Role of Reactive Oxygen Species in Collagen-Induced Platelet Activation and the Protective Effects of Antioxidants

Figures

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  Figure 1. Dose-dependent effects of collagen treatment on EMPO adducts under collagen-induced platelet aggregation in human PRP. (A) Collagen (0–2 μg/mL) was added to PRP, and platelet aggregation was calculated as a percentage relative to vehicle-treated controls (0.9% NaCl, 0 μg/mL). Data are presented as mean ± SD (n = 4–6). (B) Representative ESR spectra of EMPO adducts formed in human PRP treated with collagen (1 μg/mL) or vehicle (0 μg/mL). PRP was pre-incubated with 25 mM EMPO for 2 min before collagen addition, and spectra were recorded 2 min post-treatment. (C) Dose-dependent increases in EMPO adduct formation () and platelet aggregation () in response to collagen (0–1 μg/mL). Statistical significance was analyzed using Student’s t-test with * p < 0.005 and ** p < 0.01 compared to the vehicle group.

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  Figure 2. ESR spectrum of CAT-1 detected at the mid-thorax of a collagen-treated mouse. (A) Representative L-band ESR spectra recorded at the mid-thorax of an anesthetized mouse 1 min after injection of collagen (i.v. 0.75 μg/g body weight). Subsequently, 100 μL of 10 mM CAT-1 solution was administered via tail vein. (B) Decay of CAT-1 ESR signal in collagen- (●) and vehicle-treated (○) mice. Collagen-treated mice received 0.75 μg/g body weight of collagen intravenously, while vehicle-treated mice were administered 0.9% NaCl. Signal intensities at the low-field peak were logarithmically plotted against time post-CAT-1 injection.

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  Figure 3. Time-dependent changes in decay rate of CAT-1 in collagen-treated mice. Collagen (●, 0.75 μg/g body weight) or vehicle (○, 0.9% NaCl) was injected intravenously into mice, followed by administration of 100 μL of 10 mM CAT-1 solution. CAT-1 decay rates were determined using L-band ESR spectroscopy at various times post-collagen administration. Data are presented as mean ± SD (n = 6–11). * Indicates significant differences between collagen- and vehicle-treated mice (p < 0.005, Student’s t-test).

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  Figure 4. Temporal changes in collagen content. (A) Dose-dependent effect of collagen on the CAT-1 decay rate. One minute after the indicated collagen doses, 100 μL of 10 mM CAT-1 solution was administered intravenously, and decay rates were recorded using L-band ESR spectroscopy. Vehicle-treated mice (0 μg collagen) received 0.9% NaCl. (B) Survival rate of collagen-treated mice. Numbers in parentheses indicate the number of mice surviving per total number tested. Data are presented as mean ± SD (n = 6–11); * and ** indicate significant differences compared to vehicle-treated mice (p < 0.01, p < 0.005, respectively, Student’s t-test).

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  Figure 5. Effect of antioxidants on survival. Collagen (0.75 μg/g body weight) was injected intravenously, followed by 100 μL of 10 mM CAT-1 solution. ESR spectra recorded 1 min post-CAT-1 injection. Antioxidants, including DMTU (3 μM/mouse), mannitol (3 μM/mouse), Tiron (4 μM/mouse), SOD (300 U/mouse), and catalase (100 U/mouse), were administered at various times relative to collagen injection. Survival rates (■) and CAT-1 decay rates (□) are expressed as percentages compared to collagen-only controls. * Indicates significant differences in survival (p < 0.01), and ** indicates significant differences in decay rates (p < 0.005, Student’s t-test).

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  Figure 6. Schematic illustration of collagen-induced ROS production and platelet activation, and the protective role of antioxidants. Collagen exposure induces reactive oxygen species (ROS) generation, which subsequently triggers platelet activation. Activated platelets aggregate, facilitating thrombus formation. Enhanced thrombus formation can ultimately lead to animal death. Antioxidants effectively mitigate these adverse effects by suppressing ROS generation, thereby inhibiting platelet activation, reducing thrombus formation, and improving survival outcomes.