Halina Stelmach

The importance of Na + /H + exchanger for the generation of procoagulant activity by porcine blood platelets

This study addresses the role of the Na + /H + exchanger (NHE) in the generation of procoagulant activity in blood platelets. It was found that monensin (simulating the action of NHE) and gramicidin (causing sodium influx without concomitant H + efflux) produced a dose- and time-dependent increase in platelet procoagulant activity. Alkalinization of platelet cytosol by NH 4 Cl failed to evoke a procoagulant response. Collagen-induced procoagulant response was diminished in the absence of external Na + and in the presence of EIPA (NHE inhibitor) or GF 109203X (protein kinase C inhibitor). Phorbol ester (PMA) produced a dose- and time-dependent generation of procoagulant response which was inhibited in the absence of the external Na+ and in the presence of EIPA. Platelets stimulated by collagen and PMA accumulated 22 Na + , a phenomenon inhibited in the presence of EIPA. The data indicate that development of procoagulant activity in platelets may occur as a result of Na + influx via Na + /H + exchanger.

Vasopressin acts on platelets to generate procoagulant activity.

The aim of our study was to examine whether arginine vasopressin (AVP) is able to evoke in human platelets a procoagulant response due to activation of an Na+/H+ exchanger. It was found that treatment of platelets with AVP (20–100 nmol/l) results in generation of a weak calcium signal, activation of Na+/H+ exchanger, aggregation, and development of a procoagulant response. The AVP-evoked procoagulant response was dose and time dependent, weaker than that produced by collagen or monensin (mimics Na+/H+ exchanger), and less pronounced following the inhibition of Na+/H+ exchanger by 5-(N-ethyl-N-isopropyl) amiloride or genistein. Flow cytometry studies reveal that in-vitro platelet treatment with AVP results in an unimodal left shift in the forward and side scatter of the entire platelet population, indicating morphological changes on the plasma membrane. The shift was dose related, weaker than that evoked by collagen, similar to that produced by monensin and strongly reduced in the presence of 5-(N-ethyl-N-isopropyl) amiloride or genistein. Using flow cytometry, we demonstrated enhanced expression of phosphatidylserine on the AVP-treated platelets. AVP-evoked phosphatidylserine exposure was dose dependent, inhibited by 5-(N-ethyl-N-isopropyl) amiloride or genistein and weaker than that produced by collagen. AVP in a dose-dependent manner produced a rise in platelet volume. The swelling was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, and its kinetics was similar to that observed in the presence of monensin. We conclude that prolonged treatment of platelets with AVP results in a procoagulant response, which may occur as a consequence of Na+ influx mediated by Na+/H+ exchanger.

The involvement of the Na(+)/H(+) exchanger in the formation of microvesicles by porcine platelets.

Activated platelets release microvesicles, which express procoagulant activity. The mechanism by which vesicles are formed is not entirely clear. This study was undertaken to determine whether a link exists between the operation of the plasma membrane Na(+)/H(+) exchanger (NHE) and vesiculation. It was found, that platelets treated with NHE-simulating monensin and the sodium influx-inducing gramicidin (without concomitant H+ efflux) produced vesicles demonstrating procoagulant activity. Alkalinization of platelet cytosol by NH4Cl failed to evoke vesicle release. Collagen and phorbol ester (PMA)-evoked vesiculation was diminished in the presence of 5-(N-ethyl-N-isopropyl amiloride) (EIPA, inhibitor of NHE) or GF 109203X (inhibitor of protein kinase C). Vesicle formation induced by collagen, PMA, and the calcium ionophore A23187 was less pronounced in the absence of external Na+. In comparison with collagen, thrombin was a stronger inducer of vesiculation. Platelets stimulated by thrombin, collagen, and PMA accumulated 22Na+, a phenomenon inhibited in the presence of EIPA. Collagen-evoked vesicle formation started with aggregation but culminated after its completion. The data indicate a significant contribution of the Na(+)/H(+) exchanger in the formation of microvesicles by porcine platelets.