The Antiplatelet And Antithrombotic Effects Of DMXAA And The Mechanism Of NOD2 Potentiating Platelet Activation And Thrombosis

Part Ⅰ:Tumor vascular disrupting agent 5, 6-dimethylxanthenone-4-acetic acid inhibits platelet activation and thrombosis via inhibition of thromboxane A2 signaling and phosphodiesteraseBackground:5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a tumor vascular disrupting agent under clinical trials as an adjacent antitumor agent. DMXAA is structurally similar to flavone-8-acetic acid (FAA), an old tumor vascular disrupting agent with antiplatelet and antithrombotic effects. In contrast to FAA, which causes bleeding in tumor patients, no bleeding has been reported in patients receiving DMXAA. Whether DMXAA also affects platelet function is not clear. Objectives:To determine the effects of DMXAA on platelet function and explore the underlying mechanisms.Methods and Results:DMXAA concentration-dependently inhibited human platelet aggregation and ATP release induced by U46619, arachidonic acid, ADP, collagen or ristocetin. Furthermore, DMXAA inhibited phosphorylation of Erkl/2 and Akt downstream of thromboxane A2 signaling inhibition. DMXAA also inhibited human platelet phosphodiesterase (PDE). The antiplatelet effects were further confirmed using mice intravenously given DMXAA. DMXAA dramatically inhibited thrombus formation in FeCl3-injured mouse mesenteric arterial thrombus model and laser-injured mouse cremaster arteriole thrombus model. Notably, at a dose exhibiting antithrombotic effects similar to clopidogrel in mice, DMXAA did not significantly increase bleeding.Conclusions:For the first time, we found that tumor-vascular disrupting agent, DMXAA has potent antiplatelet and antithrombotic effects without any bleeding diathesis. As DMXAA inhibits platelet activity with safe profile, DMXAA could be used as an efficacious and safe antiplatelet drug.Part Ⅱ:NOD2 potentiates platelet activation and thrombosisBackground:Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity. We have previously shown that both human and mouse platelets express NOD2. NOD2 activation potentiates mouse platelet aggregation and secretion induced by low concentration of thrombin or collagen, as well as clot retraction. However, the roles of NOD2 on human platelets and in vivo thrombosis as well as mechanisms of NOD2 have never been explored.Objectives:To determine the roles of NOD2 on human platelets and in vivo thrombosis and explore the underlying mechanisms.Methods and Results:MDP induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates human platelet aggregation and secretion induced by low concentration of thrombin or collagen, as well as clot retraction. Using intravital microscopy, we found that MDP administration accelerated in vivo thrombosis in FeCl3-injuried mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. Finally, we found that platelets express RIP2 (receptor-interacting protein 2), and provided evidence suggesting that MAPK and cGMP/PKG pathways downstream of RIP2 mediate the role of NOD2 in platelets.Conclusion:NOD2 is expressed in platelets and potentiates platelet activation and arterial thrombosis via RIP2/MAPKs and RIP2/cGMP/PKG, possibly during the conditions of inflammation. To our knowledge, this is the first study on NOD-like receptors in platelets which links thrombotic events to inflammation.