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 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 physiopathological requirements of this process.

Cellular Mechanisms Underlying the Formation of Circulating Microparticles

“…Another study has suggested that Na ϩ loading via Na ϩ /H ϩ exchange is involved in the regulation of PS scrambling in activated platelets. 73 These results clearly indicate that cytosolic calcium ions levels are not a unique player leading to the collapse of the membrane asymmetry. Whether these mechanisms may have a role in modulating the release of MPs in vivo deserves further scrutiny.…”

Mitochondrially Mediated Integrin αIIbβ3 Protein Inactivation Limits Thrombus Growth

“…Alternatively, activation of the Na ϩ /H ϩ exchanger could facilitate cytosolic alkalinization. Interestingly, multiple studies have suggested the involvement of Na ϩ /H ϩ exchanger activity in mediating procoagulant activity and, presumably, procoagulant platelet formation (38,39).…”

Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel

“…It should be emphasized, however, that a decreased [Na ϩ ] may have 2 major consequences: it reduces the catalytic efficiency of thrombin, 15 and it likely causes inhibition of the Na ϩ /H ϩ exchanger, which may be involved in PS exposure during platelet activation. 16 We therefore compared the procoagulant response induced by collagen plus thrombin in low [Na ϩ ] solutions (ie, HEPES/choline buffer and HEPES/KCl buffer) with the response evoked in high [Na ϩ ] solution (ie, HEPES buffer). In these initial experiments, the procoagulant response was determined by the number of annexin-binding platelets measured by flow cytometry.…”