Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

Abstract

Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3-38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.

Fig 1. Effect of thrombin (Thr), OxPhos inhibitors and uncouplers in platelet oxygen consumption.

Oxygen consumption in platelet-rich plasma was determined as described under “Material and Methods”. Arrows show Thr (0.5 U/ml), ADP (10 μM); CCCP (2.5 μM), oligomycin (Oligo, 5 μM) or antimycin (Antim, 5 μM) additions. Numbers under the traces indicate the rate of oxygen consumption in nanogram atom oxygen/min/10⁹ platelets. The lines over each trace indicate the change in oxygen consumption before (dashed lines) and after (solid lines) the addition of thrombin. Experimental traces are representative of at least 4 independent measurements.

Fig 2. Effect of thrombin (Thr) in platelet aggregation and mitochondrial function.

(A) Platelet aggregation; (B) platelet oxygen consumption; (C) mitochondrial membrane potentialin the presence of increasing concentrations of thrombin (Thr) as described in “Material and Methods” section. CCCP was added at 2.5 μM. AFU, arbitrary fluorescence units.

Fig 3. Effect of PAR-1 activation or GPIb inhibition on OxPhos stimulation induced by thrombin.

Platelet aggregation (A,B) and oxygen consumption (C,D) were measured in thrombin (Thr) or Trap-6 stimulated platelets. Platelet rich plasma was incubated for 3 min with 1.5 mg/mL heparin (Hep, B,D) at 37°C under constant stirring. Afterwards, 0.5 U/mL thrombin or 22 μM Trap-6 was added as indicated by arrows. In (C), thrombin (a-c) was added in the presence of tirofiban (70 mg/ml) (b) or aspirin (1 μM) (c). In (d) only Trap-6 was added.

Fig 4. Total and phosphorylated PI3K and Akt contents in thrombin and other agonist-activated platelets.

Non-phosphorylated and phosphorylated PI3K and Akt were detected with specific antibodies as described under “Material and Methods”. (A) PRP was incubated at 37°C for 5 min under stirring in the presence of thrombin, Thr (0.5 U/mL); arachidonic acid, Arach (0.5 mM), collagen, Coll (2 μg/ml), epinephrine, Epi (50 μM), ADP (10 μM), or Trap-6 (22 μM). (B) PRP was incubated in the presence of the indicated concentrations of thrombin (U/mL, lane 1–4), or thrombin (0.5 U/ml) and PP1 0.5 μM (lane 5), wortmannin 20 nM (lane 6), heparin 0.1 mU/mL (lane 7) or ristocetin 1.5 mg/mL (lane 8). Data represent mean ± SD of at least 4 independent determinations. *P<0.05 vs. control (non-activated platelets); **P<0.01 vs. control.