Calcium, calmodulin and protein kinase C dependence of platelet shape change

Platelet shape change (PSC) represents the initial phase of platelet activation and is normally investigated in ethylene diamine tetraacetic acid (EDTA) containing platelet rich plasma (PRP); EDTA is a potent chelator of calcium and therefore reduces ionized calcium to negligible levels. It is therefore assumed that it is a process independent of calcium. To test the hypothesis that PSC may be dependent upon intracellular calcium, we examined the effect of 8-(N,N-Diethylamino) octyl 3,4,5-Trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of intercellular calcium mobilization on PSC. It produced a dose dependent inhibition of PSC. We then examined whether PSC was dependent upon calmodulin and protein kinase C, a calcium dependent enzyme which is cardinal to platelet aggregation. Both calmidazolium, a specific inhibitor of calmodulin, and H-9, a specific inhibitor of protein kinase C, produced dose dependent inhibition of PSC. Finally, we investigated whether GP IIb/IIIa receptor which binds fibrinogen was involved in PSC; DMP 728 [(cyclic [D-2-amino-butyryl-N2-methyl-L-arginyl-glycyl-L-aspartyl-3- (a min o-methyl-benzoic acid], methanesulfonic acid salt] a potent GP IIb/IIIa receptor antagonist was without any effect on PSC. We conclude that PSC is a calcium, calmodulin and protein kinase C dependent process like platelet aggregation but that it does not require extracellular calcium or the participation of platelet GP IIb/IIIa complex.