) served as controls.

Statistical Analysis

Data are presented as vertical scatter plots with medians and reported as medians with 25th and 75th percentile ranges (median [25th–75th percentile range]), or presented as bar graphs (mean ± SD). Comparisons between groups were performed using the unpaired, two-tailed Mann-Whitney test or one-way ANOVA followed by Tukey’s Multiple Comparison Test. Correlation was analyzed using Spearman’s correlation coefficient (r). P values ≤ 0.05 were considered significant (*), with values ≤ 0.01 designated (**), and values ≤ 0.001 designated (***). Statistical analyses were performed using GraphPad Prism 5.

RESULTS

Aβ42 promotes thrombin generation in plasma

To determine if Aβ42 is prothrombotic, we quantified thrombin generation in human plasma using the Calibrated Automated Thrombogram (CAT) [41] in the presence of oligomeric Aβ42, a toxic assembly that correlates with disease severity [47]. The oligomeric composition of Aβ42, which is stable for 24 hrs at RT and 37°C (Supplemental Fig. 1), was confirmed by electron microscopy (Fig. 1A). In the absence of exogenous activators, a small thrombin burst is detectable after a long lag period (Vehicle, Fig. 1B). Addition of Aβ42 to PRP promoted thrombin generation in a dose-dependent manner, as indicated by a shortening of the lag time to thrombin burst and an increase in peak height (maximum thrombin formed) (Fig. 1B). A similar prothrombotic effect was observed in PPP (Fig. 1C), indicating that platelets are not required for the effect. However, Aβ42 had no effect in microparticle-free plasma (Fig. 1D). Supplementing microparticle-free plasma with phospholipids restored Aβ42’s ability to trigger thrombin generation (Fig. 1D), indicating that the presence of phospholipid surfaces (found on platelets and microparticles) is required for Aβ42-mediated thrombin generation. The prothrombotic effect is specific to Aβ42, since amylin, another amyloid-forming peptide, failed to induce thrombin generation (Fig. 1E).

Fig. 1. Aβ42 triggers thrombin generation in human plasma.

Fig. 1

Open in a new tab

(A) Representative TEM image of Aβ42 oligomers used. (B-E) Real-time thrombin generation was measured by CAT. (B) Platelet rich plasma (PRP) was incubated with Aβ42 at concentrations indicated or kaolin (a known activator of thrombin generation). Lag time to thrombin generation was decreased and thrombin peak was increased in the presence of Aβ42 in a dose-dependent manner. (C) As in (B), except platelet poor plasma (PPP) was used. (D) Aβ42 had no effect in platelet- and microparticle-free plasma. Addition of phospholipids (PL; 4 µM) restored Aβ42’s ability to trigger thrombin generation. (E) Lag time to thrombin generation was decreased and maximum peak height was increased in PPP with Aβ42 but not amylin. All experiments were performed in duplicate, and averaged curves are presented.

Aβ42-mediated thrombin generation is FXII-dependent

Thrombin is generated through the activation of the intrinsic (FXII-driven) or extrinsic (tissue factor; TF-driven) coagulation pathways. To determine which pathway is activated by Aβ42, CAT experiments were performed in the presence of a FXIIa function blocking antibody [42] (to block the intrinsic pathway), or with active site-inhibited factor VII (ASIS; to block the extrinsic pathway). The FXIIa antibody abolished Aβ42-induced thrombin generation (Fig. 2A), whereas ASIS had no inhibitory effect (Fig. 2B), indicating that Aβ42 is prothrombotic via the FXIIa-driven intrinsic coagulation pathway. The FXIIa antibody specifically blocks FXIIa-mediated thrombin generation, since it abolished thrombin generation initiated by kaolin (a FXII activator) but did not interfere with TF-initiated thrombin generation. As expected, ASIS inhibited TF-initiated thrombin generation (Supplemental Fig. 2).

Fig. 2. Aβ42 promotes thrombin generation in a FXII-dependent manner.

Fig. 2

Open in a new tab

Thrombin generation was measured by CAT. (A) Aβ42-induced thrombin generation was blocked by a monoclonal antibody against FXIIa [69] (4 µM), but not by IgG. (B) Aβ42’s enhancement of thrombin generation was not inhibited by the extrinsic coagulation pathway inhibitor ASIS (60 nM). (C) Thrombin generation was not enhanced in human plasma from a FXII-deficient individual in the presence of Aβ42. Deficiency of FXII in this plasma was confirmed by Western blot (WB; inset). Aβ42 had no effect when thrombin generation was triggered by 3 pM FXIa. (D) Thrombin generation was enhanced in WT mouse plasma but not FXII−/− mouse plasma in the presence of Aβ42. Mouse plasma contained 240 nM ASIS to block TF-mediated thrombin generation stemming from TF contamination during blood draw. ASIS does not affect Aβ42-mediated enhancement of thrombin generation (Fig. 2B). All experiments were performed in duplicate, and averaged curves are presented.

To further examine the role of FXII in Aβ42-mediated thrombin generation, we analyzed the effect of Aβ42 in FXII-deficient human plasma (with no detectable plasma FXII antigen; Fig. 2C inset). Aβ42 failed to trigger thrombin generation in FXII-deficient plasma (Fig. 2C, dashed curves). To examine the role of Aβ42 in a system where FXII is completely absent, we tested plasma from mice that do not express any FXII (FXII−/−). While Aβ42 promoted thrombin generation in WT mouse plasma, no effect was seen in FXII−/− mouse plasma (Fig. 2D).

