Thrombin stimulates glucose transport in human platelets via the translocation of the glucose transporter GLUT-3 from alpha-granules to the cell surface

Abstract

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most alpha-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the alpha-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant alpha-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of alpha-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in alpha-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (approximately 15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.

Figure 1

Glucose transport activity in human platelets. Isolated platelets were either nonstimulated or stimulated with 10 μM ADP or 0.05, 0.2, and 0.5 U/ml thrombin, respectively. The [¹⁴C]2-DG uptake was measured as described in Materials and Methods. The increase in 2- DG uptake upon stimulation is expressed relative to the basal 2-DG values of nonstimulated platelets. The values represent the mean ± SEM of three separate experiments.

Figure 2

Ultrastructural localization of GLUT-3 in human platelets. Frozen thin-sections of resting platelets immediately fixed in whole blood were immunolabeled with an antibody specific for GLUT-3, followed by protein A conjugated to 10 nm gold. The platelets still have their discoid shape. The majority of the label is associated with the limiting membrane of the α-granules. Note that the gold particles are often situated at the cytoplasmic face of the membrane, and occasionally in a grazing section with the tip of an α-granule membrane (arrow). Some label is also associated with the plasma membrane (arrowheads). (Stars) OCS. Bar, 200 nm.

Figure 3

Distribution of GLUT-3 in activated platelets. Activation with both 0.2 U/ml thrombin (a) and 0.5 U/ml thrombin (b) results in a significant loss of the α-granules and a remarkable shape change to a ruffled cell surface. Some platelets had retained α-granules, a finding which was more often encountered at the lower dose of thrombin (a). The translocation of GLUT-3 to the OCS (stars) and to the cell surface is obvious, particularly to the pseudopods. (b, arrowheads). Bar, 250 nm.

Figure 4

GLUT-3 expression after stimulation with 10 μM ADP. The shape changes characteristic for ADP stimulation are observed (i.e., centralization of α-granules and some cell surface ruffling ). A similar morphology is observed when the low dose of 0.05 U/ml thrombin is used (not shown). The majority of the label is still associated with the membranes of the α-granules. Some label is observed at developing pseudopods. Bar, 200 nm.