John H. Hartwig

Extracellular DNA traps promote thrombosis

Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.

Filamins as integrators of cell mechanics and signalling

Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.

Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin

Gelsolin, an 82 kDa actin-binding protein, has potent actin filament-severing activity in vitro. To investigate the in vivo function of gelsolin, transgenic gelsolin-null (Gsn-) mice were generated and found to have normal embryonic development and longevity. However, platelet shape changes are decreased in Gsn- mice, causing prolonged bleeding times. Neutrophil migration in vivo into peritoneal exudates and in vitro is delayed. Gsn- dermal fibroblasts have excessive actin stress fibers and migrate more slowly than wild-type fibroblasts, but have increased contractility in vitro. These observations establish the requirement of gelsolin for rapid motile responses in cell types involved in stress responses such as hemostasis, inflammation, and wound healing. Neither gelsolin nor other proteins with similar actin filament-severing activity are expressed in early embryonic cells, indicating that this mechanism of actin filament dynamics is not essential for motility during early embryogenesis.

Actin-binding proteins.

Much new information on the sequence, structure, and function of filament crosslinking, capping, and severing proteins is now known. Other significant findings include identification of a new abundant monomer-sequestering protein in platelets, and evidence that many actin-binding proteins interact with phosphoinositides and that this interaction may have metabolic consequences.

The biogenesis of platelets from megakaryocyte proplatelets

Platelets are formed and released into the bloodstream by precursor cells called megakaryocytes that reside within the bone marrow. The production of platelets by megakaryocytes requires an intricate series of remodeling events that result in the release of thousands of platelets from a single megakaryocyte. Abnormalities in this process can result in clinically significant disorders. Thrombocytopenia (platelet counts less than 150,000/microl) can lead to inadequate clot formation and increased risk of bleeding, while thrombocythemia (platelet counts greater than 600,000/microl) can heighten the risk for thrombotic events, including stroke, peripheral ischemia, and myocardial infarction. This Review will describe the process of platelet assembly in detail and discuss several disorders that affect platelet production.

The Clearance Mechanism of Chilled Blood Platelets

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.

Paneth cells as a site of origin for intestinal inflammation

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn’s disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1HM mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn’s disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1ΔIEC) or autophagy function (Atg16l1ΔIEC or Atg7ΔIEC) in intestinal epithelial cells results in each other’s compensatory engagement, and severe spontaneous Crohn’s-disease-like transmural ileitis if both mechanisms are compromised. Xbp1ΔIEC mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn’s disease as a specific disorder of Paneth cells.

N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.

The Wiskott–Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling

FilGAP is a newly recognized filamin A (FLNa)-binding RhoGTPase-activating protein. The GTPase-activating protein (GAP) activity of FilGAP is specific for Rac and FLNa binding targets FilGAP to sites of membrane protrusion, where it antagonizes Rac in vivo. Dominant-negative FilGAP constructs lacking GAP activity or knockdown of endogenous FilGAP by small interference RNA (siRNA) induce spontaneous lamellae formation and stimulate cell spreading on fibronectin. Knockdown of endogenous FilGAP abrogates ROCK-dependent suppression of lamellae. Conversely, forced expression of FilGAP induces numerous blebs around the cell periphery and a ROCK-specific inhibitor suppresses bleb formation. ROCK phosphorylates FilGAP, and this phosphorylation stimulates its RacGAP activity and is a requirement for FilGAP-mediated bleb formation. FilGAP is, therefore, a mediator of the well-established antagonism of Rac by RhoA that suppresses leading edge protrusion and promotes cell retraction to achieve cellular polarity.