Since FXII-deficient or -neutralized plasmas have normal levels of downstream coagulation factors, the results also indicate that thrombin generation is not driven through direct activation of these factors by Aβ42. However, Aβ42 may potentiate downstream factors when they are in the activated state, which may be produced by low-level, well surface-mediated FXII activation (e.g. the background thrombin signal in Fig. 1A). To address this possibility, thrombin generation in FXII-deficient human plasma was measured following activation with FXIa, which activates downstream members of the coagulation cascade. Aβ42 had no effect on thrombin generation in plasma activated with low levels of FXIa (Fig. 2C, solid curves), indicating that it does not enhance the activity of FXIa or any downstream factors. Furthermore, Aβ42 had no effect on thrombin generation in plasma from mice that have normal levels of FXII but do not express FXI (Supplemental Fig. 3), confirming that the pathway enhanced by Aβ42 involves FXIIa-mediated activation of FXI and not FXIIa-mediated activation of another substrate.

Aβ triggers FXII-dependent FXI activation in vitro

FXII undergoes autoactivation on negatively charged surfaces. Since autoactivation of FXII has only been shown with fibrillar Aβ40 and in the presence of ZnCl2 [20], we first determined that Aβ42 oligomers can directly induce FXII autoactivation (Fig. 3A). Physiologically, contact system activation takes place in the presence of prekallikrein, which is activated by FXIIa to kallikrein, which in turn activates additional FXII, amplifying the reaction. Aβ42 dose-dependently promoted FXII activation in the presence of prekallikrein (Fig. 3B), as seen through the reduction of FXII zymogen levels (80 kDa) and the appearance of the FXIIa heavy chain (52 kDa).

Fig. 3. Aβ promotes FXII-dependent FXI activation in vitro.

Fig. 3

Open in a new tab

(A) Aβ42 (3 µM) triggered autoactivation of FXII as determined by chromogenic substrate assay. (B) In the presence of both FXII and prekallikrein (PPK), Aβ42 dose-dependently promoted activation of FXII as seen through the reduction of FXII zymogen levels at 80 kDa and the appearance of the FXIIa heavy chain at 52 kDa. Dextran sulfate 500 kDa (DS500) and kaolin were used as positive controls. (C) Aβ42 (3 µM) triggered FXII-dependent FXIa generation by chromogenic substrate assay. The signal was not due to non-specific cleavage of chromogenic substrate by FXIIa, or by autoactivation of FXI, as seen in controls where FXII or FXI were omitted. (D) FXI activation can be seen through the appearance of the 50 kDa FXIa heavy chain band following incubation of FXII, FXI, PPK, and HK with Aβ42 or kaolin. Levels of FXIa heavy chain were increased in Aβ42- (p <0.05) and kaolin- (p <0.01) treated samples compared to vehicle. All lanes presented are from the same blot. (E) Aβ42 oligomers were more potent in promoting FXII-dependent FXI activation than freshly dissolved Aβ42. Aβ42 fibrils had no effect. All Aβ42 preparations were 3 µM. (F) Aβ42 oligomers were more potent than Aβ40 oligomers in promoting FXII-dependent FXI activation. (G) Aβ42 oligomers (TEM image, top inset) promoted FXII-dependent FXI activation much more strongly than Aβ42 Dutch oligomers (TEM image, bottom inset). Chromogenic substrate assays were performed multiple times with representative results shown. Representative immunoblots are from 3 experiments.

Aβ42 led to FXIIa-dependent FXIa generation in the absence (Fig. 3C) and presence (Fig. 3D) of prekallikrein, indicating that FXII activated by Aβ42 is capable of cleaving its substrate FXI. Previously, FXIIa-dependent FXI activation and procoagulant effects were not detected in the presence of Aβ [22]. The main difference between our experiments is that the previous study used “amorphous aggregates” of Aβ42 with the Dutch mutation (E22Q) instead of the wild-type oligomeric Aβ42 used here. This discrepancy prompted us to analyze the ability of Aβ42 in different states of aggregation as well as other Aβ variants to trigger FXII-dependent FXI activation. We found that Aβ42 oligomers had a much greater ability to trigger FXII-dependent FXI activation than freshly dissolved Aβ42 (Fig. 3E) or Aβ40 oligomers (Fig. 3F), while Aβ42 fibrils produced no FXI activity at all (Fig. 3E). Furthermore, even the most active (oligomeric) form of Aβ42 Dutch was substantially less potent than oligomeric Aβ42 in stimulating FXII-dependent FXI activation (Fig. 3G), indicating that the discrepancy between our results is due to the use of Aβ42 Dutch and the different state of Aβ aggregation in the previous study.

Aβ42 oligomers trigger FXII-dependent FXI activation in plasma

We next examined FXII-mediated FXI activation by Aβ42 in human plasma. Biotinylated Aβ42 (TEM of oligomeric preparation in Supplemental Fig. 4), but not biotinylated amylin, was able to bind FXII in plasma as shown by pulldown assay (Fig. 4A), demonstrating that the Aβ42-FXII interaction is specific and occurs in the presence of plasma proteins. This interaction leads to FXII activation, since plasma incubated with Aβ42 had decreased FXII zymogen and increased FXIIa heavy chain compared to incubation with vehicle (Fig. 4B). Activation of FXI in plasma can be sensitively measured by quantifying FXIa-inhibitor complex levels, since FXIa generated in plasma is rapidly bound by inhibitors [48]. Incubation of plasma with Aβ42 but not Aβ42 Dutch oligomers resulted in increased levels FXIa-C1 inhibitor (C1inh) complex (Fig. 4C; p <0.001). The activation of FXI by Aβ42 was FXII-dependent, since Aβ42 did not promote FXIa-C1inh complex formation in FXII-deficient plasma. The levels of FXIa in complex with α1 antitrypsin (α1AT), another FXIa inhibitor, were also increased in plasma following activation with Aβ42 (Fig. 4D; p <0.0001).

Fig. 4. Aβ42 promotes FXII-dependent FXI activation in plasma.

