Study On The Inhibitory Role Of Plant Polyphenol Tannic Acid In Protein Disulfide Isomerase Activity And Thrombus Formation

Cardiovascular disease has become one of the main causes that threaten human health in the modern society.With the changes in lifestyle and environmental factors,the incidence and mortality of cardiovascular disease(CVD)in China remain high.Surpassing malignant tumors,CVD has become the leading cause of death.Platelets play an essential role in the formation of thrombus,a key event contributing to the deterioration of CVD.Under conditions such as the rupture of atherosclerotic plaques,platelets adhere to the inner wall of the damaged artery through their membrane surface receptors,and further activate and recruit platelets,as well as other blood cells from the circulating blood by secreted contents of their granules.Activated platelets bind to fibrinogen through integrin ??b?3 and promote the activation of the coagulation pathway,leading to the formation of an occlusive thrombus and causing ischemic diseases including acute myocardial infarction and stroke.Platelet activation plays a role in promoting coagulation and is also involved in the formation of venous thrombosis,contributing to the development of cerebral embolism,pulmonary embolism,and tumor-related venous thrombosis.Based on the mechanistic understanding of platelet activation,various types of antiplatelet drugs have been developed for the prevention and treatment of thrombosis.Although available antiplatelet drugs may facilitate the reduction of thrombotic events,there are still drawbacks such as the risk of bleeding and heterogeneities of effect among individuals.At present,the pursuit for safe and effective antiplatelet candidates is still intriguing and an important direction in the field of cardiovascular research.Protein disulfide isomerase(PDI)is expressed in platelets and released to the surface of cell membranes when platelets are activated.PDI binds to integrin ??b?3 and catalyzes the cleavage of disulfide bonds through its reductase activity,leading to the conformational activation of integrin ??b?3.PDI promotes platelet activation and thrombosis,and also plays a role in the activation of coagulation factors and coagulation pathways.Regarding the role of extracellular PDI as a "master switch" in thrombus formation,inhibition of PDI is expected to provide a promising strategy to combat thrombosis.Epidemiological studies have shown that diets rich in natural plant polyphenols may yield cardiovascular benefits.A large number of studies have shown that plant polyphenol extracts have anti-platelet activities,but the underlying molecules and mechanisms remain largely unclear.Recent studies have shown that plant polyphenols constitute an important source of natural PDI inhibitors.In order to explore potential antiplatelet drugs in plant polyphenols,we selected dietary polyphenol compounds with potential cardiovascular benefits to construct a molecular library of plant polyphenols.Through molecular docking simulation using this library,we screened for potential antiplatelet drugs that inhibit PDI activity.The results showed that tannic acid(TA)exhibited the highest potential to bind PDI in all screened compounds.TA has a long history in pharmaceutical application in Chinese traditional medicine.It shows antioxidant,anti-apoptotic,and anti-inflammatory activities,but the effect of TA on PDI activity and platelet activation is unknown.In this study,we determined the effects of TA on PDI activity and PDI function in platelets,and studied the inhibitory role of TA in platelet activation and thrombus formation.Aim:To identify anti-platelet natural compounds with PDI inhibitory activity from plant polyphenols based on molecular docking.Methods:1.Screening plant polyphenols with PDI inhibitory activity and the effect of TA on PDI activity.