Xuemei Fan

Paired immunoglobulin-like receptor B regulates platelet activation.

Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin β3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbβ3-mediated signaling.

Vps33b regulates Vwf‐positive vesicular trafficking in megakaryocytes

Mutations of vacuolar protein sorting‐associated protein 33b (VPS33B) cause arthrogryposis, renal dysfunction, and cholestasis syndrome, and a lack of platelet α‐granules in the affected patients. Conditional Vps33b knockout mice were developed to investigate the function(s) of Vps33b in platelet α‐granule formation. We found that early embryonic deletion of Vps33b was lethal. PF4‐Cre‐driven megakaryocyte‐targeted Vps33b gene deletion greatly diminished Vps33b expression in platelets, but had no effect on platelet α‐granule formation and protein content. Tamoxifen‐induced, haematopoietic stem cell (HSC)‐specific Vps33b deletion completely depleted Vps33b in platelets, caused the absence of α‐granules, and increased the number of vacuoles in platelets and megakaryocytes. VPS33B association with VIPAS39, α‐tubulin, and SEC22B was identified by co‐immunoprecipitation, mass spectra, and immunoblotting in human embryonic kidney 293T (HEK293T) cells. Also, pull‐down experiments revealed that VIPAS39 bound to intact VPS33B; in contrast, α‐tubulin and SEC22B separately interacted with the sec1‐like domains of VPS33B. Vps33b deficiency in megakaryocytes disturbs the redistribution of Vipas39 and Sec22b to proplatelets, and interrupted the co‐localization of Sec22b with Vwf‐positive vesicles. The data presented in this study suggest that Vps33b is involved in α‐granule formation possibly by facilitating the Vwf‐positive vesicular trafficking to α‐granule‐related vacuoles in megakaryocytes. Copyright

Extracellular signal‐regulated kinase 5 associates with casein kinase II to regulate GPIb‐IX‐mediated platelet activation via the PTEN/PI3K/Akt pathway

Essentials The mechanisms of extracellular signal‐regulated kinase 5 (ERK5) in GPIb‐IX signaling are unclear. Function of ERK5 in GPIb‐IX was tested using aggregation, western blotting, and mass spectrometry. The protein interacting with ERK5 in human platelets was identified as casein kinase II (CKII). ERK5 associates with CKII to regulate the activation of the PI3K/Akt pathway in GPIb‐IX signaling.

Platelet-Specific p38α Deficiency Improved Cardiac Function After Myocardial Infarction in MiceHighlights

Objective— MAPKs (mitogen-activated protein kinases), especially p38, play detrimental roles in cardiac diseases and cardiac remodeling post-myocardial infarction. However, the activation and function of MAPKs in coronary thrombosis in vivo and its relationship with clinical outcomes remain poorly understood. Approach and Results— Here, we showed that p38&agr; was the major isoform expressed in human and mouse platelets. Platelet-specific p38&agr;-deficient mice presented impaired thrombosis and hemostasis but had improved cardiac function, reduced infarct size, decreased inflammatory response, and microthrombus in a left anterior descending artery ligation model. Signaling analysis revealed that p38 activation was one of the earliest events in platelets after treatment with receptor agonists or reactive oxygen species. p38&agr;/MAPK-activated protein kinase 2/heat shock protein 27 and p38&agr;/cytosolic phospholipases A2 were the major pathways regulating receptor-mediated or hydrogen peroxide-induced platelet activation in an ischemic environment. Moreover, the distinct roles of ERK1/2 (extracellular signal–regulated kinase) in receptor- or reactive oxygen species-induced p38-mediated platelet activation reflected the complicated synergistic relationships among MAPKs. Analysis of clinical samples revealed that MAPKs were highly phosphorylated in platelets from preoperative patients with ST-segment–elevation myocardial infarction, and increased phosphorylation of p38 was associated with no-reflow outcomes. Conclusions— We conclude that p38&agr; serves as a critical regulator of platelet activation and potential indicator of highly thrombotic lesions and no-reflow, and inhibition of platelet p38&agr; may improve clinical outcomes in subjects with ST-segment–elevation myocardial infarction.

Drospirenone enhances GPIb-IX-V-mediated platelet activation.

Epidemiologic studies recently revealed that using drospirenone (DRSP)‐containing contraceptives is associated with an increased risk of thrombosis in women. However, the underlying causality is unclear.

Palladin is involved in platelet activation and arterial thrombosis

The dynamics of actin cytoskeleton have been shown to play a critical role during platelet activation. Palladin is an actin-associated protein, serving as a cytoskeleton scaffold to bundle actin fibers and actin cross linker. The functional role of palladin on platelet activation has not been investigated. Here, we characterized heterozygous palladin knockout (palladin+/-) mice to elucidate the platelet-related functions of palladin. The results showed that palladin was expressed in platelets and moderate palladin deficiency accelerated hemostasis and arterial thrombosis. The aggregation of palladin+/- platelets was increased in response to low levels of thrombin, U46619, and collagen. We also observed enhanced spreading of palladin+/- platelets on immobilized fibrinogen (Fg) and increased rate of clot retraction in platelet-rich plasma (PRP) containing palladin+/- platelets. Furthermore, the activation of the small GTPase Rac1 and Cdc42, which is associated with cytoskeletal dynamics and platelet activation signalings, was increased in the spreading and aggregating palladin+/- platelets compared to that in wild type platelets. Taken together, these findings indicated that palladin is involved in platelet activation and arterial thrombosis, implying a potent role of palladin in pathophysiology of thrombotic diseases.

