ABNORMAL CAMP-INDUCED PHOSPHORYLATION OF RAP-1 PROTEIN IN GRAY PLATELET SYNDROME PLATELETS

We previously demonstrated abnormal Ca2+ transport by microsomes in pl atelets from a grey platelet syndrome patient. Here, we investigated t he platelet Ca2+ ATPases that mediate this transport, as well as its p ossible regulation by rap 1 protein. We showed that grey platelet synd rome platelets expressed the same two distinct Ca2+ ATPases as those r ecently described in normal platelets; the 100 kD SERCA(2.b) isoform ( Sarco/Endoplasmic Reticulum Ca(2+)ATPase) and a new 97 kD SERCA isofor m. The two Ca(2+)ATPases formed similar amounts of transient phosphory lated intermediates. The expression of these two Ca(2+)ATPases was com pared by Western blotting using specific antibodies, which again emerg ed in similar amounts in normal and grey platelet syndrome platelets. As regards the protein phosphorylated by cAMP, it was found to be iden tical to rap 1 protein when it was immunoprecipitated with an antibody raised against a synthetic peptide specific for rap 1 protein. Althou gh the expression of rap 1 protein was similar in membranes isolated f rom grey platelet syndrome and normal platelets, its exogenous phospho rylation by cAMP was abnormal, with a concentration (10 mu g/ml) of th e catalytic subunits of the cAMP-dependent protein kinase (C.Sub.), as it decreased to half the control level. It is concluded that the abno rmal Ca2+ transport found in grey platelet syndrome platelets is not d ue to the abnormal expression of the Ca(2+)ATPases, but is associated with an abnormality of rap 1 protein phosphorylation by cAMP.