Human platelets express the SERCA2-b isoform of Ca2+-transport ATPase

Abstract

Previous biochemical studies suggested that the human platelet Ca2+ATPase system may be cell-specific. To test this hypothesis, we first undertook the molecular cloning of Ca2+ATPase from human erythroleukaemia (HEL) cells, because this human cell line exhibits megakaryocytic features and expresses a Ca2+ATPase that cross-reacts with platelet Ca(2+)-ATPase. For this cloning, an HEL-cell cDNA library was screened with a rat cardiac Ca2+ATPase cDNA probe. The insert of the longest clone isolated was 3.9 kb and its sequence displayed a 100% identity with that of the non-muscle human Ca2+ATPase 2-b isoform, termed SERCA2-b (sarco-endoplasmic-reticulum Ca2+ATPase). The 3.9 kb cDNA covered a subtotal coding region and part of the 3' non-coding end of the SERCA2-b mRNA. It cross-hybridized with the 4 kb transcript species of cardiac SERCA2-a and with non-muscle SERCA2-b mRNAs, but not with fast-skeletal-muscle SERCA1 mRNA. We next confirmed that SERCA2-b was a component of the platelet Ca2+ATPase system because (1) the platelet clones isolated from a platelet cDNA library exhibited a 100% homology with HEL-cell cDNA; (2) SERCA2-b mRNA was amplified by PCR on total platelet RNA and (3) platelet Ca2+ATPase cross-reacted with a polyclonal SERCA2-b-specific antiserum. Platelets therefore contain a Ca2+ATPase definitely identified as the SERCA2-b isoform of Ca2+ATPase, thus eliminating the possibility that they only contain a single specific Ca2+ATPase.

Inhibition of inositol 1,4,5-trisphosphate-induced Ca 2+ release by cAMP-dependent protein kinase in a living cell

“…ATPases of the sarco-endoplasmic reticulum calcium ATPase 1 and 2 classes can bind phospholamban (for review, see ref. 48), and sarco-endoplasmic reticulum calcium ATPase 2b is expressed in platelets (49). However, the key issue is whether or not the tissue of interest contains the regulatory protein phospholamban or an analogous protein.…”

Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

“…Although some controversy exists concerning the expression of InsP $ -Rs, human platelets were reported to express either InsP $ -RI and II or InsP $ -RI and III. About SERCAs, platelets account for the first cells shown to simultaneously express the ubiquitous SERCA2b [18] and SERCA3a proteins [19][20][21] as well as SERCA3b [22] and SERCA3c [23] mRNAs.…”

Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase–dependent calcium stores

“…They are found in multiple tissues, but platelets exhibit only SERCA2b and SERCA3 isoforms. [2][3][4] SERCAs maintain a Ca 21 concentration gradient between the cytosol (100 nM) and the endoplasmic reticulum (1 mM), requiring degradation of adenosine triphosphate (ATP) 5 into adenosine 59-diphosphate (ADP) then released into the cytosol. 6 SERCA enzymes share similar structures with distinct intrinsic activities: higher Ca 21 affinity for SERCA2b than for SERCA3 (K 1/2 ;0.27 mM vs 1 mM) but a lower Ca 21 uptake (7 nmol/min per mg of protein vs 21 nmol/min per mg) 7,8 allowing cytosolic Ca 21 to be maintained at low levels in the resting cells.…”