Fig. 4

Open in a new tab

(A) Western blot demonstrating that biotinylated Aβ42 oligomers pull down FXII from human plasma. (B) Incubation of Aβ42 oligomers with human plasma leads to FXII cleavage. (C) ELISA measuring FXIa-C1inh complex formation in normal and FXII-deficient human plasma. Oligomers of Aβ42 but not Aβ42 Dutch promoted the formation FXIa-C1inh (p <0.001; Aβ42 vs. vehicle). (D) ELISA measuring FXIa-α1AT complex formation in normal human plasma. Aβ42 oligomers promoted increased FXIa-α1AT complex formation (p <0.0001; Aβ42 vs. vehicle). Results are expressed as % of kaolin-activated normal plasma and presented as mean ± SD of experiments performed in triplicate.

Levels of FXI zymogen and C1inh are decreased and levels of fibrin are increased in AD patient plasma

We next investigated whether the FXII-driven intrinsic coagulation pathway is activated in AD patient plasma. Two sets of AD patient and non-demented control (ND) plasmas were obtained from two plasma banks. Group 1 consisted of 10 AD and 10 ND samples matched with respect to age, gender, and ApoE genotype (Supplemental Table 1), and Group 2 consisted of 10 AD and 10 ND samples matched with respect to age (Supplemental Table 2). Plasma was analyzed by Western blot, with results normalized to transferrin loading control, levels of which are unchanged in AD patients [49]. Increased activation of FXI in plasma can be detected as decreased plasma FXI zymogen levels, often observed in disease states accompanied by FXI activation [48, 50, 51], with decreased plasma FXI zymogen levels possibly reflecting continuous consumption of FXI due to its activation and clearance. AD plasma had decreased levels of FXI zymogen compared to ND plasma in both Group 1 (0.46 [0.36–0.50] vs. 0.69 [0.54–0.87], p = 0.008; Fig. 5A,B) and Group 2 (0.84 [0.61–1.15] vs. 1.43 [1.20–1.96], p = 0.0003; Fig. 5D,E).

Fig. 5. AD patient plasma has lower levels of FXI and C1inh and increased levels of fibrin.

Fig. 5

Open in a new tab

(A) Non-reducing Western blot analysis of FXI, C1inh, and transferrin loading control in plasma of 10 AD patients and 10 ND controls from Group 1. Lanes loaded with FXI purified protein (FXI) and FXI-deficient human plasma (FXI-def) show that the band just above the FXI band is non-specific. (B) FXI levels normalized to transferrin were lower in AD than ND plasma (p = 0.008). Levels of FXIIa normalized to transferrin in these samples were determined previously [25], and mean values for each group are designated by asterisk. (C) C1inh levels were lower in AD than ND plasma (p = 0.0008). Levels of FXIIa normalized to transferrin in these samples were determined previously [25], and mean values for each group are designated by asterisk. (D) Levels of FXI, C1inh, and transferrin were analyzed in 10 AD and 10 ND plasmas from Group 2. (E) FXI levels were lower in AD than ND plasma (p = 0.0003). (F) C1inh levels were lower in AD than ND plasma (p = 0.01). (G) Levels of fibrin monomer were analyzed under reducing conditions in 10 AD and 10 ND plasma samples from Group 2 using antibody 59D8 specific for fibrin beta chain [46]. (H) D-dimer levels were analyzed under non-reducing conditions in 10 AD and 10 ND plasma samples from Group 2. (I) Fibrin (p = 0.009) and D-dimer (p = 0.018) levels were increased in Group 2 AD plasma compared to control. (J) Fibrin (r = −0.49; p = 0.03) and D-dimer (r = −0.57, p = 0.008) levels were negatively correlated with FXI levels in samples from Group 2.

If AD plasma FXI levels are decreased due to its activation and clearance, levels of its main inhibitor C1inh [48] would also be expected to decrease. Indeed, C1inh levels were decreased in AD vs. ND plasma in both Group 1 (0.52 [0.45–0.75] vs. 1.39 [1.01–1.79], p=0.0008; Fig. 5A,C) and Group 2 (1.04 [0.91–1.11] vs. 1.18 [1.11–1.43], p=0.012; Fig. 5D,F), suggesting its consumption. Decreased levels of FXI and C1inh in AD vs. ND control plasma were accompanied by increased levels of FXIIa (FXIIa levels were derived from previous results [25] and are designated by asterisks in Fig. 5B,C). This relationship suggests that depletion of FXI and C1inh in AD patient plasma is due to FXII activation.

Activation of the intrinsic pathway of coagulation would be expected to result in increased thrombin generation and fibrin formation. Indeed, AD patients from Group 2 had elevated plasma fibrin (0.94 [0.85–1.13] vs. 0.68 [0.64–0.83], p = 0.009); and D-dimer (1.88 [1.39–2.41] vs. 1.46 [1.24–1.68], p = 0.018) levels compared to controls (Fig. 5G–I). Levels of fibrin and D-dimer were inversely correlated with FXI levels (r = −0.46, p = 0.04 for fibrin; r = −0.57, p = 0.008 for D-dimer, Fig. 5J), suggesting that activation and subsequent clearance of FXI results in thrombin generation and fibrin formation. In Group 1, there was a non-significant trend toward increased fibrin levels in AD plasma (not shown), which could be due to differences in blood draw and anticoagulation methods between the groups (see Methods). Another possible explanation is the more advanced disease stage of patients in Group 1 compared to Group 2 as determined by Clinical Dementia Rating score measuring cognitive function, where 0 = no dementia and 3 = severe dementia [45] (2.0 ± 1.1 for Group 1 vs. 1.0 ± 0.6 for Group 2, p = 0.028; Supplemental Tables 1 and 2). Since Group 1 patients are likely to have been exposed to FXI activation for longer due to more advanced disease, the fibrin formed may have been progressively deposited, thereby depleting soluble fibrin from plasma.

