Hexokinase controls platelet activation and hemostasis

Platelet activation is energy-dependent and associated with a hyperglycolytic phenotype, yet the role of key glycolytic enzymes remains unclear. We investigated the role of platelet hexokinases (HK1 and HK2) in regulating thrombin- and convulxin-stimulated bioenergetics, functions, and hemostasis using pharmacological inhibitors 2-deoxyglucose (2DG; pan-HK) and 3-bromopyruvate (3BP; HK2-preferential). Platelet activation increased HK activity, with convulxin producing a stronger response than thrombin. Using 2DG and 3BP, we found that activation-induced glucose uptake, glycolytic ATP production, and glycolysis were dependent on both HK isoforms. Platelet aggregation was reduced but not abolished by HK inhibition, indicating that platelet function is underpinned by metabolic flexibility. Integrin activation and pro-coagulant platelet formation were suppressed by both inhibitors. In contrast, α-granule secretion markers (CD62P, CD40L), platelet spreading, and convulxin-induced reactive oxygen species (ROS) generation were more sensitive to 3BP, suggesting a prominent role for HK2 in secretion, outside-in signaling, and ROS regulation. In vitro thrombus formation over collagen was significantly reduced by both inhibitors. In addition, HKs play a pivotal role in the regulation of in vivo hemostasis with increased bleeding time in mice treated with HK inhibitor. These findings identify HK as a central regulator of platelet metabolism and function, with distinct contributions of HK isoforms to specific platelet responses.