PubMed Health⌕ Search

Biomedical subjects

John H Hartwig

Publications and source records attributed to John H Hartwig.

14 recordsLinked to original sources
PubMed2004

WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells.

Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

Actins↗
PubMed2003

Glycosylation restores survival of chilled blood platelets.

Cooling of blood platelets clusters the von Willebrand factor receptor complex. Macrophage alphaMbeta2 integrins bind to the GPIbalpha subunit of the clustered complex, resulting in rapid clearance of transfused, cooled platelets. This precludes refrigeration of platelets for transfusion, but the current practice of room temperature storage has major drawbacks. We document that alphaMbeta2 is a lectin that recognizes exposed beta-N-acetylglucosamine residues of N-linked glycans on GPIbalpha. Enzymatic galactosylation of chilled platelets blocks alphaMbeta2 recognition, prolonging the circulation of functional cooled platelets. Platelet-associated galactosyltransferase produces efficient galactosylation when uridine diphosphate-galactose is added, affording a potentially simple method for storing platelets in the cold.

Acetylglucosamine↗
PubMed2003

Metalloproteinase inhibitors improve the recovery and hemostatic function of in vitro-aged or -injured mouse platelets.

Platelet transfusions are a crucial component of support for patients with severe thrombocytopenia. Storage of platelet concentrates, however, is associated with a reduction in platelet posttransfusion recovery and hemostatic function. In this study, we established a model of mitochondrial injury that resembles platelet storage lesion. Mitochondrial injury, provoked by incubation of platelets with carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to reduced posttransfusion recovery in mice, an effect that directly correlated with the duration of treatment. Damaged platelets were characterized by shape change, disruption of membrane asymmetry, surface expression of P-selectin, and profound proteolysis of GPIbalpha. Using our model, we identified a key role for endogenous metalloproteinase(s) in platelet clearance, as their inhibition markedly improved posttransfusion recovery of both the mitochondria-injured and in vitro-aged mouse platelets. Metalloproteinase inhibition also prevented proteolysis of GPIbalpha on damaged platelets, thereby improving the hemostatic function of these cells in vivo. We propose that inhibition of metalloproteinase activity during storage could significantly improve the effectiveness of platelet transfusions. Surface expression of GPIbalpha might be a powerful marker to determine the quality of platelet concentrates, because it reflects metalloproteinase activity in vitro.

Animals↗
PubMed2003

Role for phosphoinositide 3-kinase in Fc gamma RIIA-induced platelet shape change.

Platelets transform from disks to irregular spheres, grow filopodia, form ruffles, and spread on surfaces coated with anti-Fc gamma RIIA antibody. Fc gamma RIIA cross-linking leads to a tenfold increase in actin filament barbed end exposure and robust actin assembly. Activation of the small GTPases Rac and Cdc42 follows Fc gamma RIIA cross-linking. Shape change, actin filament barbed end exposure, and quantifiable actin assembly require phosphoinositide 3-kinase (PI3-kinase) activity and a rise in intracellular calcium. PI3-kinase inhibition blocks activation of Rac, but not of Cdc42, and diminishes the association of Arp2/3 complex and CapZ with polymerized actin. Furthermore, addition of constitutively active D-3 phosphorylated polyphosphoinositides or recombinant PI3-kinase subunits to octylglucoside-permeabilized platelets elicits actin filament barbed end exposure by releasing gelsolin and CapZ from the cytoskeleton. Our findings place PI3-kinase activity upstream of Rac, gelsolin, and Arp2/3 complex activation induced by Fc gamma RIIA and clearly distinguish the Fc gamma RIIA signaling pathway to actin filament assembly from the thrombin receptor protease-activated receptor (PAR)-1 pathway.

Actin-Related Protein 3↗
PubMed2003

Alpha-adducin dissociates from F-actin and spectrin during platelet activation.

Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. alpha-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activated through protease-activated receptor 1, FcgammaRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. We conclude that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form.