DISCUSSION

Our results identify Aβ as a prothrombotic factor that can trigger thrombin generation via FXII-dependent activation of FXI. Aβ42-mediated, FXII-dependent FXI activation was previously not found [22], likely because amorphous aggregates of Aβ42 with the Dutch mutation (E22Q) were used. While that study found no FXI activation with Aβ42 Dutch amorphous aggregates, our results with Aβ42 Dutch oligomers showed low levels of FXII-dependent FXI activation, highlighting the importance of the assembly state of Aβ42 Dutch in FXI activation. We also showed that wild-type oligomeric Aβ42 was a more potent FXI activator than monomeric and fibrillar preparations, further supporting the importance of Aβ aggregation state. Finally, wild-type Aβ42 oligomers were much more potent in FXI activation than Aβ42 Dutch oligomers, possibly due to differences in peptide charge (−2.7 for Aβ42 vs. −1.7 for Aβ42 Dutch at pH 7). More negative charge and/or the presence of glutamic acid at position 22 of Aβ42 therefore appears crucial for its activation of FXII and FXI, and it is possible that this region is more optimally exposed in oligomers.

Aβ’s function as a prothrombotic factor is interesting in the context of the anticoagulant properties of its parent molecule amyloid precursor protein (APP). Some isoforms of APP contain a Kunitz protease inhibitor domain that inhibits several coagulation factors [52–54]. Since protease activation is often accompanied by activation of corresponding inhibitory pathways, the release of soluble APP (sAPP) during Aβ production may attenuate the prothrombotic pathways initiated by Aβ. Dysregulation of the balance between Aβ and sAPP may lead to thrombosis or hemorrhage, depending on their abundance and/or localization. For example, an excess of Aβ over sAPP may occur in the cerebral circulation of AD patients due to transport of Aβ from the brain parenchyma [55] without corresponding transport of sAPP.

Our hypothesis that circulating Aβ triggers a prothrombotic state in AD is supported by increased levels of Aβ42 oligomers (in the nM range, similar to levels used in our thrombin generation studies) in AD patient plasma [56]. Interestingly, higher Aβ42 levels are found in sporadic and familial AD at early stages of disease, prior to and just at the onset of symptoms [33–35, 57]. It is thus tempting to speculate that the Aβ/FXII-dependent prothrombotic state suggested by our results together with Aβ/FXII-mediated proinflammatory events [25] may trigger AD-related vascular pathology when combined with age- or vascular risk factor-mediated changes.

In AD and pre-AD states, circulating Aβ may induce chronic, low-level FXII-dependent FXI activation. This is supported by increased FXIIa levels [25] and decreased levels of FXI zymogen and its inhibitor C1inh in AD patient plasma, which could reflect the clearance of the FXIa-C1inh complex following FXII-dependent FXI activation [58]. C1inh depletion in AD plasma may also stem from inhibition of FXIIa and kallikrein, which are activated in the intrinsic coagulation pathway. Our results are in agreement with decreased levels of C1inh previously found in plasma from individuals with mild cognitive impairment and AD [59, 60]. While our data suggest that FXI consumption is due to its activation by FXII, it is possible that some FXI consumption results from feedback activation by thrombin following extrinsic pathway activation.

Chronic FXI activation could mediate the production of low “idling” levels of thrombin, which may contribute to the chronic formation of fibrin, supported by increased fibrin monomer and D-dimer levels in the plasma of AD patients from Group 2. Elevated plasma fibrin monomer levels are observed in procoagulant states such as coronary artery disease [61], venous thrombosis [62], and myocardial infarction [63], among others. In AD, chronic thrombin generation could lead to the formation of persistent clots with decreased susceptibility to fibrinolysis, due to Aβ’s ability to induce the formation of a fibrinolysis-resistant fibrin network [64]. Indeed, formation of persistent clots is observed in an AD mouse model [7], and increased deposition of fibrin occurs in the brains of AD patients and mouse models [65].

Increased thrombosis in AD would have significant consequences for vascular and neuronal health. Thrombus formation in arterioles and venules decreases blood flow in surrounding capillaries [66, 67], limiting oxygen and nutrient supply to neurons. Furthermore, thrombin mediates BBB breakdown [68], and the resulting plasma protein access to the vessel wall and brain parenchyma could contribute to vascular and neuronal damage. Targeting FXII, the intrinsic coagulation pathway, thrombin activity, and thrombus formation in AD could therefore provide novel therapeutic opportunities.

Supplementary Material

Supp Table S1-2 & Fig S1-S3

Essentials.

  • *

    How the Alzheimer’s disease (AD) peptide Aβ disrupts neuronal function in the disease is unclear.

  • *

    Factor XII (FXII) initiates blood clotting via Factor XI (FXI), and thrombosis has been implicated in AD.

  • *

    Aβ triggers FXII-dependent FXI activation and thrombin generation, evidence of which is seen in AD plasma.

  • *

    Aβ-triggered clotting could contribute to neuronal dysfunction in AD and be a novel therapeutic target.

ACKNOWLEDGEMENTS

This work was supported by NIH grant NS50537; Rockefeller University Center for Clinical and Translational Science grant UL1 TR000043 from the NCATS, NIH CTSA program; the Alzheimer’s Drug Discovery, Thome Memorial Medical, Litwin, Rudin Family, Blanchette Hooker Rockefeller, and Mellam Family Foundations; the Cure Alzheimer’s Fund; and John A. Herrmann, for SS; the Nicholson Exchange program for DZ and SS; and Vetenskapsrådet (K2013-65X-21462-04-5), German Research Society (SFB841), and European Research Council grant (ERC-StG-2012-311575 F-12) for TR. The Rockefeller University Hospital is supported in part by grant UL1 TR000043 from the National Center for Research Resources and the National Center for Advancing Sciences (NCATS), NIH. We thank the Rockefeller University Electron Microscopy Resource Center for access to experimental equipment. We are grateful to P. Nelson and E. Abner from the University of Kentucky Sanders-Brown Center on Aging (NIH P30 AG028383) and A. Fagan from the Washington University ADRC for help with plasma sample selection. We acknowledge the following Washington University studies led by J.C. Morris for providing plasma samples: The Adult Children Study (NIH P01 AG02627606), Healthy Aging and Senile Dementia (NIH P01 AG003991), and ADRC (NIH P50 AG005681). We thank K. Nickel and J. Björkqvist for experimental assistance and discussion and J. A. Jämsä, E. Norris, M. Cortes-Canteli, A. Kruyer, H. J. Ahn, and P. Greengard for helpful discussion.

