Effect Of Nampt/visfatin On Platelet And Establishment Of Antithrombotic Pharmacodynamic-based Platform

OBJECTIVEPreviously,adipose tissue has only been considered simply as an energy storage organ, in recent years more and more researchers find adipose cells produced a variety of secreted proteins, which is called adipocytokines. These adipocytokines plays a very important role in the metabolic process of life. Now, adipose tissue has become the largest human endocrine organs. Nicotinamide phosphoribosyltransferase(Nampt) is the important synthetic enzyme of Nicotinamide adenine dinucleotide(NAD) in vivo. It is secreted by visceral fat with a molecular weight of 52KD and composed with 491 amino acids which is called visfatin, either. It also expresses in nucleated cells in the blood, liver, brain, heart, spleen, pancreas, muscle, bone marrow, lung and so on. Visfatin/Nampt has a wide range of biological functions, It has an important role in the metabolism,insulin sensitivity,cardiovascular function and inflammatory immunity in the process of human life.From prokaryotes to eukaryotes, the amino acid sequence of visfatin/Nampt are very conservative and has some homology.Visfatin as the adipocytokine was reported in 2005.And it was found as the important enzymes in the level of bacteria and mammalian in 2001 and 2002 respectively.Our department also found its cardiovascular field function of promote vascular smooth muscle cell proliferation and the role of stroke protection.Visfatin promote proliferation of vascular smooth muscle through its Nampt activity response and ERK,p38 signal transduction pathway,and confirmed that visfatin-SIRT1-AMPK is a new neuroprotective pathway and as a new target for prevention of stroke. General,visfatin/Nampt have an important role in the cardiovascular field with atherosclerosis, angiogenesis, proliferation and life of vascular smooth muscle, protection of stroke, meanwhile, it has an important contact with apoptosis, differentiation, inflammation, acute lung injury, biological rhythm, etc.Visfatin/Nampt play so many important biological functions in metabolism of the life, but there are not any visfatin/Nampt reports on aspects of platelet function. So we try to find the connection of visfatin/Nampt and platelet function. To study whether there is expression of visfatin/Nampt in platelet? By the addition of exogenous recombinant protein of visfatin/Nampt and its inhibitors are able to affect platelet function or not? Whether to change the platelet function through genetic interventions of visfatin/Nampt overexpression.RESEARCH DESIGN AND METHODS 1,Testing the expression of visfatin/Nampt in platelet with Western blotting.2,Platelet Rich Plasma (PRP) are centrifuged from normal rats'blood, incubated with the recombinant protein of visfatin/Nampt and visfatin/Nampt's inhibitor of FK866 respectively,then measured platelet aggregation after 30min.3,SD rats were injected intraperitoneally with visfatin/Nampt's inhibitor of FK866 (0.5mg/kg) twice a day, then test platelet aggregation after 7 days.4,The blood of Mice were incubated with visfatin/Nampt's inhibitor of FK866 in vitro then test the expression of P-Selectin which induced with ADP on platelet surface.5,Mice injected intraperitoneally with visfatin/Nampt's inhibitor of FK866 (2.8mg/kg) twice a day, bleeding time, clotting time and blood loss were tested by cutting tail after 3 days. And test the expression of P-Selectin which induced with ADP on platelet surface by flow cytometry.6,Three groups of visfatin/Nampt overexpression (NPT) and its mutant enzyme (NPTA),wild type (WT) mice were test the bleeding time, clotting time and blood loss by cutting tails . And then test the expression of P-Selectin which induced with ADP on platelet surface by flow cytometry.RESULTS1,We have detected the expression of visfatin/Nampt in platelet by western blot.2,The platelet aggregation was significantly reduced by incubating PRP in vitro with visfatin/Nampt's inhibitor of FK866,And the platelet aggregation was significantly enhanced by incubating PRP in vitro with recombinant protein of visfatin/Nampt.3,Rats were injected intraperitoneally with FK866 (0.5mg/kg) twice a day, Compared with the control rats,the PRP's platelet aggregation was decreased of rats which were injected intraperitoneally with FK866.4,After incubation of whole blood with FK866, the percentage of positive P-Selectin with McAb induced by ADP was significantly lower which detected by flow cytometry.5,Mice was injected intraperitoneally with FK866 (5.6mg/kg/d) twice a day,after 3 days its'bleeding time was prolonged and the amount of bleeding was increased. And the percentage of positive P-Selectin with McAb induced by ADP was lower which detected by flow cytometry.6,Three groups of visfatin overexpression (NPT) and its mutant enzyme (NPTA), wild type (WT) mice's tails were cutted and tested the bleeding time, clotting time and blood loss.The group of NPT's bleeding time was shorten and bleeding loss was decreased compared with WT's, and the percentage of positive P-Selectin with McAb induced by ADP was higher which detected by flow cytometry,while the group of NPTA did not change significantly.CONCLUSIONWe examined visfatin/Nampt and its inhibitor on platelet function and found that inhibitors can reduce platelet aggregation,and reduced the percentage of positive P-Selectin with McAb induced by ADP and inhibitted blood coagulation. But applied with the recombinant protein visfatin/Nampt increased platelet aggregation, and the percentage of positive P-Selectin with McAb induced by ADP was higher on overexpression mice's platelets than wild type mice's.Therefore, maybe there is a link of visfatin/Nampt associated with platelet activation, suggesting that it may be the role of maintain a stable in blood coagulation function.