Involvement of the Na+/H+ exchanger in membrane phosphatidylserine exposure during human platelet activation

Abstract

Platelet membrane phosphatidylserine (PS) exposure that regulates the production of thrombin represents an important link between platelet activation and the coagulation cascade. Here, we have evaluated the involvement of the Na+/H+ exchanger (NHE) in this process in human platelets. PS exposure induced in human platelets by thrombin, TRAP, collagen or TRAP+ collagen was abolished in a Na+ -free medium. Inhibition of the Na+/H+ exchanger (NHE) by 5-(N-Ethyl-N-Isopropyl) Amiloride (EIPA) reduced significantly PS exposure, whereas monensin or nigericin, which mimic or cause activation of NHE, respectively, reproduced the agonist effect. These data suggest a role for Na+ influx through NHE activation in the mechanism of PS exposure. This newly identified pathway does not discount a role for Ca2+, whose cytosolic concentration varies together with that of Na+ after agonist stimulation. Ca2+ deprivation from the incubation medium only attenuated PS exposure induced by thrombin, measured from the uptake of FM1-43 (a marker of phospholipid scrambling independent of external Ca2+). Surprisingly, removal of external Ca2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca2+ ionophore. The residual effect can be attributed to an increase in [Na+]i mediated by the ionophore due to a lack of its specificity. Finally, phosphatidylinositol 4,5-bisphosphate (PIP2), previously reported as a target for Ca2+ in the induction of phospholipid scrambling, was involved in PS exposure through a regulation of NHE activity. All these results would indicate that the mechanism that results in PS exposure uses redundant pathways inextricably linked to the physio-pathological requirements of this process.

Fig. 1

Na⁺-dependence of thrombin proteolytic activity (panel A). Filled and empty symbols represent thrombin activity in Na⁺-containing and Na⁺-free buffer, respectively, with equal concentration of chromozym substrate and after thrombin addition at a concentration 0.1 U/ml (triangles) 0.5 U/ml (diamonds) and 1 U/ml (squares). Data shown are representative of three independent experiments. Na⁺-dependence of platelet secretion after addition of thrombin (THR) or TRAP was evaluated by monitoring ATP release (panel B). Black and gray columns represent Na⁺-containing and Na⁺-free buffers, respectively. Data are means±S.D. of 3–4 experiments (^aP<0.05; ^dP<0.001 vs. the respective value in Na⁺-containing buffer). Na⁺-dependence of platelet aggregation expressed as the percentage of light transmission in suspensions activated with thrombin (panel C) or TRAP (panel D). Platelet aggregation was induced with 0.1 U/ml thrombin or 10 µM TRAP (triangles up—with extracellular Na⁺, triangles down without extracellular Na⁺), 0.5 U/ml thrombin or 50 µM TRAP (squares—with extracellular Na⁺, pointed squares—without extracellular Na⁺), 1 U/ml thrombin or 100 µM TRAP (circles—with extracellular Na⁺, pointed circles—without extracellular Na⁺). Data shown are representative of three independent experiments.

Fig. 2

Simultaneous analysis of PS exposure and Na⁺ influx in sodium green (SG) loaded platelets (suspended in buffer A supplemented with 2 mM CaCl₂) incubated for 10 min with annexin V-PE (AV-PE), after activation with thrombin (1 U/ml) (row B) or calcium ionophore (A23187; 5 µM) (row C). Data shown are representative of 4–6 independent experiments. Percentage of positive cells in each quadrant (row D). SG (+) Na⁺-positive, AV (+)-annexin V-PE positive (^aP<0.05; ^bP<0.02; ^cP<0.01; ^dP<0.001 vs. the respective value in control).

Fig. 3

Evaluation of PS exposure in calcium-containing (2 mM) and calcium-free buffers (0.1 mM EGTA), 20 min after platelet treatment with 2 µM A23187 (panel A),1 U/ml thrombin (panel B), 50 µM monensin (panel C) and 50 µM gramicidin (panel D). Remodeling of plasma membrane was assessed from FM1–43 staining. Data shown are representative of four independent experiments.

Fig. 4

Evaluation of intracellular calcium concentration in platelets loaded with Fura-2 and suspended in buffer A supplemented with 2 mM [Ca²⁺] (right and left columns) or 0.1 mM EGTA (center columns). Changes in [Ca²⁺]_i after activation with TRAP, collagen (panel A), 50 µM gramicidin or 50 µM monensin (panel B). Data are means±S.D. of 4–5 experiments (^bP<0.02; ^dP<0.001 vs. the respective value in control).