Distinct localization and function of 1,4,5IP3 receptor subtypes and the 1,3,4,5IP4 receptor GAP1IP4BP in highly purified human platelet membranes

Platelet activation is associated with an increase of cytosolic Ca++ levels. The (IP3)-I-1,4,5 receptors [(IP3R)-I-1,4,5] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of (IP3R)-I-1,4,5-type I, type II, and type III-with suggestions of distinct roles in Ca++ elevation. Specific receptors for (IP4)-I-1,3,4,5 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of (IP3R)-I-1,4,5 but in different amounts. Type I end type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors, The PM fractions were found to contain the type III (IP3R)-I-1,4,5 and GAPI1(IP4BP) in cont rast to IM, which contained type I (IP3R)-I-1,4,5. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III (IP3R)-I-1,4,5 was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors, Ca++ release activities were present with both (IP3)-I-1,4,5 and (IP4)-I-1,3,4,5 (EC50 = 1.3 and 0.8 mu mol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting (IP3)-I-1,4,5 but not (1,3),(IP4)-I-4,5-. induced Ca++ flux. In experiments with both PM and intact platelets, the (1,4,5)IP(3)Rs but not GAP1(IP4BP) were found to be substrates of cAMP-PK and cGMP-PK, Thus the Ca++ flux property of (IP4)-I-1,3,4,5 is insensitive to cAMP-PK, These studies suggest distinct roles for the (IP3R)-I-1,4,5 subtypes in Ca++ movements, with the type III receptor and GAP1(IP4BP) associated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores. (Blood. 2000;95:3412-3422) (C) 2000 by The American Society of Hematology.