Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets

A large variety of dietary phytochemicals have been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on platelet activity and function remains unclear. In this study, we evaluated phenotypes associated with irreversible protein engagement of twenty-three electrophilic phytochemicals. This revealed a novel antiplatelet selectivity profile of the isothiocyanate-containing natural product sulforaphane (SFN), whereby the response of platelets to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist was impaired without affecting thrombin and collagen-related peptide (CRP) activation. SFN also substantially reduces the formation of platelet thrombi on surfaces coated with collagen under arterial flow conditions. Activity-based protein profiling identified protein disulfide isomerase A6 (PDIA6) as a rapid kinetic responder of SFN. Mechanistic profiling studies revealed how SFN fine-tunes the enzymatic activity and substrate specificity of PDIA6. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, with a view to develop more effective and safer adjunctive treatments to improve the clot-busting power of rtPA – currently the sole approved therapeutic for stroke recanalization that has significant limitations.