Supplemental Information. Fibrinogen and β-amyloid Association Alters. Thrombosis and Fibrinolysis: a Possible

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1 Neuron, Volume 66 Supplemental Information Fibrinogen and β-amyloid Association Alters Thrombosis and Fibrinolysis: a Possible Contributing Factor to Alzheimer s Disease Marta Cortes-Canteli, Justin Paul, Erin H. Norris, Robert Bronstein, Hyung Jin Ahn, Daria Zamolodchikov, Shivaprasad Bhuvanendran, Katherine M. Fenz, and Sidney Strickland Inventory of Supplemental Information - Supplemental Data: o Figure S1, related to Figure 1. AD mice accumulate fibrinogen in their brains not because they have more fibrinogen in their plasma (A) or because their blood is more hypercoagulable (B), but because they do not clear fibrinogen efficiently (C-E and Figure 1). o Figure S2, related to Figure 2. The effects seen on clot formation/dissolution experiments (Figure 2) are due to a heterogeneous mixture of Aβ fibrils and soluble prefibrillar amyloid assemblies (A), and these species had no direct enhancement by plasmin (B), tpa (C), or thrombin (D) activity. o Figure S3, related to Figure 3. The presence of Aβ42 has no effect on the collagen network (A&B). Therefore, the effects of Aβ42 on clot structure (C&D and Figure 3) are specific to fibrin. o Figure S4, related to Figure 8. The modulation of fibrinogen levels in mice has an effect on cognitive performance. o Movie S1, related to Figure 3. Time-lapse confocal imaging of in vitro fibrinolysis with tpa. o Movie S2, related to Figure 6. Intravital imaging of FeCl 3 -induced thrombosis in a WT cortical blood vessel. Page 1 of 8

2 o Movie S3, related to Figure 6. Intravital imaging of laser-induced thrombosis in TgCRND8 mice. o Movie S4, related to Figure 7. Fibrinolysis is slowed in the AD mouse brain. - Supplemental Experimental Procedures Page 2 of 8

3 Supplemental Figures Figure S1, related to Figure 1. (A) Western blots of plasma samples show that circulating fibrinogen is similar in TgCRND8 and WT mice (n=3/group). (B) Activated partial thromboplastin time curves for TgCRND8 and WT mice show that AD mouse blood is not hypercoagulable as clotting time and turbidity values are similar. Composite of confocal images of immunofluorescent detection of fibrin(ogen) in the hippocampus of a WT (C) or TgCRND8 (D) mouse one day following stereotaxic injection with unlabelled fibrinogen show that TgCRND8 mouse brains do not clear fibrin(ogen) efficiently. Scale bar, 1 mm. (E) Exogenous fibrin(ogen) area units were calculated for mice in each group by subtracting saline-injected controls; ***p< (F) Detection of D-Dimer indicates conversion of fibrinogen to fibrin upon injection into TgCRND8 mouse brains. Lane 1 represents brain homogenate of a WT mouse injected with fibrinogen. Lane 2 represents pure fibrinogen. Lane 3 shows detectable levels of fibrin degradation products are present in the homogenized TgCRND8 mouse brain after injection with fibrinogen. Without injection, these products are only detectable by ELISA. Page 3 of 8

4 Figure S2, related to Figure 2. (A) Representative EM image of the Aβ42 solution used in the experiments described. Negative staining and transmission EM were performed to reveal the presence of fibrils and prefibrillar amyloid assemblies (e.g. protofibrils) in the Aβ42 samples used. Enzymatic assays using purified enzymes and colorimetric substrates. tpa (B), thrombin (C), and plasmin (D) show no difference in activity in the absence (black boxes) or presence (red circles) of Aβ42 (500 nm). Page 4 of 8

5 Figure S3, related to Figure 3. Confocal images of collagen fibrils using gold colloid and reflectance. No aggregates are formed in the absence (A) or presence (B) of Aβ42. Aβ42 affects clots formed with pure fibrinogen. (C) Confocal image acquired 90 min after addition of thrombin to pure fibrinogen (inverted gray levels), showing no aggregates are formed in the absence of Aβ42. (D) Confocal image of fibrin clot showing aggregates in the presence of Aβ42 after 90 min. Images are 35 μm x 35 μm. Figure S4, related to Figure 8. TgCRND8 mice implanted with pumps delivering ancrod or saline for 4 weeks (A), and mice deficient in plasminogen (plg-/-) (B) were tested in the Morris water maze. Mice with reduced fibrinogen levels spent significantly more time in the target quadrant than in the other quadrants of the pool (A), while plg-/- mice, which are predisposed to thrombosis and present fibrinogen deposits in different organs, showed memory impairment compared to WT mice (B). Bars represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001; (A, B) target quadrant vs each of the other quadrants by experimental group. Page 5 of 8

