Abstract 17868: Calcium and Calmodulin-dependent Kinase Kinase Beta Signalling Controls Human Platelet Aggregation
Background: Several agonists, thrombin (T), thromboxane (TXA2) and collagen (C) induce platelet activation and aggregation. Their effects are mediated by increase in intracellular calcium (Ca2+) and remodelling of actin cytoskeleton. Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta) controls actin cytoskeleton organization through AMP-activated protein kinase (AMPK) in epithelial cells, extending the role of AMPK beyond metabolism. Therefore, our hypothesis was that activation of CaMKKbeta-AMPK pathway by these agonists mediated platelet aggregation by regulating cytoskeletal targets.
Methods: Blood freshly drawn was collected from healthy donors. Platelet aggregation was analyzed on the platelet rich fraction (PRP) using a lumi-aggregometer (Chrono-log). For in vitro treatments and western blot analysis, PRP was purified by the OptiPrep method.
Results: 10μM STO609, a selective inhibitor of CaMKKbeta, inhibited thrombin-induced platelet aggregation (0.025U/ml T: 69±3; T + STO609: 11±4 % of aggregation, P<0.001). STO609 also reduced platelet aggregation induced by TXA2 and collagen (1 μM TXA2: 79±5; TXA2+STO609, 29±11, 2μg/ml C: 50±4; C+STO609, 19±2 % of aggregation, P<0.01). Thrombin induced an increase in acetyl CoA carboxylase (ACC) phosphorylation, a substrate of AMPK (control: 15±10; 0.1 U/ml T:81±17 A.U., P<0.05). TXA2 and collagen also increased ACC phosphorylation to a lower extent. STO609 prevented thrombin effect on ACC phosphorylation (Control: 15±10 ; Control+STO609: 16±11; 0.1U/ml T: 81±17; T+STO609: 16±6 A.U., P<0.05). Finally, inhibition of CaMKKbeta was associated with a decrease in the phosphorylation of two AMPK cytoskeletal targets, namely myosin light chains (MLC) and vasodilatator-phosphoprotein (VASP), known to be modulated during platelet aggregation.
Conclusion: Inhibition of CaMKKbeta by STO609 blocked platelet aggregation induced by thrombin, TXA2 and collagen. The molecular mechanism by which CaMKKbeta control aggregation could be a direct activation of AMPK and subsequent phosphorylation of MLC and VASP. CamKKbeta is a potentiel target for new antiplatelet therapies.
- © 2010 by American Heart Association, Inc.