5.1 Blood coagulation time assay

Blood recalcification time was measured in accordance with previous research [19]. Briefly, 19 μL of fresh plasma obtained from healthy human donors was mixed with 1 μL of L-PC (HY-113147A, MedChemExpress, USA. final concentrations of 0, 25, and 50 μM) and 40 μL of HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) in 96-well plate. The plates were then incubated for 10 min in a 37 °C incubator, followed by the addition of 40 μL of preheated (37 °C) CaCl2 solution (25 mM). The kinetic program was recorded using a Cytation 3 Cell Imaging Multi-Mode Reader (Biotek) to monitor absorbance at 650 nm every 30 s for 30 min.

Activated partial thromboplastin time (APTT) is used to evaluate the functionality and integrity of the intrinsic and common coagulation pathways, while prothrombin time (PT) is used to detect the functionality and integrity of the extrinsic and common coagulation pathways. Here, we determined the effects of L-PC on APTT and PT using commercial kits according to the provided directions. Briefly, for the APTT assay, 50 μL of APTT reagent (Beijing Reagan Biotechnology, China) and 50 μL of plasma were mixed with 5 μL of L-PC at different concentrations (0, 25, 50 μM). Clotting time was then recorded after the addition of 50 μL of preheated (37 °C) CaCl2 (25 mM), with absorbance monitored at 650 nm using a Cytation 3 Cell Imaging Multi-Mode Reader (Biotek). For the PT assay, 50 μL of plasma was mixed with 5 μL of L-PC at different concentrations (0, 25, 50 μM). Clotting time was then recorded after the addition of 100 μL of PT reagent (Nanjing Jiancheng Bioengineering Institute, China) preheated at 37 °C for 15 min, with then absorbance monitored at 650 nm using a Cytation 3 Cell Imaging Multi-Mode Reader (Biotek).

5.2 Enzyme kinetic assays

The enzymes used in this study were tissue-type fibrinogen activator and plasmin, with chromogenic substrates used for the enzyme kinetic experiments according to the method reported before [20]. In brief, the enzymatic reactions were conducted in 50 mM Tris–HCl buffer (pH 7.8) at 37 °C. Initially, the proteases (plasmin: HPlasmin, Enzyme Research Laboratory, USA (30 nM); tPA: T0831, Sigma, USA (20 nM)) and serial dilutions of L-PC (6.25–100 μM) were incubated in buffer at 37 °C for 5 min, followed by the addition of their corresponding chromogenic substrates (CS-41, Hyphen Biomed, France (0.2 mM) for plasmin; CS-05, Hyphen Biomed, France (0.2 mM) for tPA) to initiate the reaction. The rate of substrate hydrolysis was monitored continuously at 405 nm.

5.3 Fibrin polymer lysis assay

The fibrin polymer lysis assay was used to examine the effects of L-PC on the activity of plasmin and tPA towards their physiological substrates according to the method reported before[21]. Fibrin clots were formed by incubating fibrinogen with thrombin in buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, and 5 mM CaCl2) at room temperature until turbidity reached a stable level. Plasmin or tPA plus plasminogen in the presence or absence of L-PC was gently layered on top of the fibrinogen polymer reaction mixture, with changes in turbidity at 450 nm monitored using a Cytation 3 Cell Imaging Multi-Mode Reader (Biotek).

5.4 Surface plasmon resonance (SPR) analysis

SPR analysis was performed according to the manufacturer’s instructions. In brief, a CM5 sensor chip (29149603, Cytiva, USA) was first activated with 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 mM N-hydroxysuccinimide at a flow rate of 5 μL/min for 20 min. Plasmin (HPlasmin, Enzyme Research Laboratory, USA) or tPA (HY-P71051, MedChemExpress, USA), diluted to a concentration of 100 μg/mL using sodium acetate buffer (10 mM, pH 5), flowed across the activated surface, allowing coupling with the chip until a target response value (RU) of 12 000 was reached. The remaining activated sites on the chip were blocked by 75 μL of ethanolamine (1 M, pH 8.5). Real-time detection was recorded using a Biacore S200 instrument (USA) at a flow rate of 10 μL/min. Subsequently, to detect the equilibrium dissociation constant (KD), serially diluted L-PC (12.5, 25, 50, 100, 200 μM in 0.01 M phosphate-buffered saline (PBS), pH 7.4) was applied to analyze its interactions with plasmin or tPA coupled on the chip surface using the BIA evaluation program.

