title: Molecular and functional characterization of the human platelet Na(+) /Ca(2+)  exchangers.
abstract: BACKGROUND AND PURPOSE The Na(+) /Ca(2+)  exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+)  ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+)  exchangers: K(+) -independent Na(+) /Ca(2+)  exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+)  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(+) /Ca(2+)  exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](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 Ca(2+)  efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](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 [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+)  exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+)  influx, whereas NCKX1 continues to operate in the Ca(2+)  efflux mode to reduce [Ca(2+) ](i) .
authors.affiliation: Department of Pharmacology and Therapeutics; University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Roberts
authors.forename: Toshio
authors.initials: T
authors.lastname: Matsuda
authors.forename: Ratna
authors.initials: R
authors.lastname: Bose
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009206:Myocardium / Q000378:metabolism; D010647:Phenyl Ethers / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D000814:Aniline Compounds; D004791:Enzyme Inhibitors; D010647:Phenyl Ethers; D012333:RNA, Messenger; C430918:SEA 0400; D019831:Sodium-Calcium Exchanger; C054391:5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil; D000584:Amiloride; D000255:Adenosine Triphosphate; D003094:Collagen; D013890:Thiourea; D002118:Calcium