Investigation of the Effect of Synthetic Nanoparticles and Endogenous Tumor Cell-Derived Extracellular Vesicles on Platelet Function and Coagulation in vitro.
Open Access DepositedPlatelets are highly sensitive and reactive cells. They play a critical role in maintaining hemostasis and are the first responders to arrest bleeding during an injury. Therefore, any aberration in their function can have severe consequences resulting in bleeding or occlusion of blood vessels due to thrombus formation. Synthetic or endogenous nanoparticles (NPs) when present in the bloodstream can interact with the different components of blood and have been shown to modulate platelet functions. However, these interactions are not well understood. Here, we aim to understand the influences of the nanoscale entities on normal platelet function as well as in context with cancer and delineate the underlying mechanism of interaction. We first studied the effects of the iron-oxide nanoparticles as our synthetic model, as they are being developed for several biomedical applications. Specifically, we focused on the Superparamagnetic Iron Oxide Nanoparticles (SPIONs) coated with a biocompatible polymer, polyvinyl alcohol (PVA), on normal platelet function and plasma coagulation, ex vivo. We studied the effect of 6 formulations of SPIONs exhibiting different surface charges and polymer packing. We observed that only the positively charged SPIONs induced a significant change in platelet GPIIb-IIIa conformation, indicating increased platelet activation at the dose of 500 ug/mL. Whereas all other formulations, irrespective of the PVA charge and molecular weight, induced a dose-dependent inhibitory effect on platelet function of aggregation. After characterizing the effects of the SPIONs on platelet function we assessed multiple hypotheses to understand the mechanism by which SPIONs induced the antiplatelet effect. We concluded that the presence of PVA-SPIONs induced conformational changes in the plasma protein fibrinogen which is essential for bridging platelets during platelet aggregation resulting in the observed dose-dependent anti-platelet effect. Next, we studied the effects of tumor cell-derived extracellular vesicles (EVs) on platelet function and plasma coagulation. Tumor cell-derived EVs circulating in the bloodstream have been implicated as the causative agents in the occurrence of cancer-associated thrombosis, as they may interact with platelets or trigger the coagulation cascade, leading to clot formation. Since the interaction of EVs with the hemostatic components is not well characterized, here we aim to determine the mechanistic aspects of their interaction with platelets and their role in initiating coagulation. Given the wide variation of protein and integrin expression between different cancer cell types, we selected three cancer cell lines; lung (A549), breast (MDA-MB-231), and glioblastoma (LN18) which are known to have a high risk of cancer-associated thrombosis. We isolated and characterized the EVs in terms of size, zeta potential, and key surface markers. Phosphatidylserine was detected on the surface of EVs isolated from all three cell lines whereas tissue factor (TF) was detected only on the surface of the MDA-MB-231 and LN18 EVs. We observed that the effect of the tumor cell-derived EVs on platelet functions varied based on the cell line, the concentration, and incubation time with platelets. Our data shows that both A549 and MDA-MB-231 EVs at 50 ug/mL significantly increase platelet aggregation as compared to control when incubated with the platelets for 10 min in the presence of an agonist (20 uM ADP). Whereas LN18 EVs significantly induced platelet aggregation only after 1 h incubation with platelets in the presence of 20 uM ADP. Further, MDA-MB-231 and LN18 EVs generated a significant amount of thrombin as compared to control with LN18 EVs generating the highest amount of thrombin when incubated in platelet-poor plasma for 1 h. The EVs also had a significant effect on the coagulation cascade. A549 and MDA-MB-231 EV’s reduced coagulation time via the extrinsic coagulation pathway. In contrast, LN18 EVs reduced coagulation time via the intrinsic coagulation pathway. To elucidate the role of phosphatidylserine and tissue factor (TF) in modulating the common pathway of coagulation we blocked phosphatidylserine and TF using lactadherin and anti-TF antibody respectively. The thrombin generation potential of the MDA-MB-231 and LN18 EVs was significantly reduced when blocked with both lactadherin and the anti-TF antibody individually, as well as concomitantly. To further elucidate the mechanism by which the EVs modulate platelet function future studies will be conducted to understand the role of different ligands/integrins present on the surface of EVs in enhancing platelet aggregation. To further understand the interaction of cancer cell-derived EVs with the hemostasis system, we carried on and present a preliminary study focusing on the impact of platelets on the thrombogenicity of cancer cell-derived EVs using a co-culture model.
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