DISCLOSURE OF CONFLICT OF INTEREST:

T. Renné reports grants from Vetenskapsrådet, German Research Society, and the European Research Council during the conduct of the study.

D. Zamolodchikov reports grants from Nicholson Exchange Program during the conduct of the study.

S. Strickland reports grants from Nicholson Exchange Program, NIH, Alzheimer's Drug Discovery Foundation, Thome Memorial Medical Foundation, Litwin Foundation, Rudin Family Foundation, Blanchette Hooker Rockefeller Foundation, Mellam Family Foundation, John A Hermann, and Nicholson Exchange Program, during the conduct of the study.

Footnotes

AUTHORSHIP: D. Zamolodchikov designed the study, performed experiments, analyzed data, and wrote the manuscript; T. Renné and S. Strickland designed the study, analyzed data, and participated in manuscript preparation.

REFERENCES

  1. Walsh DM, Teplow DB. Alzheimer's disease and the amyloid beta-protein. Prog Mol Biol Transl Sci. 2012;107:101–124. doi: 10.1016/B978-0-12-385883-2.00012-6. [DOI] [PubMed] [Google Scholar]
  2. Suh J, Choi SH, Romano DM, Gannon MA, Lesinski AN, Kim DY, Tanzi RE. ADAM10 Missense Mutations Potentiate beta-Amyloid Accumulation by Impairing Prodomain Chaperone Function. Neuron. 2013;80:385–401. doi: 10.1016/j.neuron.2013.08.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Kalaria RN. The role of cerebral ischemia in Alzheimer's disease. Neurobiol Aging. 2000;21:321–330. doi: 10.1016/s0197-4580(00)00125-1. [DOI] [PubMed] [Google Scholar]
  4. Attems J, Jellinger KA. The overlap between vascular disease and Alzheimer's disease--lessons from pathology. BMC medicine. 2014;12:206. doi: 10.1186/s12916-014-0206-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Humpel C. Chronic mild cerebrovascular dysfunction as a cause for Alzheimer's disease? Exp Gerontol. 2011;46:225–232. doi: 10.1016/j.exger.2010.11.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cortes-Canteli M, Zamolodchikov D, Ahn HJ, Strickland S, Norris EH. Fibrinogen and altered hemostasis in Alzheimer's disease. J Alzheimers Dis. 2012;32:599–608. doi: 10.3233/JAD-2012-120820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cortes-Canteli M, Paul J, Norris EH, Bronstein R, Ahn HJ, Zamolodchikov D, Bhuvanendran S, Fenz KM, Strickland S. Fibrinogen and beta-Amyloid Association Alters Thrombosis and Fibrinolysis: a Possible Contributing Factor to Alzheimer's Disease. Neuron. 2010;66:695–709. doi: 10.1016/j.neuron.2010.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jarre A, Gowert NS, Donner L, Munzer P, Klier M, Borst O, Schaller M, Lang F, Korth C, Elvers M. Pre-activated blood platelets and a pro-thrombotic phenotype in APP23 mice modeling Alzheimer's disease. Cellular signalling. 2014;26:2040–2050. doi: 10.1016/j.cellsig.2014.05.019. [DOI] [PubMed] [Google Scholar]
  9. Brundel M, de Bresser J, van Dillen JJ, Kappelle LJ, Biessels GJ. Cerebral microinfarcts: a systematic review of neuropathological studies. J Cereb Blood Flow Metab. 2012;32:425–436. doi: 10.1038/jcbfm.2011.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. van Rooden S, Goos JD, van Opstal AM, Versluis MJ, Webb AG, Blauw GJ, van der Flier WM, Scheltens P, Barkhof F, van Buchem MA, van der Grond J. Increased number of microinfarcts in Alzheimer disease at 7-T MR imaging. Radiology. 2014;270:205–211. doi: 10.1148/radiol.13130743. [DOI] [PubMed] [Google Scholar]
  11. Chi NF, Chien LN, Ku HL, Hu CJ, Chiou HY. Alzheimer disease and risk of stroke: a population-based cohort study. Neurology. 2013;80:705–711. doi: 10.1212/WNL.0b013e31828250af. [DOI] [PubMed] [Google Scholar]
  12. Hooshmand B, Solomon A, Kareholt I, Leiviska J, Rusanen M, Ahtiluoto S, Winblad B, Laatikainen T, Soininen H, Kivipelto M. Homocysteine and holotranscobalamin and the risk of Alzheimer disease: a longitudinal study. Neurology. 2010;75:1408–1414. doi: 10.1212/WNL.0b013e3181f88162. [DOI] [PubMed] [Google Scholar]
  13. Vermeer SE, Prins ND, den Heijer T, Hofman A, Koudstaal PJ, Breteler MM. Silent brain infarcts and the risk of dementia and cognitive decline. N Engl J Med. 2003;348:1215–1222. doi: 10.1056/NEJMoa022066. [DOI] [PubMed] [Google Scholar]
  14. Kokmen E, Whisnant JP, O'Fallon WM, Chu CP, Beard CM. Dementia after ischemic stroke: a population-based study in Rochester, Minnesota (1960–1984) Neurology. 1996;46:154–159. doi: 10.1212/wnl.46.1.154. [DOI] [PubMed] [Google Scholar]
  15. Honig LS, Tang MX, Albert S, Costa R, Luchsinger J, Manly J, Stern Y, Mayeux R. Stroke and the risk of Alzheimer disease. Arch Neurol. 2003;60:1707–1712. doi: 10.1001/archneur.60.12.1707. [DOI] [PubMed] [Google Scholar]
  16. Ratner J, Rosenberg G, Kral VA, Engelsmann F. Anticoagulant therapy for senile dementia. J Am Geriatr Soc. 1972;20:556–559. doi: 10.1111/j.1532-5415.1972.tb00758.x. [DOI] [PubMed] [Google Scholar]
  17. Walsh AC, Walsh BH, Melaney C. Senile-presenile dementia: follow-up data on an effective psychotherapy-anticoagulant regimen. J Am Geriatr Soc. 1978;26:467–470. doi: 10.1111/j.1532-5415.1978.tb03326.x. [DOI] [PubMed] [Google Scholar]
