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

A large variety of
dietary phytochemicals has 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 the platelet activity and function remains unclear. This study
explores the irreversible engagement of 23 electrophilic phytochemicals
with platelets, unveiling the unique antiplatelet selectivity of sulforaphane
(SFN). SFN impairs platelet responses to adenosine diphosphate (ADP)
and a thromboxane A2 receptor agonist while not affecting thrombin
and collagen-related peptide activation. It also substantially reduces
platelet thrombus formation under arterial flow conditions. Using
an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6)
was identified as a rapid kinetic responder to SFN. Mechanistic profiling
studies revealed SFN’s nuanced modulation of PDIA6 activity
and substrate specificity. 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, aiming to enhance the clot-busting
power of rtPA, currently the only approved therapeutic for stroke
recanalization that has significant limitations.