Slc:ICR mice (6-8 weeks old males; weight, 30-33 g) were purchased from Jungang Animal Co., Seoul, Korea. Thrombosis was induced in mice using a previously described carotid artery injury model [11]. Three mice were used for each concentration. After an intraperitoneal injection containing both 2.5 mL/kg Zoletil (Virbac Animal Health Co., Carros, France) and 0.3 mL/kg Rompun (Bayer Korea, Anseong, Korea) for anesthesia, the skin on the upper central neck area was directly incised and the left common carotid artery was exposed. Carotid artery blood flow was measured with a miniature Doppler flow probe (diameter: 2 mm) for photoplethysmography (BioBud Inc., Seoul, Korea). Total occlusion was induced by applying a strip of filter paper (1×1 mm) saturated with 25% FeCl₃ proximal to the surface of the carotid artery for 3 min. The carotid blood flow was monitored from time 0 (before the application of the FeCl₃ paper) up to the time of occlusion. Various concentrations (0, 0.05, 0.07, and 0.1 mg/kg body weight) of fucoidan (Sigma Chemical Co., St. Louis, MO, USA) or high-molecular-weight heparin (100 KU, Grade I-A, Sigma Chemical Co) were intravenously injected just before blood flow monitoring. The concentrations of heparin were 0, 0.05, 0.07, 0.1, 0.13, 0.16, and 0.2 mg/kg body weight.

The platelet aggregation assay was performed in human platelet-rich plasma (PRP). The platelet concentrate was diluted to 300,000 cells/µL of platelet-poor plasma (PPP). Ten microliters of samples (fucoidan, heparin, and phosphate-buffered saline [PBS]) was added to 450 µL of plasma and incubated for 2 min in an incubation well of the platelet aggregometer (Chrono-Log Co., Havertown, PA, USA) at 37℃. The final concentrations of fucoidan and heparin in this experiment were 0, 0.2, 0.4, 0.6, 0.8, and 1 µg/mL. The impedance was recorded, and ADP (20 µM) was added in order to initiate platelet aggregation. The inhibition of platelet aggregation was measured at the maximum aggregation response and the IC₅₀ value was calculated by the least squares method.

The anti-thrombin and anti-factor Xa activities of fucoidan were determined by chromogenic assay methods. In a 1.5 mL tube that contained thrombin (0.1 units/mL) or factor Xa (0.005 units/mL) in a buffer (pH 8.8) containing 50 mM Tris-HCl and 38 mM NaCl and antithrombin (0.1 µg/mL), various concentrations (0, 1, 5, 10, 50, 100, and 500 µg/mL) of fucoidan or heparin (180 IU/mg) were added, and the tubes were incubated at 37℃ for 20 min. After incubation, 25 µL of the solution from each 1.5 mL tube was transferred to a well of a 96-well plate. Then, 200 µL of 50 mM Tris-HCl (pH 8.8) buffer and 25 µL of thrombin substrate (0.3 mM/mL) or factor Xa substrate (0.3 mM/mL) were added to each well of the 96-well plate and incubated for 60 min at 37℃. After incubation, amidolytic activities were measured using a spectrophotometer at 405 nm.

PPP was obtained by centrifugation of citrated human blood at 1,200×g for 15 min. The plasma was incubated with fucoidan dissolved in PBS (plasma : PBS=1 : 4), heparin dissolved in PBS, or only PBS for 2 min at 37℃. The plasma clotting times, activated partial thromboplastin time (aPTT) (Hyphen-BioMed, Andresy, France), prothrombin time (PT) (Hyphen-BioMed), and thrombin time (TT) (Hyphen-BioMed) were measured using the blood coagulometer Thrombotimer 2 (Behnk Elektronik, Norderstedt, Germany).

RAoSMCs were obtained from BioBud Inc. (Seoul, Korea). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco Life Technologies, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Welgene Co., Daegu, Korea), 3.7% sodium bicarbonate, and 1% penicillin-streptomycin (10,000 IU/mL; Gibco Life Technologies). RAoSMCs were used between the fourth and eighth passages. The cells were incubated in 96-well plates (Corning Costar, Cambridge, MA, USA) at a density of 5×10⁴ cells/well. After 24 h, the medium was changed with 100 µL fresh media containing several different concentrations (0, 50, 100, 500, and 1,000 µg/mL) of fucoidan and high-molecular-weight heparin. After changing the media, cells were further incubated for 24, 48, and 72 h at 37℃. Assays for cell proliferation were performed using a CCK-8 kit (Dojindo Molecular Technologies Inc, MA, USA). The samples were read at 450 nm using a spectrophotometer VERSAmax™ Tunable Microplate Reader (Molecular Devices Corp., CA, USA).

