Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo.

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

Vascular surveillance by haptotactic blood platelets in inflammation and infection.

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.

Rivaroxaban Reduces Arterial Thrombosis by Inhibition of FXa-Driven Platelet Activation via Protease Activated Receptor-1.

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.

Role of RXRß in platelet function and arterial thrombosis.

OBJECTIVE: Retinoid X receptors (RXR) are a family of nuclear receptors that play critical roles in the regulation of numerous fundamental biological processes including cell proliferation, differentiation, and death. Earlier studies suggested that treatment with RXR agonists attenuates platelet activation in all adults (male and femal) and mice; however, the underlying molecular mechanisms have remained insufficiently understood. To elaborate further on this issue, we characterized megakaryocyte and platelet-specific RXR knockout mice to study platelet function in vitro and arterial thrombosis in vivo. APPROACH AND RESULTS: First, we identified RXRß as the dominant RXR receptor in mouse platelets, prompting us to generate a megakaryocyte and platelet-specific PF4Cre ;RXRßflox/flox mouse. Second, we studied activation, spreading, and aggregation of platelets from C57Bl/6 wild-type mice (WT), PF4Cre+ ;RXRßflox/flox mice, and PF4Cre- ;RXRßflox/flox littermate controls in the presence or absence of RXR ligands, that is, 9-cis-retinoic acid (9cRA) and methoprene acid (MA). We found that in vitro treatment with RXR ligands attenuates spreading and aggregation of platelets and increases proplatelet particle formation from megakaryocytes (MK). However, these effects are also observed in RXRß-deficient platelets and MKs and are thus independent of RXRß. Third, we investigated arterial thrombus formation in an iron chloride (FeCl3)-induced vascular injury model in vivo, which is also not affected by the absence of RXRß in platelets. CONCLUSIONS: Absence of the most abundant RXR receptor in mouse platelets, RXRß, does not affect platelet function in vitro and thrombus formation in vivo. Furthermore, RXR agonists' mediated effects on platelet function are independent of RXRß expression. Hence, our data do not support a significant contribution of RXRß to arterial thrombosis in mice.

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3.

The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3-deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell-specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a (Regnase-1), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages.

The Actin Regulator Coronin-1A Modulates Platelet Shape Change and Consolidates Arterial Thrombosis.

Coronin-1A (Coro1A) belongs to a family of highly conserved actin-binding proteins that regulate cytoskeletal re-arrangement. In mammalians, Coro1A expression is most abundant in the haematopoietic lineage, where it regulates various cellular processes. The role of Coro1A in platelets has been previously unknown. Here, we identified Coro1A in human and mouse platelets. Genetic absence of Coro1A in mouse platelets inhibited agonist-induced actin polymerization and altered cofilin phosphoregulation, leading to a reduction in spreading and low-dose collagen induced aggregation. Furthermore, Coro1A-deficient mice displayed a defect in ferric chloride-induced arterial thrombosis with prolonged thrombus formation and reduced thrombus size. Immunofluorescence analysis revealed a less compact thrombus structure with reduced density of platelets and fibrinogen. In summary, Coro1A has a role in platelet biology with impact on spreading, aggregation and thrombosis.

Cathelicidins prime platelets to mediate arterial thrombosis and tissue inflammation.

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.

Histological comparison of arterial thrombi in mice and men and the influence of Cl-amidine on thrombus formation.

AIMS: Medical treatment of arterial thrombosis is mainly directed against platelets and coagulation factors, and can lead to bleeding complications. Novel antithrombotic therapies targeting immune cells and neutrophil extracellular traps (NETs) are currently being investigated in animals. We addressed whether immune cell composition of arterial thrombi induced in mouse models of thrombosis resemble those of human patients with acute myocardial infarction (AMI). METHODS AND RESULTS: In a prospective cohort study of patients suffering from AMI, 81 human arterial thrombi were harvested during percutaneous coronary intervention and subjected to detailed histological analysis. In mice, arterial thrombi were induced using two distinct experimental models, ferric chloride (FeCl3) and wire injury of the carotid artery. We found that murine arterial thrombi induced by FeCl3 were highly concordant with human coronary thrombi regarding their immune cell composition, with neutrophils being the most abundant cell type, as well as the presence of NETs and coagulation factors. Pharmacological treatment of mice with the protein arginine deiminase (PAD)-inhibitor Cl-amidine abrogated NET formation, reduced arterial thrombosis and limited injury in a model of myocardial infarction. CONCLUSIONS: Neutrophils are a hallmark of arterial thrombi in patients suffering from acute myocardial infarction and in mouse models of arterial thrombosis. Inhibition of PAD could represent an interesting strategy for the treatment of arterial thrombosis to reduce neutrophil-associated tissue damage and improve functional outcome.