6 Supplemental Experimental Procedures Automated activated partial thromboplastin time: TgCRND8 and WT mouse plasma was obtained by cardiac puncture at time of sacrifice. Blood was centrifuged at 4,000 x g for 5 min, and plasma was collected and added to a reaction mix according to manufacturer s protocol (Biomerieux). Absorbance at 405 nm was measured over a 5 min period. Western blot analysis was performed to determine plasma fibrinogen concentration. Plasma (1 μl) was resolved using SDS PAGE under reducing conditions. Gels were transferred overnight, and membranes were stained with a polyclonal HRP-conjugated antibody to fibrinogen (Dako) for 2 hr at room temperature and developed by enhanced chemiluminescence. Stereotactic fibrin injections: Purified human unlabelled fibrinogen (Calbiochem) was injected into 6-month-old TgCRND8 and WT mice as described in Experimental Procedures. Mice were perfused after one day, and 20 µm-thick coronal brain sections were prepared for immunofluorescence using a FITC-conjugated antibody for fibrin(ogen) (Dako). Salineinjected TgCRND8 and WT mice were used as controls. To determine the clotting of injected fibrinogen, we checked for D-Dimer. One day after stereotaxic injection of fibrinogen, brains were dissected and homogenized in PBS. Tissue suspensions were spun at 14,000 x g for 10 min at 4 C. Protein concentrations were determined by the Lowry method. For Western blot analysis of D-dimer content, 40 µg of supernatant protein was resolved by SDS PAGE with purified fibrinogen (Sigma) as protein standards. Gels were transferred, and membranes were treated as described above. Clot turbidity: To check for transglutaminase activity and clotting efficiency, purified fibrin clots were formed as described in Experimental Procedures. After 10 min, clots were centrifuged at 14,000 x g for 10 min, and the supernatant was collected for protein quantification by the Lowry method. Clots were solubilized in reducing buffer and analyzed under reducing conditions by SDS-PAGE. Tranglutaminase-dependent formation of D- dimers was detected as a 94 kd band. Electron microscopy: To visualize the nature of the amyloid species present in the Aβ42 solution, we used a standard negative staining protocol. Samples were adsorbed onto carboncoated grids (Electron Microscopy Sciences) and stained with 1% Uranyl Acetate for 1 min. Excess stain was removed with filter paper, and samples were allowed to air dry. Scanning Page 6 of 8

7 was performed using a FEI Tecnai D1201 Transmission Electron Microscope at 80KV, and images were captured with a Gatan 895 Ultrascan Digital camera. Enzymatic assays: To control for enzyme activity, plasmin (50 μg/ml; Sigma), tpa (25 nm; Genentech), or thrombin (2 U/ml; Sigma) was incubated with Aβ42 for 10 min and added to Pefachrome PL, Pefachrome tpa, or Pefachrome TH, respectively. Absorbance at 405 nm was measured for 5 min. Intravital imaging of thrombosis: Cranial window preparation: Twenty min prior to surgery, mice were given a 250 µl IP bolus of 5 mg/ml 2 MDa FITC-conjugated dextran (Sigma) dissolved in PBS, anesthetized by IP injection of 500 mg/kg avertin and 0.04 mg/kg atropine, and placed in a stereotaxic device (Kopf Instruments). A 2.5-mm circular craniotomy was prepared using 5-10 circular brush strokes with a fine dental drill bit. The dura mater was peeled away (only during administration of FeCl 3 ), and a 4 mm plastic ring surrounding the window was attached with dental acrylic and cyanoacrylate adhesive. Immediately following the surgery, mice were injected with buprenorphine as an analgesic. Sterile saline was applied periodically to protect the brain surface and prevent drying. The mouse was placed under the microscope in a custom built restraint system, and a heating pad was used during the entire imaging session. Laser-induced thrombosis: Imaging was performed using an excitation wavelength of 780 nm. The emission from the Methoxy-X04 and FITC-conjugated dextran was separated using a 485 nm dichroic mirror and collected in two non-descanned detectors with bandpass filters BA and BA First, a Z-stack of the vasculature of the area where the vessels selected for injury were located was obtained. In our system, an irradiation protocol of 1000 repetitions of 75% laser power (~ 0.01 mw/µm 2 ) at 780 nm induced a reproducible and reliable degree of clot formation among 6-month-old TgCRND8 mice. The brain circulation in the area targeted was recorded several seconds before the irradiation procedure and ~ 45 min after it, and therefore the clot formation process was entirely recorded. Behavioral analysis-morris water maze: The mice were tested for a total of 5-6 days in a circular pool filled with opaque water maintained at 20 C. The pool contained a transparent platform that was flagged during the visual platform days and submerged 1 cm during the hidden platform test. Experiments were performed in a sound-attenuated room under soft illumination, and visual cues were placed on the walls of the testing room. The mice were Page 7 of 8

8 first evaluated for swimming and visual abilities with two consecutive days of the visual platform test to familiarize them with the task. Two days later, the visual cues and the platform location were changed, and mice underwent 3-4 consecutive days of the hidden platform test. Mice were given 4 trials of 90 sec each to find the platform with a maximum inter-trial interval of 30 min. The starting point was altered among trials, and escape latencies were measured (data not shown). To analyze if mice were using a spatial learning strategy to locate the platform, the platform was removed from the pool and the mice were given a 60 sec probe-trial 1 hr after the last hidden platform session. Page 8 of 8