(1)Molecular docking screening and simulation:In order to find an anti-platelet compound with PDI inhibitory activity from plant polyphenols,we first selected dietary polyphenol compounds with potential cardiovascular benefits to construct a compound library,and obtained the structure of each compound from the PubChem database.Human PDI molecular structure of different redox states(reduced state:4EKZ,oxidized state:4EL1)were obtained from the protein database bank(PDB).The high-throughput network molecular docking platform systemsDock(http://systemsdock.unit.oist.jp/iddp/home/index)was used to analyze the binding ability of plant polyphenols to PDI molecules,yielding TA as the most strongly bound molecule by the rank of binding scores,as well as to estimate the binding site of TA on PDI surface.After obtaining the binding site,we further analyzed the pattern of the binding between TA and PDI using AutoDock Vina software,and explored the spatial conformation,amino acid residue,and intermolecular distance of the binding between TA and PDI.(2)Binding of TA and PDI molecules in vitro:We used a Biacore T200 system to analyze the binding ability and mode of liquid phase TA to immobilized PDI protein by surface plasmon resonance assay.(3)PDI reductase activity:We incubated recombinant PDI in vitro with TA or vehicle,respectively,and then detected the ability of PDI to cleave the fluorescent substrate Di-Eosin-Glutathione disulfide(Di-E-GSSG).The increase in the fluorescence intensity of the eosin after the cleavage of the disulfide bond was detected using a spectrophotometer,which reflected PDI reductase activity.2.Assessing the effect of TA on PDI function on platelet surface.(1)Free thiol formation on the surface of platelets:PDI released upon platelet activation reduces the disulfide bond in protein substrates on cell membrane and generates free thiols.The free thiols can be labeled using 3-(N-Maleimidopropionyl)-biocytin(MPB).We labeled human platelets treated with TA or vehicle,stimulated platelets with thrombin,neutralized remaining MPB with glutathione,and detected the surface level of free thiols using fluorescein-crosslinked avidin immunoblotting.(2)Binding of PDI to platelet surface integrin ??b?3:PDI released during platelet activation binds to integrin ??b?3 on platelet surface and regulates platelet activation.We incubated human platelets with Alexa Fluor-488 fluorescein-conjugated recombinant PDI in vitro,pretreated the platelets with TA or vehicle before adding manganese(8mM)to promote the conversion of integrin ??b?3 to an open conformation.Flow cytometry was used to assess the fluorescence intensity to evaluate the level of PDI bound to platelet surface.3.Studying the effects of TA on platelet activation.(1)Platelet aggregation:Gel-filtered human platelets were pretreatment with TA or vehicle before stimulation by platelet agonists including collagen and thrombin.Platelet aggregation was measured on a CHRONO-LOG platelet aggregometer with magnetic stirring for 5 min.Light transmittance-based aggregation curve was recorded to evaluate the changes in platelet aggregation.(2)Integrin ??b?3 activation:PDI mediates platelet activation and thrombus formation by promoting the open conformation change of integrin ??b?3.We pretreated human or mouse platelets with TA or vehicle before stimulation with thrombin or collagen-related peptides(CRP).Fluorescent-conjugated soluble human fibrinogen or an anti-activated mouse ??b?3 antibody(JON/A)were used to label activated integrin ??b?3 on platelet surface.The level of activated integrin ??b?3 was analyzed by flow cytometry.(3)Platelet alpha granule secretion:Human platelets were isolated and pretreated with TA or vehicle for 10min in vitro,and the expression of P-selectin on platelet surface was measured by flow cytometry to determine the effect of TA on platelet alpha granule secretion.(4)The effect of TA on platelet reactive oxygen species(ROS)production:Human platelets were pretreated with TA or vehicle and DCFDA was used to label ROS in platelets before stimulation with CRP.DCF fluorescence intensity was measured by flow cytometry to reflect the ROS levels in platelets.(5)Platelet spreading:High-affinity integrin ??b?3 binds to ligands to activate downstream cellular kinases,calcium signaling,and platelet cytoskeletal changes.Isolated human platelets were pretreated with TA or vehicle and allowed to spread on fibrinogen-coated surface.Spreading areas were calculated to reflect the early "outside-in" signal.(6)Clot retraction:Isolated human platelets were with TA or vehicle before the addition of thrombin to induce clot formation.The area of forming clot were observed continuously to evaluate the effect of TA on platelet "outside-in" signal.4.Evaluating the effects of TA on arteriolar thrombosis.