Antiplatelet and Antithrombotic Activities of Lespedeza cuneata via Pharmacological Inhibition of Integrin αIIbβ3, MAPK, and PI3K/AKT Pathways and FeCl3-Induced Murine Thrombosis

Cardiovascular diseases (CVDs) have been the major cause of mortality all around the globe. Lespedeza cuneata abbreviated as L. cuneata with the authority name of Dumont de Courset (G. Don) is a perennial flowering plant commonly grown in Asian countries such as Korea, Japan, China, and Taiwan. We aimed to investigate the L. cuneata extract's antiplatelet and antithrombotic properties as GC-MS analysis indicated that the extract contained short-chain fatty acids, which have been reported to possess beneficial cardiovascular effects. L. cuneata was extracted using water, 50% EtOH, 70% EtOH, and 100% EtOH. For in vitro antiplatelet analysis, washed platelets were prepared and incubated with L. cuneata with 200 μg/mL of 50% EtOH in the presence of 1 mM of CaCl2 for 1 minute followed by agonist (collagen 2.5 μg/mL or ADP 10 μM or thrombin 0.1 U/mL) stimulation for 5 minutes over light transmission aggregometer. Scanning electron microscopy was performed to assess platelet shape change. ATP release and intracellular calcium mobilization were quantified to assess the granular content. Fibrinogen-binding assay and clot retraction assay assessed integrin αIIbβ3-mediated inside-out and outside-in signaling. Protein phosphorylation expression was investigated by western blot analysis. Finally, the in vivo antithrombotic efficacy was investigated by oral dosage of L. cuneata 200 and 400 mg/kg and aspirin 100 mg/kg for 7 days, and tail bleeding and FeCl3-induced murine thrombus model were performed. In vitro platelet aggregation and platelet shape change were dose-dependently suppressed by L. cuneata. Calcium mobilization, dense granules secretion, integrin αIIbβ3-mediated inside-out and outside-in signaling, and protein phosphorylation of MAPK and PI3K/Akt pathways were significantly inhibited. In vivo assays revealed that L. cuneata prevents side effects of synthetic drugs via nonsignificantly increasing bleeding time and improving coronary artery blood flow and animal survival. Our results demonstrate that L. cuneata exhibited potent antiplatelet and antithrombotic effects and can be considered a potential herbal medicine with cardioprotective effects.


