Effects Of Phthalocyanine On Platelet Aggregation And Its Mechanism And The Expression And Function Of NOD2 In Platelets

Part I:The antiplatelet effect of NBP and its mechanismObjectiveArterial thrombotic diseases, such as heart attack and stroke, are the leading cause of morbidity and mortality worldwide. Platelet activation triggered by atherosclerotic plaque disruption or endothelium injury caused by percutaneous coronary intervention (PCI) and the consequent intravascular arterial thrombogenesis is the common pathological basis of heart attack and stroke; therefore antiplatelet drugs are effective for prevention and treatment of coronary artery disease and stroke.1-3-n-butylphthalide (1-NBP), was first extracted from seeds of Apium graveolens Linn. Approximately three decades ago, dl-3-n-butylphthalide (NBP), a racemate of1-3-n-butylphthalide, was synthesized and developed to treat cerebral ischemia. It was approved by SFDA of China in November2002and has become the first drug with independent intellectual property rights in the cerebral vascular area in China.It was demonstrated clinically that NBP has multiple cerebral protective effects for the patients after ischemic stroke attack through mitochondrial protective effects, improving brain energy metabolism, etc. The previous studies showed that1-NBP and dl-NBP inhibited platelet aggregation and reduced the thrombus formation in rats under both in vitro and ex vivo. However, the role and the mechanism of these compounds on human platelets were not yet clear. In this paper, we studied the antiplatelet effects of NBP on human platelets and the potential targeting sites of NBP isomers.MethodsWe first measured the effect of NBP on ADP induced platelet PAC-1binding in the whole blood by flow cytometry. We also measured the effect of NBP on platelet aggregation and ATP release induced by ADP, thrombin, collagen, arachidonic acid and U46619in human washed platelets using aggregometry. Then we investigated the effect of NBP on intracelluar cAMP level in human washed platelets using chromagraphy and the activity of PDEs extracted from human platelets by HPLC. Moreover, the effect of NBP on TXA2production in non-ASA treated human washed platelets induced by ADP, thrombin and collagen were assayed using TXB2ELISA kits; Finally, the effects of NBP on ADP induced intracellular Ca2+release in fluo-3AM loaded, ASA treated human washed platelets were evaluated.ResultsWe found that NBP or1-NBP (300μM) pretreatment substantially inhibited platelet PAC-1binding in human whole blood stimulated with ADP (10μM); in the range of10-300μM, NBP or1-NBP dramatically inhibited ADP (10μM) induced platelet aggregation and ATP release in non-ASA treated human washed platelets; NBP or1-NBP also inhibited ADP-induced platelet aggregation in ASA treated human washed platelets, and the inhibitory effect of NBP was much weaker in the ASA-treated platelets. NBP or1-NBP inhibited thrombin (0.05U/mL) induced platelet aggregation and ATP release in non-ASA treated or ASA-treated human washed platelets. In platelets pretreated with or without ASA, NBP or1-NBP also drastically inhibited platelet aggregation and ATP release induced by U46619(1μM), collagen (lμg/mL) or AA (0.5mM) concentration-dependently. NBP or1-NBP (300μM) did not inhibit ADP (10μM) induced cAMP decrease in ASA treated human washed platelets significantly. At300μM NBP or1-NBP substantially inhibited the activity of PDEs extracted from human platelets, showing its PDE inhibition role. Similar to aspirin and indometacin, NBP and1-NBP concentration-dependently inhibited thromboxane B2formation of human washed platelets induced by ADP (5μM), thrombin (0.05U/mL) and collagen (1μg/mL), demonstrating its inhibition on TXA2production. NBP100μM or1-NBP100μM significantly decreased intracellular calcium level in human platelet stimulated with ADP (10μM)Conclusion1. NBP inhibites human platelet activation induced by multiple platelet agonists in vitro.2. NBP exerts its antiplatelet effect through inhibiting the activity of PDEs and TXA2formation. Part II:NOD2is functionally expressed in plateletsIn the past few years, NOD2(nucleotide-binding oligomerization domain containing protein2) was identified as an intracellular pattern recognition receptor functioning in microbial pathogens recognition and playing a critical role in the regulation of the host innate immune response. NOD2is mainly expressed on antigen-presenting cells and epithelial cells. The importance of this molecule is underlined by the fact that NOD2mutaions have been linked to Blau syndrome or to increased susceptibility to Crohn’s disease. NOD2senses muramyl dipeptide (MDP), a small peptide which is derived from peptidoglycan of nearly all Gram-positive and Gram-negative organisms. Stimulation of NOD2result in the activation of NF-kB and MAPKs as well as the production of cytokines, while the mechanisms regulating NOD2are not fully understood.Here, for the first time, we found that NOD2is expressed on human and mice platelets and that NOD2activation potentiates mouse platelets activation responses. Intraperitoneal injection of MDP, the specific NOD2agonist, to wild type mouse increased platelet aggregation and ATP release induced by ADP, thrombin or collagen, while NOD2knockout abolished the effects of MDP. MDP pretreatment also increases platelet clot retraction in wild type mice but not in NOD2knockout mice. Interesingly, we did not observe the effects of MDP on platelets spreading on fibrinogen. Using transmission electron microscopy, we demonstrated that platelets from MDP-pretreated wild type mice have more expanded open canalicular system, less number of a-granule and lighter color of dense granules characterizing the release of platelet granules, which were not noticed in NOD2knock out mice.We conclude that NOD2is functionally expressed in platelets. We speculate that MAPK pathway mediates the role of NOD2in platelet activation, which is under study.