Molecular and functional characterization of the human platelet Na+/Ca2+exchangers

BACKGROUND AND PURPOSE The Na+/Ca2+ exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca2+ ([Ca2+](i)) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na+/Ca2+ exchangers: K+-independent Na+/Ca2+ exchanger (NCX) and K+-dependent Na+/Ca2+ exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na+/Ca2+ exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca2+](i) with calcium-green/fura-red in response to: changes in the Na+ and K+ gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca2+ efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca2+](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca2+](i). CONCLUSIONS AND IMPLICATIONS Human platelets express K+-independent Na+/Ca2+ exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca2+ influx, whereas NCKX1 continues to operate in the Ca2+ efflux mode to reduce [Ca2+]