RIP3 (Receptor-interacting Protein Kinase 3) Promotes Platelet Aggregation And Thrombosis

?Objective?RIP3?Receptor-interacting protein kinase 3?is a protein kinase that has an N-terminal Ser/Thr kinase domain similar to that found in other RIP?Receptor-interacting protein kinase?family member.RIP3 is a potent apoptosis-inducing protein involved in the tumor necrosis factor receptor?TNFa?signaling pathway.Little is know about RIP3'role in thrombosis and hematosis.?Methods?Fresh blood from health volunteers and wild-type and RIP3 knock-out mouse were collected and centrifugated,then washed platelets were prepared.Washed platelets?3x10⁸/ml?were incubated with different concentrations of GSK?872 or vehicle dimethyl sulfoxide?DMSO?at 37?for 30min.Platelet aggregation assay were performed by addition of U46619?350nmol/L?and thrombin?0.1U/ml?into washed platelets at 37?,and examined by turbidometric platelet aggregometer at a stirring speed of 1200 rpm.In the ferric chloride?FeCl₃?-induced mesenteric arteriole thrombosis model experiment,platelets were isolated from donor mice and labeled with calcein-AM?5?g/ml?.Male mice were injected i.v.with calcein-labeled platelets?5×10⁶/g?of matching genotype.The recipient mice were anesthetized.In inhibition experiments,GSK?872?96?M?or vehicle?DMSO?was retrobulbarly injected and then circulated for 30 min.The mesentery vascular bed was exteriorized,and one arteriole was chosen and visualized with an inverted fluorescent microscope?Leica Microsystems?,and recorded on videotape.Thrombus formation was induced by topical application of a 3-mm² filter paper soaked with 6%FeCl₃.The vessel occlusion time was defined the time to as complete cessation of blood flow.Washed platelets?3×10⁸/ml?from WT mice were stimulated with 0.006 U/ml thrombin for the indicated times at 37°C under stirring.The platelets were lysed with equal volumes of 2×Nonidet P-40 lysis buffer containing protease inhibitor mixture tablets on ice for 30 min.After centrifugation at 17,000×g and 4°C for 10 min,the supernatants were immunoprecipitated with anti-G?13 antibody or IgG?control?overnight.After incubation with Protein A/G Plus Agarose beads at 4°C for 2 h,the proteins were analyzed by Western blot analysis with anti-RIP3 and anti-G?13 antibodies.?Results?We found that GSK?872 dose-dependently inhibited U46619-and thrombin-induced human and mouse platelet aggregation,but had no effect on the aggregation of RIP3^-/-platelets.Compared with vehicle-injected controls,the mice injected with GSK?872exhibiteddelayedanddiminishedthrombusformation.RIP3couldbe co-immunoprecipitated with G?13,but not with Gq or Gi,from the lysates of platelets.The interaction between RIP3 and G?13 is dynamically regulated on thrombin induced activation.?Conclusion?RIP3 inhibitor GSK?872 blocks human and WT mouse platelet aggregation and in vivo thrombus formation.RIP3 has interaction with G?13,and this interaction is dynamically regulated on thrombin induced activation.