(1)Laser-induced cremaster arteriolar thrombosis:C57BL/6 mice were intraperitoneally injected with TA(5mg/kg)or vehicle and allowed to circulate for 30min before isolation of cremaster muscle arterioles after anesthesia.3,3'-dihexyloxacarbocyanine iodide(DIOC6)was injected through the jugular vein to label blood cells.An intravital microscope was used to observe the target vessel,which was then injured by pulsed laser to induce acute thrombosis.The dynamic process of thrombus formation was recorded under the intravital microscope,and the total and peak areas of thrombus were analyzed.(2)Mouse tail bleeding time:Mice were pretreated with TA(5mg/kg)or vehicle via intraperitoneal injection(i.p.)and allowed to circulate for 45min.5mm of mouse tail was then cut off after anesthesia,and the proximal truncated site was immediately immersed in normal saline at 37?.The time from initial bleeding to the first bleeding cessation was recorded as tail bleeding time.(3)Jugular vein bleeding time:Mice were pretreated with TA(5mg/kg)or vehicle via intraperitoneal injection and allowed to circulate for 30min.The jugular vein was exposed after anesthesia and pierced at one side using a 28G needle tip under a stereo microscope.The time from initial bleeding to the first bleeding cessation was recorded as jugular vein bleeding time.5.Evaluating the potential toxicity and drug likeness of TA.(1)Effect of TA on platelet viability:Inhibiting intracellular PDI may lead to endoplasmic reticulum stress,increase of ROS,cell injury,and even cell death.To explore whether TA may be toxic to platelets in this way,we used Alamar Blue reagent to detect changes in mitochondrial function in human platelets after TA or vehicle pretreatment,which reflected platelet viability.(2)Effect of TA on platelet apoptosis:The phosphatidylserine on the surface of platelet membrane was detected by fluorescein isothiocyanate(FITC)-conjugated Annexin V.The fluorescence intensity of platelets was measured by flow cytometry to evaluate the effect of TA on platelet apoptosis.(3)Effect of TA on the pH value of buffer:The pH values of different concentrations of TA-Tyrode's buffer were measured using a Sartorius benchtop pH meter and compared with Tyrode's buffer.(4)Drug likeness of TA:The druglikeness of TA was evaluated using the S WISSADME platform(http://swissadme.ch/index.php).Results:1.TA interacts with PDI and inhibits PDI reductase activity.(1)Interaction between TA and PDI:The molecular docking of 42 dietary polyphenolic compounds with human PDI protein showed that TA had the highest binding score in all compounds docked with PDI at different redox status.Binding simulation showed that TA binds PDI near its active site in the a and a' domains.Further analysis of the binding between TA and its binding site on PDI by AutoDock Vina software revealed that TA forms two hydrogen bonds with the Cys400 residue in the a' domain of the PDI molecule,with distances of 1.492A and 2.797A,respectively.(2)In vitro binding of TA to PDI protein:SPR results showed that liquid phase TA interacted with immobilized PDI with high affinity.(3)TA inhibits the reductase activity of PDI:Results of the Di-E-GSSG assay showed that TA(10?M)significantly inhibited PDI reductase activity compared with vehicle.2.TA inhibits PDI function on platelet surface.(1)TA inhibits PDI reductase function on platelet surface:When platelets are activated,PDI is released to the surface of the cell membrane and reduces its protein substrates on cell membrane,catalyzing disulfide bond cleavage to form a free thiol.Labeling of free thiols on platelets surface by MPB showed that TA significantly inhibited thrombin-induced platelet surface free thiol formation compared to vehicle.(2)TA inhibits the binding of PDI to platelet integrin ??b?3:Released PDI binds to the integrin ??b?3 to dock on platelet surface and regulates platelet activation.Flow cytometry results showed that TA(30?M)significantly inhibited the binding of recombinant PDI to Mn2+-stimulated integrin ??b?3 on platelet surface.3.TA inhibits platelet activation.