The effect of fucoidan on SMC migration was examined by performing a modified Boyden chamber assay in Transwell cell culture chambers using a polycarbonate membrane with 8 µm pores in a 6-well plate. RAoSMCs were suspended in serum-free DMEM at a concentration of 5×10⁵ cells/mL. Cells were then pretreated with several concentrations of fucoidan and heparin for 30 min at 37℃. Serum-free DMEM was added to the lower compartment. Cell suspensions were added to the upper compartment and were then incubated for 6 h at 37℃. The cells were fixed and stained with crystal violet. The filter membranes were dried and the migrated cells were counted.

Ninety-six-well plates were precoated with vitronectin, fibronectin, and laminin (500 µg/mL) at 4℃ overnight. SMCs (3×10⁵ cells/well) were preincubated with fucoidan and heparin for 30 min at 37℃. After incubation, the cells were transferred to each well and incubated for 1 h at 37℃ in 5% CO₂. Unattached cells were removed by washing with PBS. Attached cells were fixed and stained with crystal violet. The plates were read using a spectrophotometer at 490 nm to determine the relative number of cells.

HUVECs were plated onto 96-well cell culture plates coated with 0.5% gelatin. After 24 h, the serum-free media containing fucoidan and heparin were changed and the plate was incubated for 8 h at 37℃ in a CO₂ atmosphere; the cells were then stimulated with tumor necrosis factor-alpha (TNFα) (10 ng/mL) for 16 h in an incubator. After incubation, supernatants were collected for the determination of the inflammatory cytokine levels using a Multiplex cytokine assay system (Luminex 100™ IS Total System, XMAP technology, Millipore, MA, USA). Sixteen kinds of inflammatory cytokines and/or chemokines (Eotaxin, granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon (IFN)-α2, IFNγ, interleukin (IL)-10, IL-12, IL-1α, IL-1β, IL-1Rα, IL-2, IL-6, IP-10, monocyte chemoattractant protein (MCP)-1, RANTES, TNF-α, and TNF-β) were quantitatively determined.

A FeCl₃-induced mouse carotid artery occlusion model was developed and well optimized to evaluate the antithrombotic activity of anticoagulants and thrombolytic agents (Fig. 1). Before drug treatment, the average total occlusion time was determined as 12.5 min. Antithrombotic activity (effective dose, 50% [ED₅₀]) was calculated as the concentration required to double the total occlusion time. The ED₅₀ values of fucoidan and heparin were approximately 0.54 and 1.24 mg/kg body weight, respectively. Therefore, fucoidan showed approximately 2.3 times stronger antithrombotic effect than heparin in vivo (Fig. 2).

Fucoidan strongly inhibited ADP-induced human platelet aggregation in vitro in a concentration-dependent manner. The 50% inhibitory concentrations (IC₅₀ values) of fucoidan and heparin for platelet aggregation were determined as 0.36 and 1.0 µg/mL, respectively (Fig. 3). Therefore, the inhibitory effect of fucoidan on platelet aggregation is approximately 2.8 times superior to that of heparin.

The anti-thrombin and anti-factor Xa activities of fucoidan were interestingly weaker than those of heparin (Fig. 4), although the overall antithrombotic effect of fucoidan in vivo was even more potent than that of heparin (data not shown).

The anticoagulation activities of fucoidan, as determined by aPTT (Table 1), PT (data not shown), and TT (data not shown), were revealed to be dose dependent, although heparin showed considerably higher inhibitory activity on PT, aPTT, and TT than fucoidan.

We tested the inhibitory effects of fucoidan on RAoSMC proliferation, migration, and adhesion. In proliferation assays, we found that fucoidan had a stronger inhibitory effect on SMC proliferation than heparin (Fig. 5). In addition, fucoidan inhibited SMC migration and SMC adhesion to extracellular matrix to a greater extent than heparin (data not shown). Specially, fucoidan inhibited SMC adhesion to laminin in a dose-dependent manner.

Sixteen kinds of cytokines were simultaneously determined by the Multiplex assay system. We confirmed that the levels of some specific inflammatory cytokines and chemokines were reduced in HUVECs when we treated the cells with fucoidan in the presence of TNF-α. Specifically, fucoidan treatment reduced the amounts of GM-CSF, IL-6, MCP-1, and RANTES (Fig. 6). In addition, fucoidan showed a stronger inhibitory effect on the proinflammatory cytokines than heparin.