Glucose Transporter 3 in Platelets Facilitates Alpha-Granule Mediated Glucose Uptake, Driving Intragranular Glycolysis That Is Required for Platelet Degranulation and Activation

Patients with diabetes display increased thrombosis and platelet activation. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. In vitro studies of platelets under hyperglycemic conditions suggest that glucose metabolism may lead to increased platelet activation. Platelets import glucose via two glucose transporters GLUT1, which is expressed on the plasma membrane, and GLUT3, which is expressed on the plasma membrane (15%) and the remaining 85% on α-granule membranes.  Following stimulation, platelet α-granules translocate to the plasma membrane and release their cargo. To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. GLUT3 KO platelets displayed a 23% reduction in basal glucose uptake compared to littermate controls. Control platelets stimulated with thrombin displayed a significant increase in glucose uptake whereas KO platelets failed to show any change. Additionally, platelet glycogen content and glycolysis intermediates were significantly reduced in KO platelets, which exhibited reduced glycolytic rates following metabolic stress (mitochondrial uncoupling). Because GLUT3 KO platelets had only a minor decrease in glucose uptake under basal conditions, but platelet glucose metabolism was dramatically altered when stimulated, we hypothesized that under basal conditions, GLUT3 facilitates glucose uptake into α-granules, to generate glycogen and fuel intragranular glycolysis that generates the energy required for α-granule degranulation. To test this hypothesis we permeabilized platelet plasma membranes, but not α-granule membranes using saponin and incubated the platelets with C¹³-glucose.  Under these conditions, control platelets produced 2.5-fold more C¹³-lactic acid than KO platelets. In vitro, GLUT3 knockout platelets display a 90% reduction in spreading on fibrinogen and collagen matrices and significant reductions in α-granule degranulation as marked by CD62p surface translocation, platelet factor 4 release, and the persistence of α-granules observed in electron micrographs of stimulated platelets. In vivo in a KBx/N model of rheumatoid arthritis, which is dependent in part on platelet activation, GLUT3 KO mice exhibited significantly reduced severity of disease. Analysis of GLUT3 mice in models of arterial thrombosis, deep vein thrombosis and tail-bleeding indicated no alteration in thrombosis between littermate controls and KO mice. Together these data indicate that GLUT3 mediated α-granule glucose uptake is essential for platelet activation and degranulation. Moreover reducing platelet GLUT3 may ameliorate the course of rheumatoid arthritis.