EXPRESSION OF HPMCA4B, THE MAJOR FORM OF THE PLASMA-MEMBRANE CALCIUM-PUMP IN MEGAKARYOBLASTOID CELLS IS GREATLY REDUCED IN MATURE HUMAN PLATELETS

Antibodies 5F10 and JA3 (raised against the erythrocyte Ca2+ pump) wer e used to identify hPMCA4b as the major form of the plasma membrane Ca 2+ pump in human platelets and in three human megakaryoblastoid cell l ines, MEG 01, DAMI and CHRF 288-11. 5F10 was used because it has been shown to recognize all known isoforms of the hPMCA and JA3 because it reacts exclusively with hPMCA4b [Caride A.J., Filoteo A.G., Enyedi A., Verma A.K., Penniston J.T. Detection of isoform 4 of the plasma membr ane calcium pump in human tissues by using isoform-specific monoclonal antibodies. Biochem J 1996; 316: 353-359]. In addition to hPMCA4b, hP MCA1b was also detected in the megakaryoblastoid cells by using isofor m-specific polyclonal antibodies. The apparent size of this isoform, h owever, was smaller than that seen in HeLa and COS-7 cell membranes in dicating the presence of a modified form of hPMCA1b. In platelets, no evidence of the expression of hPMCA1b could be found. The amount of PM CA in these cells was compared with that of the constitutive form of t he sarco/endoplasmic reticulum Ca2+ pump in non-muscle cells (SERCA2b) and also with the amount of PMCA in human erythrocytes. A very low le vel of the plasma membrane Ca2+ pump was found in platelets while in t heir precursor cells the expression of this Ca2+ pump was much more ab undant. Whereas the expression level of PMCA decreased dramatically in mature human platelets, the expression of SERCA2b did not change subs tantially upon megakaryocytic differentiation.