Sterile-20 Kinase Misshapen/NIK-related Kinase 1 (MINK1) Is Involved In Platelet Function,Hemostasis,and Thrombus Formation

Platelets play important role in hemostasis and thrombosis.Impaired platelet function has been associated with an increased propensity for hemorrhage,and excessive activation of platelets can lead to thrombotic diseases.The exploration of molecular mechanism of platelet activation will provide basis for developing novel therapeutic targets and strategies to those diseases.Upon platelet activation,MAPKs are involved in regulating platelet functions such as adhesion,secretion and aggregation,promoting thrombus formation.In a typical signaling pathway,MAPK activation cascade is a sequential activated triple-kinase module:MAP3K phosphorylates and activates MAP2K,and then MAP2K phosphorylates and activates MAPK.However,much less is known about how MAPKs are specifically and selectively regulated in platelets.Previous studies have shown that Sterile-20 kinases play a role as MAP4K to mediate the activation of MAPK pathway.Sterile-20 kinase superfamily,according to the structure,is divided into two groups:the p21-activated kinases(PAKs)and the germinal center kinases(GCKs).MINK1,which was first identified in 2000,belongs to GCK Ⅳ subfamily,and is involved in many important cellular processes.It plays a role as MAP4K to participate in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells and negative selection of autoreactive thymocytes.However,it is not clear whether MINK1 is involved in hemostasis and thrombosis.Because of the essential role of MAPK pathway in platelet activation and thrombus formation,our hypothesis is:MINK1 participates in platelet activation,consequently thrombus formation by regulating MAPKs.In this study,with MINK 1-deficient(MINK1-/-)mice,we investigate the role of MINK1 in platelet function,hemostasis and thrombus formation.The results can be summarized as follows:(1)In the tail-bleeding assay,MINK1-/-mice exhibited a longer bleeding time than wild-type(WT)mice,suggesting that MINK1 is involved in hemostasis.(2)In a model of ferric chloride-induced mesenteric arteriolar thrombosis,vessel occlusion time in MINK1-/-micewaslargely prolonged,and was twice as long as in WT mice.To simulate in vivo thrombosis,an in vitro microfluidic whole-blood perfusion assay was performed.Thrombus formation on a collagen matrixunder arterial shear conditions was significantly reduced in MINK1-/-platelets,demonstrating that the positive role of MINK1 in thrombus formation largely relies on its participation in platelet activation.(3)After detecting platelet functions,we found that adhesion of platelets to collagen was not affected by MINK1 deficiency,however,MINK1-/-platelets demonstrated impaired aggregation and dense granule secretion in response to low doses of thrombin and collagen,while a-granule secretion and thromboxane A2 synthesis were normal in MINK1-/-platelets.Moreover,MINK1-/-platelet showed decreased spreading on fibrinogen,but normal clot retraction.(4)When ADP was hydrolyzed by apyrase,the aggregation and spreading of WT platelets was reduced to a level comparable to MINK-/-platelets.And the defective aggregation and spreading of MINK1-/-platelets can be rescued by exogenous ADP.Hence,impaired ADP secretion was responsible for the defective function of MINK1-/-platelets.(5)In the exploration of molecular mechanism of MINK1 in platelet activation,we found that inside-out and outside-in signaling of integrin αⅡbβ3 were not affected by MINK1 deficiency,and in low doses of collagen and thrombin induced aggregation,the phosphorylation of ERK,p38 and Akt was largely decreased in MINK1-/-platelets.And the regulation of MINK1 to those molecules was independent of secreted ADP and integrin αⅡbβ3 mediated outside-in signaling.Furthermore,MINK1 also regulate the phosphorylation of MEK1/2 and MKK3/6,which are upstream molecules of ERK and p38.And when platelets were incubated with ERK inhibitor U0126 or p38 inhibitor SB203580,the difference in aggregation and dense granule secretion,in response to low doses of collagen and thrombin,between WT and MINK1-/-platelets was abolished.Therefore,decreased ERK and p38 phosphorylation contribute to the impaired dense granule secretion and aggregation of MINK1-/-platelets.In summary,MINK1 is a novel player in platelet activation,hemostasis and thrombosis.Via the regulation of MAPKs and Akt,MINK1 plays a highly specific role in controlling platelet dense granule secretion.