Actins↗
PubMed2003

WIP participates in actin reorganization and ruffle formation induced by PDGF.

Platelet-derived growth factor (PDGF) is a chemotactic factor for fibroblasts that triggers actin cytoskeleton reorganization by increasing the level of GTP-Rac, the activated form of a small Rho family GTPase. GTP-Rac induces membrane ruffling and lamellipodium formation that are required for adhesion, migration and macropinocytosis, among other functions. We have shown that WIP interacts with members of the Wiskott-Aldrich syndrome protein family and is essential for filopodium formation regulated by Cdc42 GTPase. In this report, we show that WIP participates in the actin reorganization that leads to ruffle formation. WIP overexpression in murine fibroblasts (3T3 cells) enhances ruffle formation in response to PDGF stimulation, as shown by immunofluorescence and electron and video microscopy. More importantly, microinjection of anti-WIP antibody or absence of WIP in murine fibroblasts results in decreased ruffle formation in response to PDGF treatment. Finally, overexpression of a modified form of WIP lacking the actin-binding site blocks PDGF-induced membrane ruffling. These data suggest a role for WIP in actin reorganization to form PDGF-induced ruffles. This is the first in vivo evidence in mammalian cells for a function of WIP dependent on its ability to bind actin.

Actin Cytoskeleton↗
PubMed2003

Mechanisms and implications of platelet discoid shape.

The platelet marginal band consists of a single peripheral microtubule (MT) that is wound in 8 to 12 coils and maintains discoid cell shape. About 90% of beta-tubulin in the marginal band is of the divergent, megakaryocyte (MK)/platelet-restricted beta1 isoform. beta1-tubulin-null mice show reduced proplatelet formation, thrombocytopenia, and platelet spherocytosis. Here, we show that structural abnormalities in resting beta1-tubulin-/- platelets include frequent kinks and breaks in the marginal band. Platelets derived from mice lacking the transcription factor GATA1 show similar defects, probably as a direct consequence of absent beta1-tubulin. beta1-tubulin+/- platelets have normal ratios of beta-tubulin isotypes but the marginal band is half the normal thickness, which is sufficient to maintain elliptical cell shape. Thus, a threshold 50% or less of the normal amount of beta1-tubulin is required to preserve marginal band integrity and cell shape. beta1-tubulin-/- platelets have normal size and contents and show no defects in serotonin release or aggregation. Accordingly, the apparently isolated spherocytosis allows investigation of the role of discoid platelet shape in hemostasis. On agonist stimulation, the disorganized MTs in beta1-tubulin-/- platelets fail to condense into central rings and instead are dispersed in short bundles and linear arrays. Nevertheless, intravital microscopy and flow chamber studies demonstrate full functionality of these spherocytic platelets under physiologic shear conditions. Together, these findings highlight the essential requirements of the MK/platelet-restricted beta1-tubulin isoform in platelet structure and suggest that spherocytosis does not impair many aspects of platelet function.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

Filling gaps in signaling to actin cytoskeletal remodeling.

A recent publication in the April 4 issue of Cell advances our understanding of stimulus response coupling leading to actin remodeling. It describes the identification of a novel membrane component Mig-2 that engages filamin A through a new intermediary, migfilin, to stimulate actin assembly and cell spreading on a substrate of extracellular matrix.

Actins↗
PubMed2003

Dynamic regulation of microtubule coils in ADP-induced platelet shape change by p160ROCK (Rho-kinase).

