Winfried Siffert

Na+/H+ exchange as a modulator of platelet activation

Abstract Many cells respond to stimulation with activation of a Na + /H + exchanger, located in the plasma membrane. This results in an increase in the cytosolic pH, which has a major impact on cellular metabolism. Recent investigations in human blood platelets have revealed that Na + /H + exchange plays an important role in stimulus-response coupling.

Thrombin stimulates Na+-H+ exchange across the human platelet plasma membrane.

We have investigated the release of protons from thrombin‐stimulated platelets. Addition of thrombin to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.1 mM amiloride, a potent inhibitor of the Na+/H+ transport system, the amount of protons liberated was decreased by about 50%, and was further reduced to about 15% by 1 mM amiloride. Similar inhibition of H+ release was observed after Na+ in the incubating medium had been replaced by choline. We conclude that one of the earliest events in thrombin‐stimulated platelets consists of the activation of an Na+/H+ countertransport, which leads to an increase in intracellular pH.

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets.

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin‐induced Ca2+ mobilization and (ii) turnover of 32P‐labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 thrombin, whereas increasing pHi by NH4C1 enhanced the thrombin‐induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin‐induced cleavage of phosphatidylinositol 4,5‐bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus‐induced hydrolysis of PIP2.

Evidence for A role of Na+H+ exchange in platelets activated with calcium-ionophore A 23187

We have investigated the release of protons from human platelets and platelet aggregation induced by the calcium ionophore, A 23187. Addition of the ionophore to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.2 mM amiloride, a potent inhibitor of Na+/H+ countertransport, the amount of protons liberated was decreased by 50% and was further reduced to about 10% by 1 mM amiloride. Similar inhibition of H+-release was observed after decreasing Na+ in the incubation medium. Both results suggest that increasing internal Ca2+ by the ionophore induces Na+/H+ exchange in human platelets. Platelet aggregation could be induced by adding the ionophore to the platelet suspension. This aggregation was inhibited by amiloride, at least when induced by low ionophore concentrations. The results suggest that stimulation of Na+/H+ exchange, and the concomitant increase in intraplatelet pH, are important mechanisms in platelet activation.

A phorbol ester and 1-oleoyl-2-acetylglycerol induce Na+/H+ exchange in human platelets.

This study aimed at investigating the mechanisms by which stimulation of human platelets results in activation of Na+/H+ exchange. Platelets were suspended in a slightly buffered medium and the stimulus-induced, amiloride-sensitive H+ release, reflecting Na+/H+ exchange, was estimated from changes in the medium pH. H+ release could be evoked by thrombin and by activators of protein kinase C such as 1-oleoyl-2-acetylglycerol (OAG) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the thrombin-and the OAG-induced Na+/H+ exchange could be blocked by trifluoperazine, a protein kinase C inhibitor. The thrombin-induced H+ release was also sensitive to increased intracellular cAMP levels, probably due to inhibition of phospholipase C activation, whereas the OAG-induced activation of Na+/H+ exchange was unaffected. Our data suggest that activation of Na+/H+ exchange is mediated by protein kinase C.

The effect of carbonic anhydrase inhibition on the velocity of thrombin-stimulated platelet aggregation under physiological conditions

We have studied the effect of ethoxzolamide , a specific carbonic anhydrase inhibitor, on the velocity of thrombin-stimulated platelet aggregation. After preincubation of platelet rich plasma with 10(-6) M ethoxzolamide the velocity of platelet aggregation was reduced by about 40%. Between 10(-11) M and 10(-10)M ethoxzolamide was necessary to achieve a half-maximal diminution of the aggregation velocity. An identical maximal reduction of the velocity of aggregation as with ethoxzolamide could be achieved by a nearly complete removal of CO2 from the platelet rich plasma. These results suggest that the intracellular CO2 hydration-dehydration reaction is involved in the activation of human platelets by thrombin. It is possible that the cytosolic carbonic anhydrase of platelets provides a rapid source of the protons that are transferred across the plasma membrane during the activation process.

Role of Cytoplasmic pH for Stimulus-Induced CA2+ Mobilization in Human Platelets

Stimulation of human platelets by thrombin resulted in cytoplasmic alkalinization due to activation of Na+/H+ exchange. Inhibition of this Na+/H+ exchange suppressed the thrombin-induced Ca2+ mobilization. Artificial cytoplasmic alkalinization, on the other hand, markedly enhanced the stimulus-induced rises in the cytosolic free Ca2+ concentration. It is concluded that activation of Na+/H+ exchange is needed for Ca2+ mobilization and thus constitutes an essential step in platelet activation.

Significance of Carbonic Anhydrase in the Function of Skeletal Muscle

To look into the function of CA, we have started our work with the excised frog gastrocitemius muscle with its nerve supply intact (Scheid and Siffert 1985). The muscle was suspended in air in an apparatus that allowed measurement of single-twitch isometric force. After a control, the muscle of one body side was incubated in frog Ringer’s solution containing one of the CA inhibitors, acetazolamide, ethoxzolamide, or methazolamide; the muscle of the other body side was incubated, as control, in Ringer’s solution without inhibitor.