Introduction
Cardiovascular diseases (CVDs) have afected the majority of population worldwide [1]; among them, coronary heart disease is the most common type of heart disease, causing mortality of around 375,476 people in 2021.According to the heart disease and stroke statistics update of 2021, heart diseases are responsible for 1 in 4 fatalities in the United States [2].About 1 in 20 adults aged 20 and older have CVD (about 5%) [3].Statistics from China in 2023 reported that two out of every fve deaths are due to CVD [4].Over the past 50 years, CVD mortality has declined, but cardiovascular events such as heart attacks and strokes remain by far the leading cause of death in the European Union, accounting for 36% of all deaths and impacting the lives of 60 million people who live with CVD.More broadly, CVD accounts for 47% and 39% of all deaths in women and men, respectively, in the wider European Regions [5].Although the total mortality rate of cardiovascular disease (CVD) in South Korea has signifcantly decreased, heart disease remains the second leading cause of death, and ischemic heart disease (IHD) mortality has continuously increased until recently [6].
Te pathophysiology of CVDs involves several factors; among them, hyperactive platelets are the main ofenders, leading to platelet plug formation and vascular stenosis, which may result in ischemic stroke [7].Originating from megakaryocytes, platelets express various receptors, adhesion molecules on their surface, and granules in the inner compartments, which contain downstream efector molecules [8].Glycoprotein Ib/V/IX interacts with von Willebrand factor after vascular damage, resulting in platelet receptors GPVI and integrin αIIbβ3 binding to collagen, causing outside-in signaling, platelet activation, phosphatidyl serine exposure, and release of granule contents [9].Subsequent thrombin generation, platelet binding to integrin αIIbβ3, and stable platelet thrombus fasten the injured site and make a basis to heal the damaged vascular site.Moreover, platelets contain mitogen-activated protein kinases (MAPKs), such as ERK1/2 and JNK1, which are involved in apoptosis, migration, and proliferation.Many agonists, including collagen, ADP, and thrombin, phosphorylate MAPKs, which are crucial for both "inside-out" and "outside-in" signaling [10].In addition, essential for platelet activation and aggregation is the PI3K/Akt signaling pathway.Moreover, tyrosine phosphorylation-based signaling pathways triggered by GPVI or αIIbβ3 require PI3Ks [11].
Te majority of the countries have a long history of employing medicinal plants for diagnosis, treatment, and maintenance of personal hygiene as well as for use as food supplements, cosmetics, and scents.Te world's medicinal and aromatic plant diversity is highest in the Asia-Pacifc region.Global demand for herbal medicine has seen a tremendous increase as evident from a report released by the Research and Information System (RIS) for Developing Countries.According to this report, the herbal sector is expected to reach a global market size of US$746.9 billion in 2022, compared to a projected US$657.5 billion in 2020 [12].Meanwhile, synthetic drugs have been successfully used to treat and prevent CVDs, but no cures are without serious side efects.For instance, clopidogrel occasionally causes aplastic anemia and thrombocytopenic purpura, and the most commonly used antiplatelet drug aspirin sometimes causes severe stomach ulcers or persistent bleeding with high levels of resistance to these drugs [13].Considering this, the ethnomedical approach could be a promising strategy for preventing CVDs and their complications [14].Natural herbal components and the Mediterranean diet contain bioactive compounds that can modulate platelet activity.Tese natural approaches may help lower the risk of thrombosis [15].
Lespedeza cuneata G. Don belongs to the family Fabaceae and the genus Leguminosae.L. cuneata is a fowering plant commonly grown in Asian countries and has been reported for its antioxidative and anti-infammatory efects [16], scalp-improving properties [17], prostatic hyperplasia [18], early atherosclerosis [19], and whitening skin [20].However, its pharmacological action in preventing platelet aggregation and antithrombotic properties has not been explored yet.Terefore, this study aimed to explore the pharmacological action of L. cuneata on attenuating platelet aggregation via in vitro and in vivo models.To the best of our knowledge, this will be the frst study to unravel antiplatelet and antithrombotic properties of L. cuneata via inhibition of integrin αIIbβ3, MAPK, PI3K/AKT pathways, and FeCl 3induced murine thrombosis model.  .Trombin from human plasma was purchased from Sigma-Aldrich (Lot # SLCF9776 CAS no.9002-04-4 647-014-00-9).Fura 2-AM (2acetoxymethyl) (CAS number: 108964-32-5) and Alexa Fluor 488-conjugated fbrinogen (Cat # A32723) were obtained from Invitrogen (Eugene, OR, USA).All antibodies were supplied by Cell Signaling (Beverly, MA, USA).

Extraction and Procurement of Plant Samples.
Plant samples were extracted as described previously [21].In brief, L. cuneata was extracted for 2 h with distilled water and 50%, 70%, and 100% EtOH at 100 °C for water and 80 °C for EtOH in a 1 : 20 ratio (plant: solvent w/v).After extraction, these samples were fltered using flter paper (Whatmann ™ no.4).
Ten, the samples were evaporated via rota vapor and placed at −70 °C overnight to freeze.Te samples were then freezedried for 3-4 days at −55 °C to obtain a powdered form.Ultrapure water was used to dissolve the water extract and dimethyl sulfoxide (DMSO) for ethanol extracts in particular concentrations for the evaluation of the samples.Te characterization of the extract was done with GC-MS as explained in the following.

GC-MS Analysis.
Te GC-MS analysis was performed using an Agilent 7890A GC instrument (Agilent Technologies, Santa Clara, CA, USA).Te instrument was equipped with a 30 m × 0.

Preparation of Washed Rat Platelets.
To prepare washed platelets, SD rat blood was obtained.Blood was drawn by cardiac puncture with a syringe containing the anticoagulant solution acid citrate dextrose (ACD) and then transferred to a round bottom tube containing Tyrode's bufer and ACD in a 1 : 4 ratios under light anesthesia with diethyl ether.To obtain platelets from the whole blood, initially, it was centrifugated at 170 × g for 7 min and then another round of centrifugation at 350 × g for 10 min to get washed platelets.For platelet aggregometry analysis, the collected platelets were balanced to 3 × 10 8 cells/mL by adding Tyrode's bufer.

