These data suggest the feasibility and procedural safety of myoblast transplantation performed via the trans-coronary-venous approach using the TransAccess catheter system.

Platelets play a critical role in vascular inflammation through the podoplanin and collagen/fibrin receptors, C-type-lectin-like-2 (CLEC-2) and glycoprotein VI (GPVI), respectively. Both receptors regulate endothelial permeability and prevent peri-vascular bleeding in inflammation. Here we show that platelet-specific deletion of CLEC-2 but not GPVI leads to enhanced systemic inflammation and accelerated organ injury in two mouse models of sepsis–intra-peritoneal lipopolysaccharide and cecal ligation and puncture. CLEC-2 deficiency is associated with reduced numbers of podoplanin-expressing macrophages despite increased cytokine and chemokine levels in the infected peritoneum. Pharmacological inhibition of the interaction between CLEC-2 and podoplanin regulates immune cell infiltration and the inflammatory reaction during sepsis, suggesting that activation of podoplanin underlies the anti-inflammatory action of platelet CLEC-2. We suggest podoplanin-CLEC-2 as a novel anti-inflammatory axis regulating immune cell recruitment and activation in sepsis.

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemI-TAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-␤-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A 2 (TxA 2 ). The role of ADP and TxA 2 in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVIFcR␥-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation. (Blood. 2010; 115(14):2938-2946) IntroductionDectin-1, C-type lectin-like receptor 2 (CLEC-2), and CLEC-9A are C-type lectin receptors which have been shown to signal through an immunoreceptor tyrosine-based activation motif (ITAM)-like pathway via a single YxxL in their short cytoplasmic tails known as a hemITAM. [1][2][3][4] Tyrosine phosphorylation of the conserved tyrosine and both SH2 domains of the tyrosine kinase Syk have been shown to be essential for activation of Syk by CLEC-2 and dectin-1, favoring a model in which the tyrosine kinase is activated by the cross-linking of 2 receptors. 2 This is consistent with the observations that CLEC-9A is a disulphide-linked dimer and that CLEC-2 is expressed as a noncovalent dimer. 1,5 CLEC-2 is highly expressed in platelets and at lower levels in other hematopoietic cells, including neutrophils and dendritic cells. 6,7 It is a type II transmembrane protein and a nonclassical C-type lectin that lacks the conserved amino acids that mediate binding to glycans. 7 CLEC-2 was identified by affinity chromatography as a ligand for the snake venom protein rhodocytin, purified from the Malayan pit viper, Calloselasma rhodostoma. 3 Independent crystal structures of rhodocytin show that it assembles as a tetramer, which leads to the suggestion that it mediates activation of CLEC-2 through clustering. 8,9 Consistent with this clustering model of activation, whole antibodies and F(ab) 2 fragments have been reported to activate CLEC-2, whereas antibody Fab fragments were inhibitory. 3,10,11 Physiologic roles of CLEC-2 are now beginning to emerge. Recently, CLEC-2 has been implicated in tumor metastasis via its interaction with the type I sialoglycoprotein podoplanin, which is also expressed on the surface of kidney podocytes, lung type I alveolar cells, and lymphatic endothelium. 12-14 CLEC-2 has also been shown to play a role in neutrophil phagocytosis. 15 Evidence obtained using antibody depletion of CLEC-2...

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.

BackgroundIncreasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.Methodology/Principal FindingsIn this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.ConclusionsThese results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.