Proteomic Analysis Of Inhibitory Effects Of Curdione On Thrombin-induced Platelet Activation

Thrombosis is the formation of blood clots or deposits on the surface of the blood vessels or the inner lining of the heart.In the case of physiology,when the endothelial cell wall damage,platelets attached,resulting in platelet activation,causing platelet aggregation,can play a hemostatic effect and repair the site of injury.Platelets are not only helpful for haemostasis and thrombus but also crucial to abnormal bleeding and thrombotic diseases.Pathological thrombus occurs under conditions of excessively activate platelets and is considered to be an important pathological basis of cardiovascular disease.It can be seen that thrombosis is closely related to platelet aggregation.Curdione,isolated from the essential oils of Curcuma aromatica Salisb.,is one of the major sesquiterpene compounds.Our previous study proved that curdione has a significant anticoagulant and antithrombotic effect.Moreover,we also found that curdione resulted in the obvious attenuation of thrombin-induced platelet aggregation;however,the underlying mechanism was not fully elucidated.As a high throughput screening technology,proteomics has been widely used in the search for drug target proteins and protein function.Platelet function depends on protein expression and dynamic changes in post-translational modifications(PTM)in the context of enucleated cells.Platelet proteomics can map the platelet proteome and characterize the protein profiles and PTM to reveal the differences in platelet function.In the present study,we exploited the proteomic approach to investigate the potential targets of curdione on thrombin induced platelet activation.Aim of study: This study was designed to determine the molecular mechanism of curdione action on platelet activation and thus provide more useful information for its anti-platelet therapy.Materials and methods: Platelet proteins extracted from washed human platelets,including normal group(treated with normal saline),thrombin group and curdione group and ultramicrocuts obtained from rat platelets were used for morphological observation of the ?-granule.The three groups were digested and analyzed by nano ESI-LC-MS/MS.For protein acquisition,the data were analysed by Sequest HT search engine,against the Uni Prot human proteome database under the Proteome Discoverer 1.3 software.Proteins were detected at a false discovery rate < 0.05.The following protein identification data were aligned by Chrom Align software.Label-free peptides eluted between 0 and 100 min were conducted by SIEVE 1.3 software.Furthermore,possible mechanisms involved were explored by Ingenuity Pathway Analysis(IPA)Software and validated by western blot experiments.Results: The 100 ?M curdione seemed to inhibit 0.3 U thrombin-induced ?-granule secretions completely.22 differentially proteins between normal group and thrombin group were identified.Compared with the thrombin group,only 2 proteins(Talin1 and ?1-tubulin)were shown significantly down-regulated in the context of curdione treatment.Bioinformatics analysis showed that Talin1 and ?1-tubulin could be involved in the integrin signalling pathway,blood coagulation and cytoskeletal regulation.These results of western blot analysis were consistent with that of the proteomics data.The down-regulation of ?1-tubulin facilitated the decrease in vinculin/Talin1 and attenuated the secretion of?-granule.Conclusion: The results indicated that ?1-tubulin/vinculin/Talin1 signaling pathway might be a new mechanism in the progress of platelet activation.The ?1-tubulin may be the potential target of curdione underlying thrombin induced human platelet inhibition.