Platelet Aggregation Assay and SEM Analysis.
As reported previously [22], light transmission aggregometry was performed to access platelet aggregation and inhibition by the plant sample.In brief, washed platelets were obtained from rat blood and incubated for 1 min with 1 mM calcium chloride and varying concentrations of L. cuneata (50 μg/mL, 100 μg/mL, and 200 μg/mL) or DMSO.After 1 min, collagen, ADP, and thrombin (collagen 2.5 μg/mL or ADP 10 μM or thrombin 0.1 U/mL) were added to stimulate platelets and aggregation was stopped after 6 min.Light transmission through the glass tube was read as percent transmission by the light transmission aggregometer.
For SEM, after incubation for 5 min with L. cuneata and agonists, 0.5% paraformaldehyde and 0.5% osmium tetroxide were used for platelet fxation, and platelets were then dehydrated with increasing concentrations of EtOH from 50% to 100%, followed by freeze-drying at −55 °C.Platelet shape change was assessed by ultrastructure pictures captured using a feld transmission electron microscope (SU8220; Hitachi, Japan).

[Ca 2+
] i Mobilization.Fura-2/AM at a concentration of 5 M is incubated with platelet-rich plasma (PRP) for 1 h at 37 °C.Fura-2-loaded platelets, at a concentration of 3 × 10 8 /mL, were then stimulated with collagen for 5 min after preincubation with a plant sample in the presence of 1 mM CaCl 2 .Fura-2 fuorescence in the cytosol is calculated using the formula [Ca 2+ ] i � 224 nM (F F min )/(F max F), where F is the dissociation constant and F min and F max are the fuorescence intensities.

ATP Release Assay.
Collagen was used to stimulate washed platelets after they have been preincubated with various doses of plant extract for 5 min at 37 °C.After the aggregation reaction, the platelet mixture was centrifuged at 12000 rpm to extract the supernatant, and the amount of ATP secreted (in the supernatant) was then determined using an ATP Assay kit and a luminometer.
2.9.Fibrinogen-Binding Assay.Washed platelets were pretreated with L. cuneata and an antifbrinogen antibody.Ten, paraformaldehyde 0.5% was used to fx the platelets.A fow cytometer (FACS Aria III) was used in the cytometric analysis.

Clot Retraction.
By evaluating clot retraction as previously described [23], after incubating PRP (250 L) with the vehicle, L. cuneata, or Y27632 (Rock inhibitor) for 2 min, the volume was increased to 1 mL by adding red blood cells (RBCs, 5 L) and Tyrode's bufer.Te injection of thrombin (1 U/mL) caused the clot to retract, which was then monitored for 90 min at room temperature.To evaluate clot retraction, clot weight was lastly assessed.

Western Blotting.
Various quantities of L. cuneata were preincubated with washed platelets for 1 min and stimulated with collagen for 5 min.Platelet aggregation was stopped by adding a lysis bufer (PRO-PREP; iNtRON Biotechnology, Seoul, Korea), and protein concentration was determined using the BCS assay (PRO-MEASURE; iNtRON Biotechnology).Total platelet proteins were isolated, separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difuoride (PVDF) membranes.Te membranes were blocked with 5% skim milk, probed with appropriate antibodies (phospho-ERK, phospho-JNK, phospho-p38MAPK, phospho-PI3K, phospho-Akt, etc.), and visualized using enhanced chemiluminescence.

In Vivo Tail Bleeding and the FeCl 3 -Induced Trombus
Model.To evaluate tail bleeding assay and FeCl 3 -induced thrombus formation, four treatment groups were treated orally for 7 days (group 1 � saline, group 2 � ASA, and group 3 and 4 � low and high dose of the 50% EtOH extract of L. cuneata) using C57BL/6J and ICR mice, respectively.A Evidence-Based Complementary and Alternative Medicine tail bleeding assay was carried out as previously reported 1 h after the last treatment while the FeCl 3 -induced thrombus formation assay was carried out 20 min after the last treatment as previously reported [24,25].C57BL/6J mice have been established for the tail bleeding assay [22] but here, we used ICR mice for the FeCl 3 -induced thrombus model as Shim et al. recently reported that ICR mice showed better dose responses in thrombus formation and stability compared to the C57BL/6N mice [26].

