Platelet Ca 2+ ATPases

Abstract

Abstract —Gaining insight into nonmuscle Ca 2+ signaling requires basic knowledge of the major structures involved. We investigated the expression of platelet Ca 2+ ATPases in normal and hypertension-associated abnormal Ca 2+ signaling. First, overall identification of normotensive Wistar-Kyoto rat Ca 2+ ATPases was attempted by looking for newly described human platelet 3′-end alternatively spliced sarco/endoplasmic reticulum Ca 2+ ATPases (SERCA) 3b mRNA and plasma membrane Ca 2+ ATPase (PMCA) 1b and 4b proteins, in addition to SERCA2b and SERCA3a isoforms. For SERCAs, comparative analyses of human and Wistar-Kyoto rat SERCA3 platelet mRNA by reverse transcription–polymerase chain reaction (RT-PCR) followed by sequencing established that human platelets coexpressed SERCA3b and a third SERCA3c, while rat cells were devoid of them but expressed a still unknown splice variant that we termed rSERCA3b/3c. Its identification using 3′-end SERCA3 gene and rapid amplification of cDNA ends (RACE)–PCR studies showed that it results from an additional SERCA3 alternative splicing process, which uses a second alternative polyadenylation site located in the last intron. For PMCAs, with the use of gene-specific RT-PCR followed by sequencing and Western blotting using 5F10 monoclonal antibody, expression of human and rat platelet PMCA1b and PMCA4b was similar. Second, comparative analysis of these newly identified Ca 2+ ATPases and SERCA3a in age-matched spontaneously hypertensive rat platelets demonstrated (1) a marked downregulation of rSERCA3b/3c, which became null, and a 1.71-fold increase in SERCA3a and (2) an opposite regulation of the 2 PMCAs, namely, a 3.3-fold decrease in PMCA1b mRNA and a 3.7-fold increase in PMCA4b mRNA. Hence, platelets coexpress multiple, diverse, and species-specific Ca 2+ ATPases, including a novel fourth SERCA3. Moreover, expression of PMCA (1b and 4b), SERCA3a, and rSERCA3b/3c was modulated in rat hypertension. Hence, Ca 2+ ATPases should be regarded as constituting a new rational basis for the understanding of nonmuscle cell Ca 2+ signaling.