  18. Bergamaschini L, Rossi E, Storini C, Pizzimenti S, Distaso M, Perego C, De Luigi A, Vergani C, De Simoni MG. Peripheral treatment with enoxaparin, a low molecular weight heparin, reduces plaques and beta-amyloid accumulation in a mouse model of Alzheimer's disease. J Neurosci. 2004;24:4181–4186. doi: 10.1523/JNEUROSCI.0550-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Timmer NM, van Dijk L, van der Zee CE, Kiliaan A, de Waal RM, Verbeek MM. Enoxaparin treatment administered at both early and late stages of amyloid beta deposition improves cognition of APPswe/PS1dE9 mice with differential effects on brain Abeta levels. Neurobiol Dis. 2010;40:340–347. doi: 10.1016/j.nbd.2010.06.008. [DOI] [PubMed] [Google Scholar]
  20. Shibayama Y, Joseph K, Nakazawa Y, Ghebreihiwet B, Peerschke EI, Kaplan AP. Zinc-dependent activation of the plasma kinin-forming cascade by aggregated beta amyloid protein. Clin Immunol. 1999;90:89–99. doi: 10.1006/clim.1998.4621. [DOI] [PubMed] [Google Scholar]
  21. Bergamaschini L, Donarini C, Foddi C, Gobbo G, Parnetti L, Agostoni A. The region 1–11 of Alzheimer amyloid-beta is critical for activation of contact-kinin system. Neurobiol Aging. 2001;22:63–69. doi: 10.1016/s0197-4580(00)00174-3. [DOI] [PubMed] [Google Scholar]
  22. Maas C, Govers-Riemslag JW, Bouma B, Schiks B, Hazenberg BP, Lokhorst HM, Hammarstrom P, ten Cate H, de Groot PG, Bouma BN, Gebbink MF. Misfolded proteins activate factor XII in humans, leading to kallikrein formation without initiating coagulation. J Clin Invest. 2008;118:3208–3218. doi: 10.1172/JCI35424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yasuhara O, Walker DG, McGeer PL. Hageman factor and its binding sites are present in senile plaques of Alzheimer's disease. Brain Res. 1994;654:234–240. doi: 10.1016/0006-8993(94)90484-7. [DOI] [PubMed] [Google Scholar]
  24. Renné T, Schmaier AH, Nickel KF, Blomback M, Maas C. In vivo roles of factor XII. Blood. 2012;120:4296–4303. doi: 10.1182/blood-2012-07-292094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zamolodchikov D, Chen ZL, Conti BA, Renne T, Strickland S. Activation of the factor XII-driven contact system in Alzheimer's disease patient and mouse model plasma. Proc Natl Acad Sci U S A. 2015;112:4068–4073. doi: 10.1073/pnas.1423764112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ashby EL, Love S, Kehoe PG. Assessment of activation of the plasma kallikrein-kinin system in frontal and temporal cortex in Alzheimer's disease and vascular dementia. Neurobiol Aging. 2012;33:1345–1355. doi: 10.1016/j.neurobiolaging.2010.09.024. [DOI] [PubMed] [Google Scholar]
  27. Bergamaschini L, Donarini C, Gobbo G, Parnetti L, Gallai V. Activation of complement and contact system in Alzheimer's disease. Mech Ageing Dev. 2001;122:1971–1983. doi: 10.1016/s0047-6374(01)00311-6. [DOI] [PubMed] [Google Scholar]
  28. Deane R, Bell RD, Sagare A, Zlokovic BV. Clearance of amyloid-beta peptide across the blood-brain barrier: implication for therapies in Alzheimer's disease. CNS & neurological disorders drug targets. 2009;8:16–30. doi: 10.2174/187152709787601867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kokjohn TA, Van Vickle GD, Maarouf CL, Kalback WM, Hunter JM, Daugs ID, Luehrs DC, Lopez J, Brune D, Sue LI, Beach TG, Castano EM, Roher AE. Chemical characterization of pro-inflammatory amyloid-beta peptides in human atherosclerotic lesions and platelets. Biochimica et biophysica acta. 2011;1812:1508–1514. doi: 10.1016/j.bbadis.2011.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Skovronsky DM, Lee VM, Pratico D. Amyloid precursor protein and amyloid beta peptide in human platelets. Role of cyclooxygenase and protein kinase C. J Biol Chem. 2001;276:17036–17043. doi: 10.1074/jbc.M006285200. [DOI] [PubMed] [Google Scholar]
  31. Schreiber S, Bueche CZ, Garz C, Kropf S, Angenstein F, Goldschmidt J, Neumann J, Heinze HJ, Goertler M, Reymann KG, Braun H. The pathologic cascade of cerebrovascular lesions in SHRSP: is erythrocyte accumulation an early phase? J Cereb Blood Flow Metab. 2012;32:278–290. doi: 10.1038/jcbfm.2011.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rogers J, Li R, Mastroeni D, Grover A, Leonard B, Ahern G, Cao P, Kolody H, Vedders L, Kolb WP, Sabbagh M. Peripheral clearance of amyloid beta peptide by complement C3-dependent adherence to erythrocytes. Neurobiol Aging. 2006;27:1733–1739. doi: 10.1016/j.neurobiolaging.2005.09.043. [DOI] [PubMed] [Google Scholar]