Statistical Analysis.
Acquired data were subjected to one-way analysis of variance and post hoc Dunnett's test (SAS Institute Inc., Cary, NC, USA) to determine the statistical signifcance of the observed diferences.Te provided information is displayed as the mean ± standard deviation (SD).Statistical signifcance was defned as a p value of 0.05 or lower.

Results for the In Vitro Study
4.1.L. cuneata Inhibits Agonist-Induced Platelet Aggregation.
After extracting plant samples using diferent solvents such as 50% EtOH, 70% EtOH, 100% EtOH, and water, antiplatelet efects by inhibiting platelet aggregate formation during light transmission aggregometry was determined.Among them, 50% EtOH showed the highest platelet aggregation inhibition at 200 μg/mL (Figure 3).Terefore, L. cuneata with 50% EtOH was selected for further evaluation for its mechanistic pathways to present antiplatelet and antithrombotic activities.

L. cuneata Reduces [Ca 2+
] i Mobilization and ATP Release from Alpha Granules.Te amount of intracellular calcium release was quantifed by the following: Here, F min and F max are the fuorescence intensities at extremely low and very high Ca 2+ concentrations, respectively, and F is the dissociation constant of the Fura-2-Ca 2+ complex.Our results demonstrated that the dose regimen for L. cuneata signifcantly and dose-dependently inhibited calcium mobilization, whereas 200 μg/mL completely abolished the increase in calcium concentration induced by collagen stimulation (Figure 5(a)).
ATP release was measured using supernatant from the aggregation reaction using an ATP ELISA kit.Our results demonstrated that 50, 100, and 200 μg/mL dose-dependently inhibited ATP release (Figure 5(b)).

L. cuneata Reduced Fibrinogen Release from δ-Granule Secretion and Attenuated Inside-Out and Outside-In
Signaling.Te efects of the L. cuneata extract at various doses on collagen-induced platelet signaling were investigated.Our results revealed that L. cuneata reduced fbrinogen interaction to integrin αIIbβ3 signifcantly to inhibit inside-out signaling (Figure 6).In addition, the L. cuneata with 50% EtOH extract signifcantly and dosedependently decreased clot retraction and outside-in signaling to prevent platelet shape change (Figure 7).

L. cuneata Attenuates MAPK and PI3K/Akt
Phosphorylation.Protein phosphorylation during platelet aggregation inhibition was investigated using Western blot.Te results demonstrated signifcant inhibition of the phosphorylation of MAPK and PI3K/AKT by treatment with diferent doses of L. Cuneata with 50% EtOH (Figure 8).

L. cuneata Prevents Trombosis and Regulates Hemostasis.
To evaluate the side efects caused by remedial substances on thrombosis and hemostasis, a FeCl 3 -induced thrombus model and a tail bleeding assay was performed using acetylsalicylic acid (ASA) as a positive control.After induction of thrombus with 35% FeCl 3 , L. cuneata not only improved the blood fow but also the survival rate of mice similar to the ASA group.On the other hand, L. cuneata does not possess the side efect of aspirin by preventing increased bleeding time (Figure 9).

