Linking triphenylphosphonium cation to a bicyclic hydroquinone improves their antiplatelet effect via the regulation of mitochondrial function.

Platelets are the critical target for preventing and treating pathological thrombus formation. However, despite current antiplatelet therapy, cardiovascular mortality remains high, and cardiovascular events continue in prescribed patients. In this study, first results were obtained with ortho-carbonyl hydroquinones as antiplatelet agents; we found that linking triphenylphosphonium cation to a bicyclic ortho-carbonyl hydroquinone moiety by a short alkyl chain significantly improved their antiplatelet effect by affecting the mitochondrial functioning. The mechanism of action involves uncoupling OXPHOS, which leads to an increase in mitochondrial ROS production and a decrease in the mitochondrial membrane potential and OCR. This alteration disrupts the energy production by mitochondrial function necessary for the platelet activation process. These effects are responsive to the complete structure of the compounds and not to isolated parts of the compounds tested. The results obtained in this research can be used as the basis for developing new antiplatelet agents that target mitochondria.

Assessing mitochondria-targeted acyl hydroquinones on the mitochondrial platelet function and cytotoxic activity: Role of the linker length.

INTRODUCTION: The use of triphenylphosphonium cation (TPP+) linked to phenolic compounds by alkyl chains has a significant relevance as a mitochondrial delivery strategy in biomedicine because it affects mitochondrial bioenergetics in models of noncommunicable diseases such as cancer and cardiovascular-related conditions. Studies indicate that a long alkyl chain (10-12 carbon) increases the mitochondrial accumulation of TPP+-linked drugs. In contrast, other studies show that these compounds are consistently toxic to micromolar concentrations (as observed in platelets). In the present study, we evaluated the in vitro effect of three series of triphenylphosphonium-linked acyl hydroquinones derivates on the metabolism and function of human platelets using 3-9 carbons for the alkyl linker. Those were assessed to determine the role of the length of the alkyl chain linker on platelet toxicity. METHODS: Human platelets were exposed in vitro to different concentrations (2-40 µM) of every compound; cellular viability, phosphatidylserine exposition, mitochondrial membrane potential (ΔΨm), intracellular calcium release, and intracellular ROS generation were assessed by flow cytometry. An in silico energetic profile was generated with Umbrella sampling molecular dynamics (MD). RESULTS AND CONCLUSIONS: There was an increase in cytotoxic activity directly related to the length of the acyl chain and lipophilicity, as seen by three techniques, and this was consistent with a decrease in ΔΨm. The in silico energetic profiles point out that the permeability of the mitochondrial membrane may be involved in the cytotoxicity of phosphonium salts. This information may be relevant for the design of new TPP+ -based drugs with a safe cardiovascular profile.

In Vitro Effect of Mitochondria-Targeted Triphenylphosphonium-Based Compounds (Honokiol, Lonidamine, and Atovaquone) on the Platelet Function and Cytotoxic Activity.

Introduction: Obtaining triphenylphosphonium salts derived from anticancer compounds to inhibit mitochondrial metabolism is of major interest due to their pivotal role in reactive oxygen species (ROS) production, calcium homeostasis, apoptosis, and cell proliferation. However, the use of this type of antitumor compound presents a risk of bleeding since the platelet activation is especially dependent on the mitochondrial function. In this study, we evaluated the in vitro effect of three triphenylphosphonium-based compounds, honokiol (HNK), lonidamine (LDN), and atovaquone (ATO), on the platelet function linked to the triphenylphosphonium cation by a lineal 10-carbon alkyl chain and also the decyltriphenylphosphonium salt (decylphos). Methods: Platelets obtained by phlebotomy from healthy donors were exposed in vitro to different concentrations (0.1-10 µM) of the three compounds; cellular viability, exposure of phosphatidylserine, the mitochondrial membrane potential (∆Ψm), intracellular calcium release, and intracellular ROS generation were measured. Platelet activation and aggregation were induced by agonists (adenosine diphosphate, thrombin receptor-activating peptide-6, convulxin, or phorbol-12-myristate-13-acetate) and were evaluated by flow cytometry and light transmission, respectively. Results: The three compounds showed a slight cytotoxic effect from 1 µM, and this was concomitant with a decrease in ∆Ψm and intracellular calcium increase. Only ATO produced a modest but significant increase in intra-platelet ROS. Also, the three compounds increased the exposure to phosphatidylserine in platelets expressed in platelets positive for annexin V. None of the compounds had an inhibitory effect on the aggregation or activation markers of platelets stimulated with three different agonists. Similar results were obtained with decylphos. Conclusion: Triphenylphosphonium derivatives showed slight platelet toxicity below 1 µM, probably associated with their effect on ∆Ψm and exposure to phosphatidylserine, but no significant effect on platelet activation and aggregation, making them an antitumoral alternative with a low risk of bleeding. However, future assays on animal models and human trials are required to evaluate if their effects with a low risk for hemostasis are replicated in vivo.