| Class â…¢ PI3K can phosphorylate the D-3 hydroxyl of PtdIns to produce PtdIns3P. By recruiting proteins containing FYVE and PX domains, it can perform distinct function, such as participating in cell autophagy, separating for new synthetic secreted protein and vesicular transportation. In the previous study, we found that under the conditions, starvation or the mTOR inhibitor rapamycin incubation, autophagy of human platelets was class â…¢ PI3K-dependent. However, the function and mechanism of Class â…¢ PI3K involved in platelet activation was unknown, requiring further elucidation. With the establishment of catalytic subunit Vps34-deficient (Vps34-/-) mouse model, and the using of 3-Methyladenine (3-MA), the common class â…¢ PI3K inhibitor, we investigated the role of class â…¢ PI3K in platelet activation, thrombus formation and the potential mechanisms. Our data demonstrated that class â…¢ PI3K play a positive role in platelet aggregation and secretion upon low doses of collagen and thrombin stimulation, adhesion on collagen, spreading on fibrinogen and clot retraction. In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on collagen matrix under arterial shear conditions was significantly reduced in Vps34-/- platelets or human platelets incubated with 3-MA. In a model of ferric chloride-induced mesenteric arteriolar thrombosis, vessel occlusion times were more than twice as long in Vps34-/- mice as in WT mice. However, the bleeding time was not affected by Vps34 deficiency. The functional differences of Vps34-/- platelets or 3-MA incubated platelets could be attributed to the impaired production of ROS. We found that membrane translocation and Thr154 phosphorylation of p40phox, a cytosolic subunit of Nox2 NADPH oxidase complex, were impaired by Vps34 deficiency or class â…¢ PI3K inhibition, and which affected the assembly of the NOX2 compound on platelet membrane and subsequent activation, leading to the reduced ROS generation. |