Clathrin-mediated integrin αIIbβ3 trafficking controls platelet spreading

Abstract Dynamic endocytic and exocytic trafficking of integrins is an important mechanism for cell migration, invasion, and cytokinesis. Endocytosis of integrin can be classified as clathrin dependent and clathrin independent manners. And rapid delivery of endocytic integrins back to the plasma membrane is key intracellular signals and is indispensable for cell movement. Integrin αIIbβ3 plays a critical role in thrombosis and hemostasis. Although previous studies have demonstrated that internalization of fibrinogen-bound αIIbβ3 may regulate platelet activation, the roles of endocytic and exocytic trafficking of integrin αIIbβ3 in platelet activation are unclear. In this study, we found that a selective inhibitor of clathrin-mediated endocytosis pitstop 2 inhibited human platelet spreading on immobilized fibrinogen (Fg). Mechanism studies revealed that pitstop 2 did not block the endocytosis of αIIbβ3 and Fg uptake, but inhibit the recycling of αIIbβ3 to plasma membrane during platelet or CHO cells bearing αIIbβ3 spreading on immobilized Fg. And pitstop 2 enhanced the association of αIIbβ3 with clathrin, and AP2 indicated that pitstop 2 inhibit platelet activation is probably due to disturbance of the dynamic dissociation of αIIbβ3 from clathrin and AP2. Further study demonstrated that Src/PLC/PKC was the key pathway to trigger the endocytosis of αIIbβ3 during platelet activation. Pitstop 2 also inhibited platelet aggregation and secretion. Our findings suggest integrin αIIbβ3 trafficking is clathrin dependent and plays a critical role in platelet spreading, and pitstop 2 may serve as an effective tool to address clathrin-mediated trafficking in platelets.

Vacuolar Protein Sorting 33B Is a Tumor Suppressor in Hepatocarcinogenesis

Polarity defects are frequently involved in liver diseases, such as chronic hepatitis and hepatocellular carcinoma (HCC). It was reported that vacuolar protein sorting 33B (Vps33b) plays critical roles in the maintenance of hepatocyte polarity; however, the functional roles and mechanisms of Vps33b in HCC occurrence and progression remain unknown. First of all, we showed that Vps33b is down‐regulated in human and mouse liver cancer samples, and the low expression levels of Vps33b correlate with the poor prognosis of many HCC patients. Liver‐specific Vps33b deficiency induces liver damage, progressive hepatitis, fibrosis, and HCC in male mice, indicating that Vps33b is a crucial contributory factor to hepatocarcinogenesis. Vps33b deficiency–caused liver damage was primarily due to the disorders of structural and functional hepatocyte polarity, which were reflected by the decreased protein levels of E‐cadherin because of inaccurate location to lysosomes and polarity defects at both apical and lateral plasma membrane proteins. The results of a mechanism study revealed that Vps33b interacts with VPS33B‐interacting protein, which is involved in polarity and apical protein restriction; vesicle‐trafficking protein Sec22b; and Flotillin‐1 in hepatocytes and is in charge of the normal distribution of polarity‐determined proteins. Expression levels of Vps33b negatively correlated with the degree of inflammatory cell infiltration in livers from diethylnitrosamine‐induced or transgenic HCC mouse models, and the inflammatory stimuli suppressed the expression of Vps33b in vitro. Conclusion: Down‐regulation of Vps33b expression is a critical step for inflammation‐driven HCC, and Vps33b serves as an important tumor suppressor in hepatocarcinogenesis.

Platelet MEKK3 regulates arterial thrombosis and myocardial infarct expansion in mice

MAPKs play important roles in platelet activation. However, the molecular mechanisms by which MAPKs are regulated in platelets remain largely unknown. Real-time polymerase chain reaction and western blot data showed that MEKK3, a key MAP3K family member, was expressed in human and mouse platelets. Then, megakaryocyte/platelet-specific MEKK3-deletion (MEKK3-/- ) mice were developed to elucidate the platelet-related function(s) of MEKK3. We found that agonist-induced aggregation and degranulation were reduced in MEKK3-/- platelets in vitro. MEKK3 deficiency significantly impaired integrin αIIbβ3-mediated inside-out signaling but did not affect the outside-in signaling. At the molecular level, MEKK3 deficiency led to severely impaired activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun NH2-terminal kinase 2 but not p38 or ERK5. In vivo, MEKK3-/- mice showed delayed thrombus formation following FeCl3-induced carotid artery injury. Interestingly, the tail bleeding time was normal in MEKK3-/- mice. Moreover, MEKK3-/- mice had fewer microthrombi, reduced myocardial infarction (MI) size, and improved post-MI heart function in a mouse model of MI. These results suggest that MEKK3 plays important roles in platelet MAPK activation and may be used as a new effective target for antithrombosis and prevention of MI expansion.