  33. Mayeux R, Schupf N. Blood-based biomarkers for Alzheimer's disease: plasma Abeta40 and Abeta42, and genetic variants. Neurobiol Aging. 2011;32(Suppl 1):S10–S19. doi: 10.1016/j.neurobiolaging.2011.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pesini P, Perez-Grijalba V, Monleon I, Boada M, Tarraga L, Martinez-Lage P, San-Jose I, Sarasa M. Reliable Measurements of the beta-Amyloid Pool in Blood Could Help in the Early Diagnosis of AD. International journal of Alzheimer's disease. 2012;2012:604141. doi: 10.1155/2012/604141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Gabelle A, Richard F, Gutierrez LA, Schraen S, Delva F, Rouaud O, Buee L, Dartigues JF, Touchon J, Lambert JC, Berr C. Plasma amyloid-beta levels and prognosis in incident dementia cases of the 3-City Study. J Alzheimers Dis. 2013;33:381–391. doi: 10.3233/JAD-2012-121147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Blasko I, Jellinger K, Kemmler G, Krampla W, Jungwirth S, Wichart I, Tragl KH, Fischer P. Conversion from cognitive health to mild cognitive impairment and Alzheimer's disease: prediction by plasma amyloid beta 42, medial temporal lobe atrophy and homocysteine. Neurobiol Aging. 2008;29:1–11. doi: 10.1016/j.neurobiolaging.2006.09.002. [DOI] [PubMed] [Google Scholar]
  37. Schupf N, Tang MX, Fukuyama H, Manly J, Andrews H, Mehta P, Ravetch J, Mayeux R. Peripheral Abeta subspecies as risk biomarkers of Alzheimer's disease. Proc Natl Acad Sci U S A. 2008;105:14052–14057. doi: 10.1073/pnas.0805902105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stine WB, Jungbauer L, Yu C, LaDu MJ. Preparing synthetic Abeta in different aggregation states. Methods Mol Biol. 2011;670:13–32. doi: 10.1007/978-1-60761-744-0_2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pauer HU, Renne T, Hemmerlein B, Legler T, Fritzlar S, Adham I, Muller-Esterl W, Emons G, Sancken U, Engel W, Burfeind P. Targeted deletion of murine coagulation factor XII gene-a model for contact phase activation in vivo. Thromb Haemost. 2004;92:503–508. doi: 10.1160/TH04-04-0250. [DOI] [PubMed] [Google Scholar]
  40. Gailani D, Lasky NM, Broze GJ., Jr A murine model of factor XI deficiency. Blood Coagul Fibrinolysis. 1997;8:134–144. doi: 10.1097/00001721-199703000-00008. [DOI] [PubMed] [Google Scholar]
  41. Hemker HC, Giesen P, AlDieri R, Regnault V, de Smed E, Wagenvoord R, Lecompte T, Beguin S. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb. 2002;32:249–253. doi: 10.1159/000073575. [DOI] [PubMed] [Google Scholar]
  42. Larsson M, Rayzman V, Nolte MW, Nickel KF, Bjorkqvist J, Jamsa A, Hardy MP, Fries M, Schmidbauer S, Hedenqvist P, Broome M, Pragst I, Dickneite G, Wilson MJ, Nash AD, Panousis C, Renne T. A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk. Science translational medicine. 2014;6:222ra17. doi: 10.1126/scitranslmed.3006804. [DOI] [PubMed] [Google Scholar]
  43. Tchaikovski SN, BJ VANV, Rosing J, Tans G. Development of a calibrated automated thrombography based thrombin generation test in mouse plasma. J Thromb Haemost. 2007;5:2079–2086. doi: 10.1111/j.1538-7836.2007.02719.x. [DOI] [PubMed] [Google Scholar]
  44. Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee LM, Vogel FS, Hughes JP, van Belle G, Berg L. The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology. 1991;41:479–486. doi: 10.1212/wnl.41.4.479. [DOI] [PubMed] [Google Scholar]
  45. Morris JC. The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology. 1993;43:2412–2414. doi: 10.1212/wnl.43.11.2412-a. [DOI] [PubMed] [Google Scholar]
  46. Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science. 1983;222:1129–1132. doi: 10.1126/science.6648524. [DOI] [PubMed] [Google Scholar]
  47. Watson D, Castano E, Kokjohn TA, Kuo YM, Lyubchenko Y, Pinsky D, Connolly ES, Jr, Esh C, Luehrs DC, Stine WB, Rowse LM, Emmerling MR, Roher AE. Physicochemical characteristics of soluble oligomeric Abeta and their pathologic role in Alzheimer's disease. Neurological research. 2005;27:869–881. doi: 10.1179/016164105X49436. [DOI] [PubMed] [Google Scholar]
  48. Wuillemin WA, Minnema M, Meijers JC, Roem D, Eerenberg AJ, Nuijens JH, ten Cate H, Hack CE. Inactivation of factor XIa in human plasma assessed by measuring factor XIa-protease inhibitor complexes: major role for C1-inhibitor. Blood. 1995;85:1517–1526. [PubMed] [Google Scholar]
  49. Squitti R, Salustri C, Siotto M, Ventriglia M, Vernieri F, Lupoi D, Cassetta E, Rossini PM. Ceruloplasmin/Transferrin ratio changes in Alzheimer's disease. International journal of Alzheimer's disease. 2010;2011:231595. doi: 10.4061/2011/231595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Minnema MC, Pajkrt D, Wuillemin WA, Roem D, Bleeker WK, Levi M, van Deventer SJ, Hack CE, ten Cate H. Activation of clotting factor XI without detectable contact activation in experimental human endotoxemia. Blood. 1998;92:3294–3301. [PubMed] [Google Scholar]
  51. Konings J, Cugno M, Suffritti C, Ten Cate H, Cicardi M, Govers-Riemslag JW. Ongoing contact activation in patients with hereditary angioedema. PloS one. 2013;8:e74043. doi: 10.1371/journal.pone.0074043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Storey E, Cappai R. The amyloid precursor protein of Alzheimer's disease and the Abeta peptide. Neuropathol Appl Neurobiol. 1999;25:81–97. doi: 10.1046/j.1365-2990.1999.00164.x. [DOI] [PubMed] [Google Scholar]
