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Thomas P Stossel

Publications and source records attributed to Thomas P Stossel.

At least 19 recordsLinked to original sources
PubMed2007

Plasma gelsolin is a marker and therapeutic agent in animal sepsis.

OBJECTIVE: Plasma gelsolin is a circulating actin-binding protein that serves a protective role against tissue injuries. Depletion of plasma gelsolin in systemic inflammation may contribute to adverse outcomes. We examined the role of plasma gelsolin in animal models of sepsis. DESIGN: Animal and laboratory experiments. SETTING: Academic research laboratory. SUBJECTS: Adult male mice. INTERVENTIONS: Mice subjected to endotoxin or cecal ligation and puncture (CLP) were treated with exogenous plasma gelsolin or placebo. MEASUREMENTS AND MAIN RESULTS: We document the depletion of plasma gelsolin (25-50% of normal) in murine models of sepsis associated with the presence of circulating actin within 6 hrs of septic challenge. Repletion of plasma gelsolin leads to solubilization of circulating actin aggregates and significantly reduces mortality in endotoxemic mice (survival rates were 88% in the gelsolin group vs. 0% in the saline group, p < .001) and in CLP-challenged mice (survival rates were 30% in the gelsolin group vs. 0% in the saline group, p = .001). Plasma gelsolin repletion also shifted the cytokine profile of endotoxemic mice toward anti-inflammatory (plasma interleukin-10 levels were 205 +/- 108 pg/mL in the gelsolin group vs. 39 +/- 29 pg/mL in the saline group, p = .02). CONCLUSIONS: We propose that circulation of particulate actin is a marker for sepsis-induced cell injury, that plasma gelsolin has a crucial protective role in sepsis, and that gelsolin replacement represents a potential therapy for this common lethal condition.

Actins↗
PubMed2006

Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin.

Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform, plasma gelsolin (pGSN). Blood concentrations of pGSN decrease in response to diverse tissue injuries. Depletion of pGSN to critical levels precedes and often predicts complications of injuries such as lung permeability changes and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. One proposed mechanism for pGSN's protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. However, no direct evidence in support of this hypothesis has been available. Here we show that recombinant pGSN modestly inhibited LPA-induced P-selectin upregulation by human platelets in the presence of albumin (P < 0.0001). However, physiologically relevant pGSN concentrations inhibit platelet-activating factor (PAF)-mediated P-selectin expression by up to 77% (P < 0.0001). pGSN also markedly inhibited PAF-induced superoxide anion (O(2)(-)) production of human peripheral neutrophils (PMN) in a concentration-dependent manner (P < 0.0001). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O(2)(-) generation (P = 0.024). Therefore, pGSN interferes with PAF- and LPA-induced cellular activation in vitro, suggesting a mechanism for the protective role of pGSN in vivo.

Blood Platelets↗
PubMed2006

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.

3T3 Cells↗
PubMed2006

Relationship of plasma gelsolin levels to outcomes in critically ill surgical patients.

OBJECTIVE: To examine the relationship between plasma gelsolin levels and mortality following surgery or trauma. BACKGROUND: Few simple predictive diagnostic tests are available to predict mortality following surgery or trauma. We hypothesize that plasma concentrations of gelsolin, a protein that responds to injured tissue, might be a predictor of patient outcomes. METHODS: We conducted a prospective, observational study in the surgical intensive units (ICU) at a tertiary care teaching hospital. A total of 31 patients were enrolled in the study. Chart abstraction was used to gather data about patient demographics, clinical characteristics, and clinical outcomes. Plasma gelsolin concentrations were assessed serially on day 0 through day 5. RESULTS: Low plasma gelsolin levels were associated with increased risk of death occurring in the ICU. Plasma gelsolin levels lower than 61 mg/L predicted longer ICU stay, prolonged ventilator dependence, and increased overall in-hospital mortality. CONCLUSION: Plasma gelsolin is a potential prognostic biomarker for critically ill surgical patients. Plasma gelsolin replacement may have therapeutic application.

Adolescent↗
PubMed2005

The structure of the GPIb-filamin A complex.

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.

Contractile Proteins↗
PubMed2005

Arp2/3 complex-deficient mouse fibroblasts are viable and have normal leading-edge actin structure and function.