5.5 Bleeding time test

Male C57BL/6 mice (6 weeks old) were randomly divided into five groups (n = 6 per group): saline-treated group, three L-PC-treated groups (concentrations of 0.25, 1, 4 mg/kg), and sodium heparin-treated positive control group (2 500 U/kg). After 10-min intravenous administration of the respective drugs via the tail vein, the tails were cut at a distance of 2 mm from the tip and immersed in saline at 37 °C. The time taken for continuous blood flow to cease was recorded as bleeding time.

5.6 FeCl3‑induced mouse thrombosis model

Male C57BL/6 J mice (6 weeks old) were anesthetized with 3% pentobarbital sodium (80 mg/kg) and fixed in a supine position. The left carotid artery (CCA) was exposed through a midline neck incision. Subsequently, four groups were established, each receiving different concentrations of L-PC (0, 0.25, 1, 4 mg/kg) administered via tail vein injections. After a 10-min interval, carotid arterial thrombosis was induced by placing a filter paper disc (diameter 5.0 mm) soaked in 10% FeCl3 onto the artery for 2 min. Blood flow was monitored using a laser-speckle blood flow imaging system (RFLSI Pro; RWD Life Science).

5.7 Transient MCAO (tMCAO)5.7.1 Assessment of functional outcome

Male C57BL/6 J mice (6 weeks old) were anesthetized with 3% pentobarbital sodium (80 mg/kg) and fixed in a supine position. The skin along the midline of the neck was incised to expose the external and internal carotid arteries. A standardized silicon rubber-coated nylon monofilament (6023910PK10; Doccol, Sharon, MA, USA) was carefully inserted into the left common carotid artery and advanced via the internal carotid artery to occlude the origin of the left middle cerebral artery, inducing ischemia. Subsequently, four groups were established, each receiving different concentrations of L-PC (0, 0.25, 1, or 4 mg/kg) administered 10 min prior to reperfusion. Reperfusion was initiated by removing the occluding filament 60 min after its insertion. After 24 h of modeling, neurological assessments, including Bederson score and grip test, were performed following previously established protocols [22].

5.7.2 Measurement of infarct size and volume

The mice were sacrificed 24 h after tMCAO and five 2-mm-thick coronal brain sections were obtained. The brain slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, USA) in a dark environment at 37 °C for 10 min. TTC reacts with intact mitochondrial respiratory enzymes, resulting in the unaffected brain regions appearing bright red and the infarct areas exhibiting a pale coloration. The extent of infarction in the brain sections was estimated by calculating the pale area. The infarct volume ratio was quantified using Image Pro Plus v6.0 (Media Cybernetics, Bethesda, MD, USA).

5.7.3 Evans blue staining

Mice were injected with 2% Evans blue (R31047, Yuanye, China) in the tail vein 2 h before sacrifice. Brain sections were taken to calculate the area of Evans blue staining using Image Pro Plus v6.0 (Media Cybernetics, Bethesda, MD, USA).

5.7.4 Enzyme-linked immunosorbent assay (ELISA)

Male C57BL/6 J mice (6 weeks old) were sacrificed 24 h after tMCAO, followed by the collection of brain tissue and blood. Mouse IL-1β ELISA kit (DG30045M-96 T, Dogesce, China), IL-6 ELISA kit (DG30062M-96 T, Dogesce, China), and TNF-α ELISA kit (DG30048M-96 T, Dogesce, China) were used to detect the levels of inflammatory factors in brain tissue and blood samples according to the manufacturer’s instructions.

5.8 Statistical analyses

Data obtained from independent experiments are presented as mean ± standard deviation (SD). For normal continuous variables, one-way analysis of variance (ANOVA) was used. All analyses were conducted using GraphPad Prism v5. Asterisks represent p-value classifications: * p < 0.05; ** p < 0.01, and *** p < 0.001.