P145	Influence of oxidized LDL on VASP phosphorylation in human platelets.
W.Schmid, A.Roth, E.Koller, I.Volf
Center of Physiology and Pathophysiology, Vienna, AT.

Atherosclerotic vascular disease and its complications represent the leading cause of death in Western countries. Under physiological conditions platelet activation is tightly regulated by platelet antagonists like nitric oxide (NO) and prostaglandins (e.g. PGI2, PGE1). Acting via cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases these vasodilators inhibit platelet function by phosphorylation of downstream proteins. A major downstream target for both cAMP- and cGMP-dependent protein kinases is the vasodilator-stimulated phosphoprotein (VASP). In vitro experiments suggest that, upon phosphorylation, VASP loses its enhancing function on actin disassembly and polymerization, thereby leading to inhibition of platelet shape change, the initial event of platelet activation. Furthermore, phosphorylation of VASP has been shown to closely correlate with inhibition of integrin αIIbβIII, the fibrinogen receptor responsible for aggregation and clot formation following pro-thrombotic stimuli. In cardiovascular disease, impairment of this regulatory system contributes to enhanced platelet reactivity. Elevated plasma levels of low-density lipoproteins (LDL) represent a well-recognized risk factor for the development of atherosclerosis and it is accepted that LDL gain their atherogenic potential upon oxidative modification (oxLDL). Several groups were able to show that oxLDL exert stimulating effects on platelet reactivity. In this study we investigated the interaction oxLDL with VASP phosphorylation in isolated human platelets by flow-cytometric measurement. Quantification of VASP phosphorylation status with a FITC conjugated monoclonal antibody directed against a VASP-phosphorylation site revealed that oxLDL reduce sodium nitroprusside induced VASP phosphorylation. The observed effects were dose-dependent and increased in parallel with the oxidation state of LDL, while antioxidants were able to reverse the effects of oxLDL. Since in platelets, VASP is located at the intersection of two major inhibitory pathways and VASP phosphorylation plays a pivotal role in platelet inhibition, the reduced VASP phosphorylation status by oxLDL could-at least in part-explain the platelet activating function of oxLDL observed in vitro and the enhanced platelet activation state observed in patients with atherosclerosis.

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