Discussion
A myriad of ailments known as CVDs afects the heart and blood vessels.Cardiovascular conditions include thrombosis, acute myocardial infarcts, and coronary heart artery disease.Platelets have an important role in the treatment and prevention of CVDs and are currently the subject of numerous investigations.Antiplatelet medications lower death rates caused by myocardial infarction.
At areas of atherosclerotic plaque rupture, changes in the blood fow promote platelet activation and arterial thrombus formation [27].Plant extracts having antiplatelet efects can be a better remedy for thrombotic and CVDs assessed by providing similar conditions to blood fow as in light transmission aggregometry.Intra-arterial coronary collateral vessels may contract because of ATP, and platelet activity in coronary arteries connected to the collateral vasculature may result in collateral vasoconstriction, limiting blood fow to the dependent myocardia [28].Te terminal stage of platelet aggregation can be recognized by inside-out signaling and the activation of integrin αIIbβ3, whose activation is crucial during this process.In other words, integrin αIIbβ3 connects itself to another integrin through a bridge network resembling fbrinogen.Te blood clot is fnally tightened and becomes stable [29].
Collagen, thrombin, and ADP by initiating subsequent signaling processes via the activation of the GPVI, PAR, and P2Y12 receptor signaling pathways cause signifcant platelet aggregation.We tested the antiplatelet activity of water, 50%, 70%, and 100% EtOH L. cuneata extracts.Figure 3     Evidence-Based Complementary and Alternative Medicine morphology from discoid to a rounded shape containing flopodia upon activation with collagen which was prevented by treatment with L. cuneata with 50% EtOH (Figure 4) [30].
Te alpha (a) granules in platelets are flled with sticky ligands including fbrinogen, fbronectin, P-selectin, and dense (d) granules such as Ca 2+ and ATP.Following platelet activation, these granules are secreted, which improves platelet adherence, shape change, and aggregation [31].By preventing Ca 2+ release and fbrinogen binding, L. cuneata with 50% EtOH was found to block the secretion of both dense and alpha granules, lowering platelet activation, adhesion, shape change, and aggregation (Figure 5) as reported previously by Holmsen [32].
Te ability of plant extracts to regulate platelet aggregation and activation can be determined by testing them for the inhibition of fbrinogen binding to αIIbβ3 using fow cytometry [33].Similarly, in our results, αIIbβ3 integrins were activated by collagen and a reduction in αIIbβ3 integrins was observed after L. cuneata treatment (Figure 6).Trough cytoskeletal alterations in actin, GTPases control platelet adherence, shape change, and clot retraction.By phosphorylating the myosin light chain, Rho kinases are downstream regulators that afect the actin cytoskeleton in response to RhoA.Y-27632 has been employed to investigate the role of Rho kinase in promoting clot retraction [34].Src kinase, a member of the SFKs family, plays a signifcant role extracts with 1 mM CaCl 2 were preincubated using washed platelets for 1 min at 37 °C before collagen stimulation for 5 min with constant stirring.By adding a lysis bufer, platelet aggregation stopped, and the protein concentration was calculated using the BCS assay (PRO-MEASURE; iNtRON Biotechnology).In a 10% SDS-PAGE, total platelet proteins were isolated, and then they were transferred to PVDF membranes.Membranes were blocked with 5% skim milk, probed with the appropriate antibodies (phospho-ERK, phospho-JNK, phospho-p38MAPK, phospho-PI3K, phospho-Akt, etc.), and then observed using enhanced chemiluminescence.* p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the collagen.
Evidence-Based Complementary and Alternative Medicine in integrin αIIbβ3-mediated signaling, which may be also implicated in clot retraction [11].Abciximab and eptifbatide are αIIbβ3 antagonists that were previously consumed for preventing occlusive vascular events in atherosclerosis [35].Similar results were obtained when platelets were incubated with thrombin from human plasma or L. cuneata (Figure 7).
MAPKs, including ERK, JNK, and p38 MAPK, and their phosphorylation result in the release of granules, which increase platelet aggregation [36].Furthermore, MAPK and PI3K/Akt play critical roles in platelet activation by infuencing calcium mobilization, granule secretion, and platelet aggregation [37].In our study, the plant extract inhibited  Evidence-Based Complementary and Alternative Medicine these molecules and signifcantly reduced the phosphorylation of MAPK and PI3K/Akt, exhibiting a potential method of the inhibition of platelet activities (Figure 8).Finally, the inhibitory efects of L. cuneata with 50% EtOH on thrombus formation and hemostasis were evaluated with a FeCl 3 -induced thrombus model and a tail bleeding assay.By inhibiting platelet activation, we found that treatment with L. cuneata with 50% EtOH signifcantly reduced thrombus formation and modestly increased bleeding duration compared with treatment with ASA control [21] (Figure 9).
We speculate that the potent antiplatelet and antithrombotic activity of the L. cuneata extract via GC-MS is due to the presence of single components similar to inositol, which has been reported to treat high-fat diet-induced cardiac dysfunction [38] and prevent vascular calcifcation [39].Active components evaluated by GC-MS analysis in our study have previously been reported for their antiplatelet and antithrombotic activities such as acetic acid [40], glycerin [41], hexanoic acid, 2-hexanoic acid [42], 3methylene [43], nonane [44], and 2,3 butanediol [45] (Figure 1 and Table 1).
Te results of our study are summarized in Figure 10, which show potent cardioprotective efects of L. cuneata.

