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

• Published in: Biochimica et biophysica acta 2006-02, Vol.1761 (2), p.195-204
• Main Authors: Bucki, Robert, Pastore, Jennifer J., Giraud, Françoise, Janmey, Paul A., Sulpice, Jean-Claude
• Format: Article
• Language: English
• Subjects: Amiloride - analogs & derivatives; Amiloride - pharmacology; Blood Platelets - drug effects; Blood Platelets - metabolism; Calcium - blood; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Lipids - blood; Na +/H + exchanger (NHE) phosphatidylinositol 4,5-bisphosphate (PIP 2); Phosphatidylinositol 4,5-Diphosphate - blood; Phosphatidylserine; Phosphatidylserines - blood; Platelet; Platelet Activation - drug effects; Platelet Activation - physiology; Receptor, PAR-1 - blood; Sodium - blood; Sodium-Hydrogen Exchangers - blood; Thrombin
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2005.12.008

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 Ca 2+, whose cytosolic concentration varies together with that of Na + after agonist stimulation. Ca 2+ 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 Ca 2+). Surprisingly, removal of external Ca 2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca 2+ 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 (PIP 2), previously reported as a target for Ca 2+ 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.