  53. Smith RP, Higuchi DA, Broze GJ., Jr Platelet coagulation factor XIa-inhibitor, a form of Alzheimer amyloid precursor protein. Science. 1990;248:1126–1128. doi: 10.1126/science.2111585. [DOI] [PubMed] [Google Scholar]
  54. Van Nostrand WE, Wagner SL, Farrow JS, Cunningham DD. Immunopurification and protease inhibitory properties of protease nexin-2/amyloid beta-protein precursor. J Biol Chem. 1990;265:9591–9594. [PubMed] [Google Scholar]
  55. Ramanathan A, Nelson AR, Sagare AP, Zlokovic BV. Impaired vascular-mediated clearance of brain amyloid beta in Alzheimer's disease: the role, regulation and restoration of LRP1. Frontiers in aging neuroscience. 2015;7:136. doi: 10.3389/fnagi.2015.00136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhou L, Chan KH, Chu LW, Kwan JS, Song YQ, Chen LH, Ho PW, Cheng OY, Ho JW, Lam KS. Plasma amyloid-beta oligomers level is a biomarker for Alzheimer's disease diagnosis. Biochem Biophys Res Commun. 2012;423:697–702. doi: 10.1016/j.bbrc.2012.06.017. [DOI] [PubMed] [Google Scholar]
  57. Rissman RA, Trojanowski JQ, Shaw LM, Aisen PS. Longitudinal plasma amyloid beta as a biomarker of Alzheimer's disease. J Neural Transm. 2012;119:843–850. doi: 10.1007/s00702-012-0772-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wuillemin WA, Bleeker WK, Agterberg J, Rigter G, ten Cate H, Hack CE. Clearance of human factor XIa-inhibitor complexes in rats. British journal of haematology. 1996;93:950–954. doi: 10.1046/j.1365-2141.1996.d01-1740.x. [DOI] [PubMed] [Google Scholar]
  59. Cutler P, Akuffo EL, Bodnar WM, Briggs DM, Davis JB, Debouck CM, Fox SM, Gibson RA, Gormley DA, Holbrook JD, Hunter AJ, Kinsey EE, Prinjha R, Richardson JC, Roses AD, Smith MA, Tsokanas N, Wille DR, Wu W, Yates JW, Gloger IS. Proteomic identification and early validation of complement 1 inhibitor and pigment epithelium-derived factor: Two novel biomarkers of Alzheimer's disease in human plasma. Proteomics Clinical applications. 2008;2:467–477. doi: 10.1002/prca.200780101. [DOI] [PubMed] [Google Scholar]
  60. Muenchhoff J, Poljak A, Song F, Raftery M, Brodaty H, Duncan M, McEvoy M, Attia J, Schofield PW, Sachdev PS. Plasma protein profiling of mild cognitive impairment and Alzheimer's disease across two independent cohorts. J Alzheimers Dis. 2015;43:1355–1373. doi: 10.3233/JAD-141266. [DOI] [PubMed] [Google Scholar]
  61. Redondo M, Caliezi C, Biasiutti FD, Lammle B, Wuillemin WA. Increased fibrin monomer plasma concentration in stable coronary artery disease in patients without oral anticoagulation. Atherosclerosis. 2001;157:417–422. doi: 10.1016/s0021-9150(00)00731-0. [DOI] [PubMed] [Google Scholar]
  62. Vogel G, Dempfle CE, Spannagl M, Leskopf W. The value of quantitative fibrin monomer determination in the early diagnosis of postoperative deep vein thrombosis. Thromb Res. 1996;81:241–251. doi: 10.1016/0049-3848(95)00241-3. [DOI] [PubMed] [Google Scholar]
  63. Giannitsis E, Siemens HJ, Mitusch R, Tettenborn I, Wiegand U, Schmucker G, Sheikhzadeh A, Stierle U. Prothrombin fragments F1+2, thrombin-antithrombin III complexes, fibrin monomers and fibrinogen in patients with coronary atherosclerosis. International journal of cardiology. 1999;68:269–274. doi: 10.1016/s0167-5273(98)00256-3. [DOI] [PubMed] [Google Scholar]
  64. Zamolodchikov D, Strickland S. Abeta delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin. Blood. 2012;119:3342–3351. doi: 10.1182/blood-2011-11-389668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Cortes-Canteli M, Mattei L, Richards AT, Norris EH, Strickland S. Fibrin deposited in the Alzheimer's disease brain promotes neuronal degeneration. Neurobiol Aging. 2015;36:608–617. doi: 10.1016/j.neurobiolaging.2014.10.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Nishimura N, Schaffer CB. Big effects from tiny vessels: imaging the impact of microvascular clots and hemorrhages on the brain. Stroke. 2013;44:S90–S92. doi: 10.1161/STROKEAHA.112.679621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Nguyen J, Nishimura N, Fetcho RN, Iadecola C, Schaffer CB. Occlusion of cortical ascending venules causes blood flow decreases, reversals in flow direction, and vessel dilation in upstream capillaries. J Cereb Blood Flow Metab. 2011;31:2243–2254. doi: 10.1038/jcbfm.2011.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Chen B, Cheng Q, Yang K, Lyden PD. Thrombin mediates severe neurovascular injury during ischemia. Stroke. 2010;41:2348–2352. doi: 10.1161/STROKEAHA.110.584920. [DOI] [PubMed] [Google Scholar]
  69. Ponitz V, Brugger-Andersen T, Pritchard D, Grundt H, Staines H, Nilsen DW, Group RS. Activated factor XII type A predicts long-term mortality in patients admitted with chest pain. J Thromb Haemost. 2009;7:277–287. doi: 10.1111/j.1538-7836.2008.03248.x. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Table S1-2 & Fig S1-S3
NIHMS741654-supplement-Supp_Table_S1-2___Fig_S1-S3.docx (5.7MB, docx)

RESOURCES