Leukocyte-released antimicrobial peptides contribute to pathogen elimination and activation of the immune system. Their role in thrombosis is incompletely understood. Here we show that the cathelicidin LL-37 is abundant in thrombi from patients with acute myocardial infarction. Its mouse homologue, CRAMP, is present in mouse arterial thrombi following vascular injury, and derives mainly from circulating neutrophils. Absence of hematopoietic CRAMP in bone marrow chimeric mice reduces platelet recruitment and thrombus formation. Both LL-37 and CRAMP induce platelet activation in vitro by involving glycoprotein VI receptor with downstream signaling through protein tyrosine kinases Src/Syk and phospholipase C. In addition to acute thrombosis, LL-37/CRAMP-dependent platelet activation fosters platelet–neutrophil interactions in other inflammatory conditions by modulating the recruitment and extravasation of neutrophils into tissues. Absence of CRAMP abrogates acid-induced lung injury, a mouse pneumonia model that is dependent on platelet–neutrophil interactions. We suggest that LL-37/CRAMP represents an important mediator of platelet activation and thrombo-inflammation.

genesis involves activation of NF-B in endothelial cells by fluid shear stress. Because this pathway involves integrins, we investigated the involvement of focal adhesion kinase (FAK). We found that FAK was not required for flow-stimulated translocation of the p65 NF-B subunit to the nucleus but was essential for phosphorylation of p65 on serine 536 and induction of ICAM-1, an NF-B-dependent gene. NF-B activation by TNF-␣ or hydrogen peroxide was FAK independent. Events upstream of NF-B, including integrin activation, Rac activation, reactive oxygen production, and degradation of IB, were FAK independent. FAK therefore regulates NF-B phosphorylation and transcriptional activity in response to flow by a novel mechanism.atherosclerosis; fluid shear stress; integrin signaling; mechanotransduction CURRENT MODELS FOR ATHEROSCLEROSIS suggest that local endothelial dysfunction results in monocyte recruitment and lipid deposition in the vessel intima (29). In addition to systemic risk factors, fluid shear stress from flowing blood plays a crucial role. Atherosclerotic lesions develop preferentially in regions of oscillatory or disturbed flow that have lower mean shear stress, multidirectionality, and flow separation (33).The proinflammatory transcription factor NF-B is thought to be a key determinant of atherogenesis (8). NF-B is activated in atheroprone regions in vivo concomitant with expression of its target genes including ICAM-1, VCAM-1 (20), monocyte chemoattractant protein 1, tissue factor, and PDGF (8). NF-B is a dimer, usually consisting of a p65 and a p50 subunit, though other combinations also exist. Inactive NF-B is sequestered in the cytoplasm by IB proteins (8,20). The IB kinase (IKK) complex phosphorylates IB, leading to its ubiquitination and proteosomal degradation. Free NF-B dimer then translocates into the nucleus and binds to B enhancer sites. Phosphorylation, acetylation, and sumoylation regulates the subsequent recruitment of cofactors and other members of the transcriptional machinery required to drive target gene expression.Integrins have been implicated in activation of NF-B and several other proinflammatory pathways in response to flow (19). Shear stress induces conversion of integrins to a high-affinity state (i.e., integrin activation), which is followed by binding of these high-affinity integrins to the extracellular matrix beneath the cells to form new integrinmatrix contacts (53). The newly ligated integrins trigger downstream signals, including RhoA, Rac, and Cdc42 activation (53-55). These GTPases mediate cell alignment, sterol regulatory element-binding protein (SREBP) cleavage (30), and . Consistent with this model, flow-induced activation of NF-B is integrin and matrix dependent, occurring in cells on fibronectin (FN) or fibrinogen but not on collagen or laminin (38).Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin signaling (43,46), is present in endothelial cells and has been implicated in responses of endothelial cells to fluid shear stress. Complete ...

The liver has a unique regenerative capability, which involves extensive remodelling of cell-cell and cell-matrix contacts. Here we study the role of integrins in mouse liver regeneration using Cre/loxP-mediated gene deletion or intravenous delivery of b1-integrin siRNA formulated into nanoparticles that predominantly target hepatocytes. We show that although short-term loss of b1-integrin has no obvious consequences for normal livers, partial hepatectomy leads to severe liver necrosis and reduced hepatocyte proliferation. Mechanistically, loss of b1-integrin in hepatocytes impairs ligand-induced phosphorylation of the epidermal growth factor and hepatocyte growth factor receptors, thereby attenuating downstream receptor signalling in vitro and in vivo. These results identify a crucial role and novel mechanism of action of b1-integrins in liver regeneration and demonstrate that protein depletion by nanoparticle-based delivery of specific siRNA is a powerful strategy to study gene function in the regenerating liver.

Rationale: A reduced rate of myocardial infarction has been reported in patients with atrial fibrillation treated with FXa (factor Xa) inhibitors including rivaroxaban compared with vitamin K antagonists. At the same time, low-dose rivaroxaban has been shown to reduce mortality and atherothrombotic events in patients with coronary artery disease. Yet, the mechanisms underlying this reduction remain unknown. Objective: In this study, we hypothesized that rivaroxaban’s antithrombotic potential is linked to a hitherto unknown rivaroxaban effect that impacts on platelet reactivity and arterial thrombosis. Methods and Results: In this study, we identified FXa as potent, direct agonist of the PAR-1 (protease-activated receptor 1), leading to platelet activation and thrombus formation, which can be inhibited by rivaroxaban. We found that rivaroxaban reduced arterial thrombus stability in a mouse model of arterial thrombosis using intravital microscopy. For in vitro studies, atrial fibrillation patients on permanent rivaroxaban treatment for stroke prevention, respective controls, and patients with new-onset atrial fibrillation before and after first intake of rivaroxaban (time series analysis) were recruited. Platelet aggregation responses, as well as thrombus formation under arterial flow conditions on collagen and atherosclerotic plaque material, were attenuated by rivaroxaban. We show that rivaroxaban’s antiplatelet effect is plasma dependent but independent of thrombin and rivaroxaban’s anticoagulatory capacity. Conclusions: Here, we identified FXa as potent platelet agonist that acts through PAR-1. Therefore, rivaroxaban exerts an antiplatelet effect that together with its well-known potent anticoagulatory capacity might lead to reduced frequency of atherothrombotic events and improved outcome in patients.