RNA interference silencing of up to 90% of Arp3 protein expression, a major subunit of the Arp2/3 complex, proportionately decreases the intracellular motility of Listeria monocytogenes and actin nucleation activity ascribable to the Arp2/3 complex in mouse embryonic fibroblasts. However, the Arp2/3-deficient cells exhibit unimpaired lamellipodial actin network structure, translational locomotion, spreading, actin assembly, and ruffling responses. In addition, Arp3-silenced cells expressing neural Wiskott-Aldrich syndrome protein-derived peptides that inhibit Arp2/3 complex function in wild-type cells retained normal PDGF-induced ruffling. The Arp2/3 complex can be dispensable for leading-edge actin remodeling.

Actin-Related Protein 2-3 Complex↗
PubMed2005

Ca2+ and calmodulin regulate the binding of filamin A to actin filaments.

Filamin A (FLNa) cross-links actin filaments (F-actin) into three-dimensional gels in cells, attaches F-actin to membrane proteins, and is a scaffold that collects numerous and diverse proteins. We report that Ca(2+)-calmodulin binds the actin-binding domain (ABD) of FLNa and dissociates FLNa from F-actin, thereby dissolving FLNa.F-actin gels. The FLNa ABD has two calponin homology domains (CH1 and CH2) separated by a linker. Recombinant CH1 but neither FLNa nor its ABD binds Ca(2+)-calmodulin in the absence of F-actin. Extending recombinant CH1 to include the negatively charged region linker domain makes it, like full-length FLNa, unable to bind Ca(2+)-calmodulin. Ca(2+)-calmodulin does, however, dissociate the FLNa ABD from F-actin provided that the CH2 domain is present. These findings identify the first evidence for direct regulation of FLNa, implicating a mechanism whereby Ca(2+)-calmodulin selectively targets the FLNa.F-actin complex.

Actins↗
PubMed2005

The macrophage alphaMbeta2 integrin alphaM lectin domain mediates the phagocytosis of chilled platelets.

alpha(M)beta(2) integrin receptors on myeloid cells mediate the adhesion or uptake of diverse ligands. Ligand binding occurs in the alpha(M) chain, which is composed of an I domain and a lectin domain. The alpha(M) I domain binds iC3b, fibrinogen, intercellular adhesion molecule-1, and other ligands and mediates the adhesion of neutrophils to platelet glycoprotein Ibalpha (GPIbalpha). alpha(M)beta(2) also recognizes beta-GlcNAc residues on GPIbalpha that are clustered on platelets after cooling. The phagocytosis of chilled platelets could be reconstituted when Chinese hamster ovary cells were transfected with alpha(M)beta(2). Replacement of the I domain or the lectin domain of the alpha(M) chain with the corresponding domain from the alpha(X) chain (p150) revealed that the activity of the alpha(M)beta(2) integrin toward chilled platelets resides within the lectin domain and does not require the I domain. Additional evidences for this conclusion are: 1) Sf9 cells expressing solely the alpha(M) lectin domain bound chilled platelets, and 2) soluble recombinant alpha(M) lectin domain inhibited the phagocytosis of chilled platelets by alpha(M)beta(2)-expressing THP-1 cells, whereas I domain substrates showed no inhibitory effect. Therefore chilled platelets are removed from blood by an interaction between beta-GlcNAc residues on clustered GPIbalpha and the lectin domain of alpha(M) chain of the alpha(M)beta(2) integrin, distinguishing this interaction from those mediated by the alpha(M) I domain.

Animals↗
PubMed2004

Effect of thrombopoietin alone and a combination of cytochalasin B and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-AM on the survival and function of autologous baboon platelets stored at 4 degrees C for as long as 5 days.

