Punicalagin, a polyphenolic compound isolated from pomegranate fruit, possess several pharmacological activities including anti-inflammatory, hepatoprotective, antigenotoxic and anticoagulant activities [13]. PDIA3/ERp57, a member of the protein disulfide isomerase family, is an endoplasmatic reticulum protein mainly involved in the correct folding of newly synthetized glycoproteins. It is associated with different human diseases such as cancer, prion disorders, Alzheimer¿s and Parkinson¿s diseases [2]. PDIA3 plays an important role during platelets activation. It is secreted upon Glycoprotein VI activation and after vascular injury, extracellular PDIA3 is accumulated in the thrombus where it induces the recruitment of other platelets. It seems PDIA3 bind ß3 integrin in thrombin-activated platelets. ß3 integrins create active fibrinogen receptor (¿IIbß3) on the surface of platelets, and this needs several conformational changes that require a new pattern of disulfide bond formation [9]. The anti-PDIA3 antibody inhibits platelets aggregation and ATP secretion, calcium mobilization and activation of ¿IIbß3 in platelets stimulated by collagen-related peptide. PDIA3-null platelets reveal decreased platelets aggregation and decreased activation of ¿IIbß3 [10]. The current research is focusing on PDIA3 inhibition in platelets aggregation as a target for seeking novel anti-thrombotic agents [5a]. In this regard, our preliminary data demonstrated that punicalagin is able to bind PDIA3 with a Kd of 8,3 µM. Moreover, punicalagin can inhibit PDIA3 redox activity up to 90%. So, I would to fix the PDIA3 involvement in platelets aggregation (defining redox status of PDIA3 during ¿IIbß3 activation and PDIA3 ability to induce ß3 activation) and test punicalagin as a possible PDIA3 inhibitor (determining Kd by ITC, Km by kinetic assay, its relative affinity to PDIA3 respect other PDIs and capability to inhibit platelets aggregation).
The PDIA3 involvement in platelet aggregation is well demonstrated but the mechanism is not completely understood. Then, the goal of this research is to better understand the PDIA3 role in platelet aggregation. In the last two decades, many groups focus the attention on the development of new antiaggregatory drugs in order to reduce the side effects of older drugs. In fact, ASA (acetylsalicylic acid) is still the well-chosen drug for coagulation but it has a lot of side effects, such as increasing bleeding and gastritis due to its lower specificity for COX-1. Many pathways, beyond COX-1, are involved in platelets activation for example the ADP or thromboxane A2 release, as well as several drugs were developed. Also in these cases, the lower affinity and specificity induces many collateral effects. Last but not least a new discovered target is the integrin ¿IIbß3 (fibrinogen receptor) and it could be a more specific target for platelet aggregation respect the COX-1 or thromboxane. ¿IIbß3 activation induces exposition of a fibrinogen and von Willebrand Factor binding domain that facilitates inter-platelet binding. There are three types of ¿IIbß3 antagonists which include monoclonal antibodies or fragments, cyclic peptides based on the von Willebrand Factor binding domain (RGD) motif, and peptidomimetic. These drugs act as fibrinogen or von Willebrand Factor antagonists. Unfortunately, these antagonists did not show minor side effects than ASA. So, the innovation of this work could be a preliminary screening of a new hypothetical ¿IIbß3 modulator that does not mimic its substrates but that can modulate the starting ¿IIbß3 activation. Since PDIA3 seems involved in ¿IIbß3 activation, PDIA3 inhibitors could be eligible candidates to regulate aggregation of the platelets. In this regard, a better knowledge of PDIA3 role in platelet aggregation becomes essential. Sequentially, the evaluation of punicalagin effects on platelet aggregation could be the first step to obtain preliminary information on its activity as a new anti-thrombotic agent for modulation of protein disulfide isomerase and its possible specificity versus PDIA3. Our research group studies the protein PDIA3 since many years then, we have the knowledge and instruments to investigate on PDIA3 in this biological process. Thanks, the collaboration with Professor Madlenka, through which I have the platelet model available, we can seek the PDIA3 involvement in platelet aggregation. Moreover, the basis for this work is also reflected in the fact that our preliminary data demonstrated that punicalagin has a dissociation constant for PDIA3 in micromolar range (8,3 µM). Moreover, I tested this substance on the neuroblastoma cellular model using wild-type (SHSY-5Y) and PDIA3-knockdown cells (SHSY-5Y ShPDIA3). After a 4-hours pre-treatment with 20 uM punicalagin and a 24 hours H2O2 treatment (0,1 mM, 0,2 mM and 0,5 mM), data showed that in SHSY-5Y the punicalagin pre-treatment increased the cellular death respect to the H2O2-only treatment. Conversely, the punicalagin pre-treatment had no effects in SHSY-5Y ShPDIA3. In this case, the SHSY-5Y ShPDIA3 died in the same manner of SHSY-5Y after H2O2-only treatment. Hence, it seems clear that the cellular death in neuroblastoma cells is correlated to PDIA3 activity and that punicalagin can modulate it. So, in this regard, I would to characterize punicalagin biological effects and investigate about the contribution of PDIA3 in platelet aggregation, testing punicalagin as a possible PDIA3 inhibitor.