It is long known that platelet aggregation and secretion are accompanied by acidification of the extracellular medium. Much of the proton extrusion results from hydrolysis of ATP generated in the glycolytic pathway and liberation of secretion granules, which are slightly acidic. Recent eyidence points at a third source for extracellular protons.Following early observations (1) that epinephrine-induced platelet functions depended on extracellular Na+ (Na+
o ), it became evident that platelets possess a Na+ /H+ antiport, which regulates the cytosolic pH (pH.) via stochiometric exchange of intracellular protons with extracellular Na+ (2). Platelet functions triggered by epinephrine, AdP or low doses of thrombin are impaired by (i) the absence of Na+
o, and (ii) the presence of EIPA, an amiloride analogue which blocks the antiport. Ionophores which enhance proton efflux enhance the platelet responses. Thus, the antiport affects platelet functions via changes in pHi, but this has been difficult to establish experimentally. Early studies by Simons based on 6-carboxyfluorescein indeed reported a rise in pHi. during platelet activation, but more precise analysis awaited the development of more sensitive pHi-indicators. Recently (3),1studies employing BCECF, have confirmed that resting platelets maintain a pH. of about 7.1 via an EIPA-sensitive mechanism.Platelet activation induces a rise of 0.1-0.2 pH units, which lasts for several minutes unless the antiport is inhibited. When Na+/H+ exchange is gradually inhibited by lowering Na+
o , EIPA-sensitive proton efflux, mobilization of Ca2+ ions and aggregation are inhibited in parallel following stimulation with a low dose of thrombin. Artificial alkalinization reverses these effects. Alkalinization alone is not a trigger for platelet functions. Furthermore, high doses of thrombin (> 0.2 U/ml) initiate Ca2+ -mobilization and aggregation independent of changes in pHi Possibly, Na+ /H+ exchange enhances Ca mobilization by inositol-P3, generated by weak stimulation of the thrombin receptor, wfiich accords with the pH profile of IP3-induced Ca2+ liberation from isolated dense tubular membranes. However, concurrent measurement of Quin-2 and BCECF-fluoresence indicate that Ca2+ mobilization slightly precedes the rise in pHi which would make Ca+ mobilization a trigger for Na+ /H+ exchange is stead of one of its effects. Recent data favour a role for protein kinase C in activation of the antiport. A rise in pHi. is seen during incubation with OAG, an activator of protein kinase C. Thrombin (low dose)-induced Na /H exchange is inhibited by TFP, an inhibitor of this enzyme. These findings are bes^explained by assuming that low doses of thrombin initiate phospholipase C-mediated formation of inositol-P3, which triggers Ca2+ mobilization. Concurrently, diacylglycerol is formed, which activates protein kinase C. The result is a rise in pHi, which enhances the mobilization of Ca2+ by inositol-P3.This scheme differs from the sequence seen during activation by ADP or epinephrine (1), where Na+ /H2+ exchange is an early step after receptor occupancy and precedes phospholipid A2-mediated PG-endoperoxides/TxA2 formation. These metabolites activate phospholipase C resulting in diacylglycerol and inositol-P3-formation at a rather late stage in signal processing. Recent evidence (4) indicates that in epinephrine-stimulated platelets Na+ /H+ exchange requires fibrinogen binding, which opens the intriguing possibility that occupancy of GPIIb-IIIa starts a process that affects signal processing pathways in platelets.Sweatt, J.D., Limbird, L.E, et al. J.B.C. 1983, 1985, 1986Siffert, W., Akkerman, J.W.N., et al. FEBS Lett 1984, 1987; Nature 1987.Zavoico, G.B., Feinstein, M.B st al. J.B.C. 1986Banga, H.D., Rittenhouse, S.E. PNAS 1986
Platelet Ca2+ responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca2+ stores