. Conclusion
Tis study used 50% EtOH extract, the most efective component, to test L. cuneata as herbal medication.As evidenced by our results L. cuneata dose dependently and signifcantly inhibited fbrinogen binding, alpha and dense granule release, protein expression, MAPK, and PI3K/Akt signaling pathways after agonist-induced platelet activation.Finally, L. cuneata did not have the negative consequences of prolonged bleeding times caused by the most commonly used antiplatelet medications (i.e., aspirin and clopidogrel).Tese results demonstrate that L. cuneata can be a potent herbal plant extract to substitute synthetic antiplatelet medications to prevent CVDs.

Figure 1 :
Figure1: Gas chromatography-mass spectrometry analysis.To separate and measure Lespedeza cuneata compounds, the GC-MS instrument was outftted with a 30 m × 0.25 mm (i.d.DB-5MS) chromatography column and an Agilent 5975C mass selective detector.At 250 °C, the extract was injected.Te source and transfer lines had temperatures of 230 °C and 280 °C, respectively.Te column temperature was initially set at 70 °C for 1 min and then increased to 300 °C at a rate of 5 °C/min, remaining at this ultimate temperature for 30 min.Scan and electron ionization modes were used to obtain mass spectrometry data.

Figure 2 :Figure 3 :
Figure 2: Chemical structures of the single compounds present in the Lespedeza cuneata.

Figure 4 :Figure 5 :Figure 6 :Figure 7 :
Figure 4: Efects of L. cuneata on agonist-induced platelet aggregation and changes in the platelet shape.(a) Agonists, such as collagen, ADP, and thrombin, were preincubated with washed platelets for 1 min in a light transmission aggregometer in the presence of 1 mM calcium chloride (CaCl 2 ).Tis has been followed by stimulation for 5 min with constant stirring at 37 °C using various agonists.* p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with agonist.(b) Te shape change and aggregation of platelets were evaluated using scanning electron microscopy.

Figure 8 :
Figure8: Western blot analysis to assess protein phosphorylation under Lespedeza cuneata stimulation.Various quantities of L. cuneata extracts with 1 mM CaCl 2 were preincubated using washed platelets for 1 min at 37 °C before collagen stimulation for 5 min with constant stirring.By adding a lysis bufer, platelet aggregation stopped, and the protein concentration was calculated using the BCS assay (PRO-MEASURE; iNtRON Biotechnology).In a 10% SDS-PAGE, total platelet proteins were isolated, and then they were transferred to PVDF membranes.Membranes were blocked with 5% skim milk, probed with the appropriate antibodies (phospho-ERK, phospho-JNK, phospho-p38MAPK, phospho-PI3K, phospho-Akt, etc.), and then observed using enhanced chemiluminescence.* p < 0.05, * * p < 0.01, and * * * p < 0.001 compared with the collagen.

Figure 9 :Figure 10 :
Figure9: L. cuneata prevents thrombosis and regulates hemostasis.To evaluate FeCl 3 -induced thrombus formation and the tail bleeding assay, four treatment groups were treated orally for 7 days (saline, ASA, or a low and high dose of the 50% EtOH extract of L. cuneata) using C57BL/6J and ICR mice, respectively.Mice were anesthetized, and FeCl 3 -induced thrombus formation and the tail bleeding assay were carried out almost 1 h after the last treatment.* * * p < 0.001 compared with the agonist.

Table 1 :
GC-MS analysis of major compounds present in the 50% EtOH L. cuneata extract.

Table 1 )
. Te structure of active compounds present in the extract is shown in Figure2.