Role of phosphoinositide 3-kinase in platelet responses to platelet-activating facto

Platelets are necessary for hemostasis but are also involved in a broad range of pathophysiologic processes such as atherosclerosis and thrombosis. Platelet activating factor (PAF), an inflammatory mediator, is an important physiological regulator of platelet function. The major objective of this work was to determine the role of phosphoinositide 3-kinase (PI 3-kinase) in platelet responses to platelet activating factor. We show, for the first time in platelets, that PAF activates PI 3-kinase over a rapid time course that correlates closely with the aggregation response. The potent PI 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) were used to probe the dependence of PAF-induced aggregation and dense granule release on PI 3-kinase. Both compounds markedly inhibited PAF-induced aggregation; however, only at low activation states giving reversible aggregation (primary phase) did this correlate with PI 3-kinase inhibition. Secretion, measured as release of $sp3$H-hydroxytryptamine, was inhibited to a maximum of 30% and only at low concentrations of PAF. We suggest that PI 3-kinase activation is important for reversible (primary) aggregation of platelets in response to PAF, perhaps by contributing to the 'inside-out' activation of platelet glycoprotein IIbIIIa, the fibrinogen receptor. PI 3-kinase only plays a minor role in PAF induced dense granule release at low activation states. Both responses are dramatically less dependent on PI 3-kinase activity when high concentrations of PAF are used, suggesting greater contribution from other pathways. Tyrosine kinases appear to be important regulators of PI 3-kinase at high PAF levels as stimulation results in a greater than 10 fold increase in PI 3-kinase activity associated with tyrosine-phosphorylated proteins. The p85 regulatory subunit of PI 3-kinase is not tyrosine-phosphorylated. Rather, the enzyme associates rapidly with a major tyrosine-phosphorylated 115 kDa protein, a potential regulator of PI 3-kinase activation in platelets. This protein was not immunoreactive with several antibodies to proteins in this molecular weight range known to be tyrosine phosphorylated and to associate with PI 3-kinase on cell activation.