Platelet shape change is an extremely rapid process mediated by both the calcium-sensitive and p160ROCK pathways. The present study examines how different features of shape change studied by scanning electron microscopy clearly correlate to changes in the pattern of light absorbance measured in an aggregometer. Platelets change shape from the initial 'disc' form by producing: membrane 'blebs', sphere formation (cell-rounding), filopodia extension, and surface membrane folding. The presentation of these features was dramatically slower in the absence of intracellular calcium mobilization. In the presence of the p160ROCK-inhibitor, Y-27632, shape change was initially normal but platelets rapidly transformed back to smooth discs with extended filopodia. The reappearance of the disc shape is reflected by an increase in the amplitude of oscillations in the aggregometer shape change tracing. The kinetics of actin/cytoskeleton association correlated with filopodia formation but not with disc to sphere transformation. Changes in the level of tubulin polymerization correlated with changes from disc to sphere morphology. These experiments are consistent with a role for a RhoA/Rho kinase-regulated pathway in the maintenance of a spherical platelet shape after agonist-dependent activation. Continued disruption of the cytoskeletal microtubule ring, appears to be a Rhokinase-dependent event involved in the transformation of discoid platelets into spheres.

Actins↗
PubMed2002

Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts.

We investigated the effect of actin filament barbed end uncapping on Arp23 complex function both in vivo and in vitro. Arp23 complex redistributes rapidly and uniformly to the lamellar edge of activated wild-type platelets and fibroblasts but clusters in marginal actin filament clumps in gelsolin-null cells. Treatment of gelsolin-null platelets with the negative dominant N-WASp C-terminal CA domain has no effect on their residual actin nucleation activity, placing gelsolin actin filament severing, capping, and uncapping function upstream of Arp23 complex nucleation. Actin filaments capped by gelsolin or the gelsolin homolog CapG fail to enhance Arp23 complex nucleation in vitro, but uncapping of the barbed ends of these actin filaments restores their ability to potentiate Arp23 complex nucleation. We conclude that Arp23 complex contribution to actin filament nucleation in platelets and fibroblasts importantly requires free barbed ends generated by severing and uncapping.

Actin-Related Protein 2↗
PubMed2002

Normal Arp2/3 complex activation in platelets lacking WASp.

Arp2/3 complex is believed to induce de novo nucleation of actin filaments at the edge of motile cells downstream of WASp family proteins. In this study, the signaling pathways leading to Arp2/3 complex activation, actin assembly, and shape change were investigated in platelets isolated from patients with Wiskott-Aldrich Syndrome (WAS), that is, who lack WASp, and in WASp-deficient mouse platelets. WASp-deficient human and mouse platelets elaborate filopodia, spread lamellae, and assemble actin, identical to control WASp-expressing platelets. Human platelets contain 2 microM Arp2/3 complex, or 8600 molecules/cell. Arp2/3 complex redistributes to the edge of the lamellae and to the Triton X-100-insoluble actin cytoskeleton of activated WASp-deficient platelets. Furthermore, the C-terminal CA domain of N-WASp, which sequesters Arp2/3 complex, inhibits by half the actin nucleation capacity of octylglucoside-permeabilized and activated WAS platelets, similar to its effect in WASp-expressing cells. Along with WASp, platelets express WAVE-2 as a physiologic activator of Arp2/3 complex and a small amount of N-WASp. Taken together, our findings show that platelets activate Arp2/3 complex, assemble actin, and change shape in the absence of WASp, indicating a more specialized role for WASp in these cells.

Actin-Related Protein 2↗
PubMed2002

WIP deficiency reveals a differential role for WIP and the actin cytoskeleton in T and B cell activation.

WIP stabilizes actin filaments and is important for filopodium formation. To define the role of WIP in immunity, we generated WIP-deficient mice. WIP(minus sign/minus sign) mice have normal lymphocyte development, but their T cells fail to proliferate, secrete IL-2, increase their F-actin content, polarize and extend protrusions following T cell receptor ligation, and are deficient in conjugate formation with superantigen-presenting B cells and anti-CD3 bilayers. In contrast, WIP-deficient B lymphocytes have enhanced proliferation and CD69 expression following B cell receptor ligation and mount normal antibody responses to T-independent antigens. Both WIP-deficient T and B cells show a profound defect in their subcortical actin filament networks. These results suggest that WIP is important for immunologic synapse formation and T cell activation.

Actins↗