(1)TA inhibits platelet aggregation induced by different agonists:Gel-filtered human platelets were incubated with TA or vehicle before stimulation by different agonists.The results showed that TA inhibited collagen(2?g/mL)-stimulated platelet aggregation in a dose-dependent manner compared with vehicle,with an IC50 of 34.2?M.TA also inhibited thrombin(0.05U/mL)-induced platelet aggregation.(2)TA inhibits platelet integrin ??b?3 activation:Activation of platelets transduces intracellular signals to integrin ??b?3,eliciting its conformational change and full activation under the effect of extracellular PDI.Flow cytometry results showed that TA(50?M)pretreatment significantly reduced the activation of human platelet surface integrin ??b?3 induced by thrombin(0.1 U/mL)or CRP(1?g/mL)compared to vehicle.The results in mouse platelets also confirmed that TA(30?M,50?M)had an inhibitory effect on the activation of platelet surface integrin ??b?3.(3)TA inhibits platelet P-selectin expression:Alpha granules released by activated platelets can be analyzed according to the level of P-selectin on platelet surface.Flow cytometric analysis showed that TA(10?M,30?M,50?M)pretreatment significantly inhibited CRP(1?g/mL)-or thrombin(0.1U/mL)-induced platelet surface P-selectin expression compared to vehicle.(4)TA inhibits platelet oxidative stress:During the activation of platelets,ROS are produced to promote platelet activation.Flow cytometry showed that TA(30?M,50?M),as a natural antioxidant,significantly inhibited CRP(1?g/mL)-induced upregulation of platelet ROS levels.(5)TA inhibits the spreading of platelets on immobilized fibrinogen:Integrin ??b?3 binds to fibrinogen and promotes platelet activation,causing cytoskeletal changes.The results of platelet spreading experiments showed that the spreading area of platelets pretreated with TA(30?M)on fibrinogen surface was significantly smaller than that of the control group.(6)TA inhibits clot retraction:platelet "outside-in" signals transduced by integrin ??b?3 can be assessed by blood clot retraction as the end stage of platelet activation.The results of the experiment showed that TA(30?M)significantly inhibited thrombin(1U/mL)-induced blood clot retraction compared to vehicle.4.TA inhibits mouse arteriolar thrombus formation and does not prolong bleeding time.(1)TA inhibits cremaster arteriolar thrombus formation in mice:Real-time quantitative analysis of arterial thrombosis by intravital microscopy showed that TA(5mg/kg,i.p.)inhibited mouse cremaster arteriolar thrombus formation induced by laser injury compared with vehicle.Quantitative analysis showed that the total area of thrombus in the TA group was significantly lower than that in the control group.(2)TA does not prolong the bleeding time of mice:We measured the tail bleeding and jugular vein bleeding time of mice to evaluate the effect of TA on physiological hemostasis.The results showed that the tail bleeding time and jugular vein bleeding time in the TA(5mg/kg,i.p.)group were not significantly prolonged compared with the control group.5.TA has no toxic effects on platelets.(1)TA does not affect platelet activity:Alamar Blue was used to detect mitochondrial function to assess cell viability.Analysis of Alamar Blue fluorescence by a spectrophotometer showed that there was no significant decrease in human platelet activity after 4 hours of incubation with different concentrations of TA(10?M,30?M,50?M,10?M)compared to vehicle.(2)TA does not induce platelet apoptosis:Increased PS on platelet surface upon apoptosis was quantified with Annexin V.Flow cytometry experiments showed that TA(30?M,50?M,100?M)did not increase platelet Annexin V levels compared to vehicle.(3)TA does not affect the pH value of buffer:The pH values of the TA-Tyrode's buffer(10,30,50,100?M)solution were not significantly different from Tyrode's buffer as measured by a pH meter.(4)Druglikeness of TA:Calculation using the SWISSADME platform showed that TA has a bioavailability score of 0.17 and Lipinski score of 3.Conclusion:1.The plant polyphenol TA binds PDI with high affinity.TA docks with the PDI molecule at Cys400.2.TA inhibits PDI activity and the binding of PDI to platelet integrin ??b?3.3.TA inhibits platelet activation and thrombus formation.4.TA has no toxic effect on platelets and shows the potential of becoming a safe antithrombotic agent.