Abnormal cAMP-induced phosphorylation of rap 1 protein in grey platelet syndrome platelets

We previously demonstrated abnormal Ca2+ transport by microsomes in platelets from a grey platelet syndrome patient. Here, we investigated the platelet Ca2+ ATPases that mediate this transport, as well as its possible regulation by rap 1 protein. We showed that grey platelet syndrome platelets expressed the same two distinct Ca2+ ATPases as those recently described in normal platelets; the 100 kD SERCA2-b isoform (Sarco/Endoplasmic Reticulum Ca2+ATPase) and a new 97 kD SERCA isoform. The two Ca2+ATPases formed similar amounts of transient phosphorylated intermediates. The expression of these two Ca2+ATPases was compared by Western blotting using specific antibodies, which again emerged in similar amounts in normal and grey platelet syndrome platelets. As regards the protein phosphorylated by cAMP, it was found to be identical to rap 1 protein when it was immunoprecipitated with an antibody raised against a synthetic peptide specific for rap 1 protein. Although the expression of rap 1 protein was similar in membranes isolated from grey platelet syndrome and normal platelets, its exogenous phosphorylation by cAMP was abnormal, with a concentration (10 micrograms/ml) of the catalytic subunits of the cAMP-dependent protein kinase (C.Sub.), as it decreased to half the control level. It is concluded that the abnormal Ca2+ transport found in grey platelet syndrome platelets is not due to the abnormal expression of the Ca2+ATPases, but is associated with an abnormality of rap 1 protein phosphorylation by cAMP.