BACKGROUND: PLTs stored at 22 degrees C have the potential for bacterial contamination, a problem that could be reduced by 4 degrees C storage. Nevertheless, PLTs stored at 4 degrees C exhibit a significantly reduced life span. This study was performed to determine whether treatment of PLTs with thrombopoietin or cytochalasin B plus ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)-AM could prevent exponential loss of PLTs stored at 4 degrees C. STUDY DESIGN AND METHODS: Autologous baboon PLTs were stored at 22 or 4 degrees C. The 4 degrees C stored PLTs were treated with 1.5 ng per mL thrombopoietin, with 1 micro mol per L cytochalasin B, and 80 micromol per L EGTA-AM (cyto-EGTA) or not treated and labeled with (111)In-oxine to study their in vivo recovery and life span. PLT function was assessed by correction of an aspirin-induced prolonged bleeding time. Aggregation responses and morphology were also assessed. RESULTS: PLTs stored at 22 degrees C had normal in vivo recovery and linear survival. PLTs stored at 4 degrees C, whether or not they were treated with thrombopoietin, had normal recovery and exponential survival. Aggregation of cyto-EGTA-treated PLTs was similar for PLTs stored at 4 degrees C and fresh PLTs, but decreased in PLTs stored at 22 degrees C for 5 days. The addition of cyto-EGTA to PLTs before 4 degrees C storage inhibited morphologic changes that occurred in PLTs stored at 22 degrees C and cold-induced PLT clumping, but did not prevent exponential disappearance of the PLTs. CONCLUSION: Addition of thrombopoietin or cyto-chalasin B and EGTA-AM to PLTs before 4 degrees C storage did not prevent exponential loss of PLTs.

Animals↗
PubMed2004

Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site.

The Ras-mitogen-activated protein (Ras-MAP) kinase pathway regulates various cellular processes, including gene expression, cell proliferation, and survival. Ribosomal S6 kinase (RSK), a key player in this pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. Here we report the characterization of the cytoskeletal protein filamin A (FLNa) as a membrane-associated RSK target. We show that the N-terminal kinase domain of RSK phosphorylates FLNa on Ser(2152) in response to mitogens. Inhibition of MAP kinase signaling with UO126 or mutation of Ser(2152) to Ala on FLNa prevents epidermal growth factor (EGF)-stimulated phosphorylation of FLNa in vivo. Furthermore, phosphorylation of FLNa on Ser(2152) is significantly enhanced by the expression of wild-type RSK and antagonized by kinase-inactive RSK or specific reduction of endogenous RSK. Strikingly, EGF-induced, FLNa-dependent migration of human melanoma cells is significantly reduced by UO126 treatment. Together, these data provide substantial evidence that RSK phosphorylates FLNa on Ser(2152) in vivo. Given that phosphorylation of FLNa on Ser(2152) is required for Pak1-mediated membrane ruffling, our results suggest a novel role for RSK in the regulation of the actin cytoskeleton.

Amino Acid Sequence↗
PubMed2004

Blasts from the past.

With this issue of the JCI, we celebrate the 80th anniversary of the Journal. While 80 years is not a century, we still feel it is important to honor what the JCI has meant to the biomedical research community for 8 decades. To illustrate why the JCI is the leading general-interest translational research journal edited by and for biomedical researchers, we have asked former JCI editors-in-chief to reflect on some of the major scientific advances reported in the pages of the Journal during their tenures.

Animals↗
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

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↗
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

Prognostic implications of declining plasma gelsolin levels after allogeneic stem cell transplantation.

The idiopathic pneumonia syndrome (IPS) represents a common and often fatal complication of hematopoietic stem cell transplantation (HSCT). Gelsolin is a highly conserved actin-binding protein normally present in plasma that may serve a basic physiological role in limiting acute lung injury of diverse etiologies. We hypothesized that depletion of circulating gelsolin following HSCT might play a permissive role in the pathogenesis of IPS. Plasma gelsolin levels were measured by immunoblotting in frozen samples obtained weekly from 24 patients undergoing allogeneic HSCT. Patients with and without IPS were similar with respect to age, diagnosis, histocompatibility differences between donor and recipient, and conditioning regimen. Mean gelsolin levels in the 9 patients with rapidly fatal IPS were significantly lower than those in patients without this complication by week 3 after HSCT (101 +/- 61 mg/L versus 221 +/- 54 mg/L; P =.0002). Seven (88%) of the 8 patients with gelsolin levels of less than 100 mg/L in the first month after HSCT died from IPS within 3 months; conversely, gelsolin levels fell to less than 100 mg/L in 7 (78%) of the 9 patients who died from IPS within 3 months of HSCT (P =.0007). These findings suggest that gelsolin levels shortly after allogeneic HSCT can predict the later development of fatal IPS. Gelsolin replacement in selected transplant patients may offer a novel strategy to prevent or reverse IPS.

Adult↗